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DIETARY REGULATION OF WHOLE BODY GLUCOSE DISPOSAL AND
CARBOHYDRATE METABOLISM IN HUMAN SKELETAL MUSCLE
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
TANYA PEHLEMAN
In partial fulfilment of requirements
for the degree of
Master of Science
August, 2000
OTanya Pehleman, 2000
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ABSTRACT
DIETARY REGULATION OF WHOLE BODY GLUCOSE DISPOSAL AND CARBOHYDRATE METABOLISM IN HUMAN SKELETAL MUSCLE
Tanya Lynn Pehlernan University of Guelph, 2000
Advisor: Dr. L.L. Spriet
This thesis investigated whole body giucose disposa1 and the adaptive
changes in skeletal muscle carbohydrate (CHO) rnetabolism after a 56-hr high
fat/low CHO diet (LCD; 5 % CHO, 73 % fat, 22 % protein). LCD increased the 90-
min area under the [glucose] and [insulin] curve (2 and 1 -25-fold) during an oral
glucose tolerance test (OGTT) (1 glkg). LCD increased resting pyruvate
dehydrogenase kinase (PDK) activity (0.1 91 0.05 vs. 0.083 + 0.02 min") and
decreased the activated f o n of pyruvate dehydrogenase (PDHa) at rest (0.38 +
0.08 vs. 0.79 + 0.1 0 mm01 acetyl-CoA/kglmin) and during OGTT (0.602 0.1 1 vs.
1 .O4 + 0.09) ( ~ 4 . 0 5 ) . LCD did not alter total GLUT-4 protein, total nexokinase
(HK) or glycogen synthase (GS) activity. It was concluded that glucose disposa1
was decreased after LCD and was related to restricted CHO oxidationi at the
level of PDH activation, secondary to increased PDK activity.
ACKNOWLEDGEMl3NTS
1 would like to thank my advisor Dr. Lawrence Spriet for his guidance and
support from start îo finish of this thesis. Lawrence's respect for his students and his
sincere involvement in their projects fosters the development of confidence to meet any
challenge with enthusiasm. 1 have considered it a pnvilege to work with him, and will be
forever grateful for everything 1 have learned in the process.
1 would also like to thank Dr, George Heigenhauser for his tirne and help over the
course of my graduate studies. In addition, 1 am grateful to Dr. Dave Dyck for always
taking the tirne to answer my questions when 1 knocked on his door dong the way!
A special thank-you to Dr. Sandra Peters who has been a wonderhl fnend and an
inspirationai role model, 1 have also been fortunate to work with Melissa Evans and
Ingrid Savasi. It is rare to work with a team of excellent students and friends.
Finaily, 1 would like to thank Steve for always believing in me, and rny family for
their encouragement.
TABLE OF CONTENTS
Acknowledgements ............................ .., ...................................... i
. . Table of Contents .....................~.....~............................................... LI
List of Tables .............................................................................. iv
List of Figures .................................................... .... ....................... v
List of Abbreviations .................................................................... vi
Introduction ................................................................................ 1
Literature Review ........................... ,... ..................................... Handling of a CHO load in normal subjects at rest ........................... Regulation of major sites involved in skeletal muscle glucose disposal at rest ................................................................................
Glucose transport ............................................................. .................................................... Glucose phosphorylation
Glucose storage ............................................................... Glucose oxidation ............................................................
Insulin Resistance .................................................................. Physiology ...................................... .. ............................. Mechanisms of insulin resistance .......................................... Tirnecourse of skeletal muscle adaptations to altered substrate availability that may downregulate insuiin-stimulated glucose
.................................... disposal .................................. .. ............................ Short-tem adaptations (min to 6-hrs)
Moderate-temz adaptations (>6-hrs tu 6-days) ................ Long-rem adaptations (> 6-days) .................... .. .........
Models used to study metabolic adaptations to altered substrate availability ........................................................................
Euglycemic hyperinsulinernic clamp + intralipid infusion ............ AnimaL Studies ...................................................... Humun Studies ......................................................
OGTT + intralipid infusion ................................................ High fat/iow carbohydrate diet ............................................
Animal Studies ...................................................... Human Studies .....................................................
Statement of the problem .............................................................. 40
.......................................... ................................... Hypotheses .. 41
Methods .................................... ... ...................................... 42 ...................................................... Pre-experimental protocol 42
........................................................... Experimental protocol 42 Bloodanalyses .......................... ... .................................. 45 Muscle analyses ...................... .... .................................... 45
...................................................................... PDK activity 45 ............................ Mitochondrial and total homogenate CS activity 47
...................................... .................... Enzyme activities ... 47 PDHa ...................................................................... 47
..................................................... GLycogen synthase 48 Hexokinase ............................................................... 49 .............................................................................. Pro tein 50
............................................................. Total GLUT-4 protein 50 ........................... ............................ Muscle metabolites .... 51
........................................................................ Calculations 51 ................................................................. Statistical analysis 52
....................................................................................... Results 53 ........................................................................ Diet analysis 53 ........................................................................ B lood results 53
Glucose and plasma insulin .................... ... ............... 53 Blood lactate, glycerol, b-hydroxybutyrate and plasma FFA ....... 54
............................................................. Muscle biopsy results 59 .......................................................... GLUT-4 protein 59 ........................................................ Enzyme activities 59
Hexokinase 59 . ....................................................... Glycogen synthase .............................................. 59
............................................................... PDK 60 PDHa .............................................................. 60
.................................................... Muscle îuels and metabolites 60 ......................................................... Muscle glycogen 60
...................................................... Muscle metabolites 60
.................................................................................. Discussion ............................................................... Insulin insensitivity
................................... Possible mediators of insulin insensitivity Sites of skeletal muscle adaptation to LCD ...................................
Glucose transport and phosphorylation .............................. ......................................................... Glucose storage
................................... Glucose oxidation .. ................ ..................................................... OGTT and acetyl-carnitine
Conclusion ........................................................................ .................................................................. Future directions
................................................................................. References Appendix .................... .... ........................................................
LIST OF TABLES
. ........................................ Table 1 Dietary analysis for CON and LCD 55
. Table 2 Muscle metabolites ........................................................... 64
Figure 1 . Potential sites of dietary adaptation leading to altered glucose ............................................................... disposal by skeletal muscle 6
Figure 2 . Regulation of glycogen synthase (GS) and glycogen phosphorylase ... 12
...................... Figure 3 . Acute regulation of pynivate dehydrogenase (PDH) 17
Figure 4 . Schematic of experimental protocol for both the CON and LCD trial .. 44
......... Figure 5 . Blood-[glucose] and plasma-[insulin] over Ume during OGlT 56
..... Figure 6 . Plasma FFA. and blood B.OH. glycerol and lactate during OGïT 57
............... Figure 7 . Western blot of total GLUT-4 protein for CON and LCD 61
Figure 8 . Basal (O-min) and insulin-stimulated (75-min) glycogen synthase .................................................................. fractional velocity (GSk) 62
...................................................... Figure 9 . PDK and PDHa activities 63
LIST OF ABBREVIATIONS
Acetyl-CoA AUC B-OH CHO CoA CON CS FFA G-6-P GFA GLYp GNG GS GS fv
GSK HK =NSP 1S&ly LCD LDH N2 NlDDM NMR OGTT PDH PDHa PDK PDP PFK PK PP1 PPARs RER TG VO2 VO2rna.x
acetyl-coenzyme A area under the curve beta-hydroxybutyrate carbohydrate coenzyme-A control citrate synthase fiee fatty acids glucose-6-phosphate glucose fatty acid cycle 2-hr area under the glucose curve during OGTT gluconeogenesis glycogen synthase glycogen synthase fiactional velocity glycogen synthase kinase hexo kinase 2-hr area under the insuiin curve during OGTT insulin sensitivity index high fat/low carbohydrate diet lactate dehydrogenase liquid nitrogen non-insulin-dependent diabetes mellitus nuclear magnetic resonance oral glucose tolerance test pyruvate dehydrogenase active pyruvate dehydrogenase pyruvate dehydrogenase kinase pyruvate dehydrogenase phosphatase phosphofiuctokinase pyruvate kinase protein phosphatase- 1 peroxisomal proliferator activating receptors respiratory exchange ratio triglyceride oxygen uptake maximum oxygen uptake
INTRODUCTION
Fat, in the form of free fatty acids (FFA), and carbohydrate (CHO) in the form of
glucose, are the two major fuel sources used by skeletal muscle to generate ATP under
most resting and exercise situations. The combination of fat and CHO used by the
muscle to meet an energy demand in resting and exercise situations is dependent on a
nurnber of factors including the training status of the individual, the oxygen uptake
required to meet the demand, and the concentration of substrates available to the muscle.
The net rate of skeletal muscle rnetabolism can increase by up to 100-fold during the
transition fiom rest to maximal exercise. However, in the resting situation, the energy
dernand and therefore the oxygen uptake by the muscie is low and relatively constant. A
function of resting muscle is to help clear a glucose load in the body by stonng glucose as
glycogen and by oxidizing some of the glucose load. The handling of a glucose load by
the resting skeletal muscle is the focus of the following work.
It is well known that increasing the acute availability of fat to the muscle
increases fat oxidation while decreasing the utilization of CHO. States of nutritional
alteration and disease where FFA levels are elevated andor CHO is restricted, are also
accompanied by a down-regulation in the handling of CHO by skeletal muscle. CHO
stores in the body are finite compared to fat stores, When CHO is not replaced in the
diet, liver glycogen depletes rapidly, within 24-hrs, and muscle glycogP ,n stores are
slowly depleted at rest. Tissues in the body that rely almost enürely on CHO for fuel
become cornprornised when CHO stores are depleted, making the ability of muscle to
preferentially oxidize fat and "spare" finite CHO stores advantageous in the face of
starvation or CHO restriction. In contrast, in a disease state such as non-insulin-
dependent diabetes mellitus -DM), CHO utilization by the muscle is downregulated
secondary to insulin resistance and contributes to the pathogenesis of the disease.
Recently in Our Iaboratory, a short-term high fat/low CHO diet was used to study
the regulation of pyruvate dehydrogenase (PDH) in skeletal muscle in response to a
decrease in CHO availability and an increase in fat availability for 3 and 6-days. In a
related pilot study, with two subjects, upregulation of pyruvate dehydrogenase kinase
(PDK) was found to be present as early as Zdays of the diet. In addition, disposai of an
oral glucose load was significantly decreased following only 2-days of LCD- This
suggested that the diet may have induced a state of decreased insuiin sensitivity, and
altered the handling of excess CHO by the resting muscle. Since our previous studies
indicated a rapid muscle adaptation at the Ievei of PDH regulation, we decided to M e r
examine the handling of glucose after a high fat/low CHO diet by investigating a number
of possible sites of CHO metabolism that may adapt and contribute to the altered
handling of glucose at rest. In the current work, a 56-hr high fatnow CHO diet (LCD)
was used to study the short-term adaptation of skeletal muscle to the disposal of a CHO
load following the alteration of substrate availability.
LITERATURE REVIEW
Handling of a CHO load in normal suhiects at rest
The postabsorptive state, defined as the period between an overnight fast and the
ingestion of a meal, is a common reference point for metabolic studies. After an
overnight fast, insulin concentrations are at basal levels, resulting in a low level of
glucose uptake by insulin-dependent tissues (20 to 25 %) (24). Non-insulin-dependent
tissues such as the brain and other tissues with an obligatory need for glucose, account for
75 to 80 % of the glucose utilizaton. Hepatic glucose output matches glucose utilization
in the postabsorptive state in order to maintain blood gIucose homeostasis. Hepatic
glucose output is a resuk of liver glycogenolysis (70 to 80 %) and glucogeogenesis
(GNG) (20 to 30 %). Liver glycogenolysis at this rate leads to the rapid depletion of
glycogen stores in this tissue during fasting. Liver glycogen is rapidly replenished with
the ingestion of CHO, and only 25 % of the repletion of liver glycogen following an oral
glucose load (lgkg) was due to splanchnic uptake of the glucose, suggesting that the
rnajority of Iiver glycogen repletion occurred via the GNG pathway (22).
When a glucose load is administered in the form of a meal or an oral glucose load,
the basal glucose disposa1 by the brain and other tissues remains constant and the increase
in peripheral glucose disposal ( ~ 3 0 0 %) is accounted for by insulin-dependent tissues. It
has been shown that after both oral (lg/kg) (22,42) and intravenous glucose
administration (18), 70 to 80 % of the glucose load is taken up by peripheral tissues,
predominantly s keletal muscle.
The metabolic fate of glucose within the muscle may not be identical after oral
and intravenous administration. Glucose storage is the major route of disposal of
intravenous glucose (18). During clamp studies the rate of glucose supply exceeds the
resting oxidative requirement and this may lead to overestimation of the role of glycogen
synthesis in the disposal of a normal glucose load (98). It is difficult to directly
quantitate changes in muscle glycogen concentration after both oral and intravenous
glucose administration due to the variability of biochemicd rneasurements in muscle
biopsies. One study used 13c-NM~ to demonstrate that fasted subjects (4 male, 4 fernale)
who ingested a 1900 kcal meal (60 8 CHO), stored 35 % of the CHO as glycogen in the
muscle (98). The increase in muscle glycogen content did not become significant until3-
hrs after the CHO meaI was ingested, despite the rapid timecourse of muscle gIucose
uptake measured after oral glucose (42).
In the basai state, the energy requirements of the muscle are supplied almost
entirely by iipid oxidation whereas the ingestion of oral glucose and subsequent
hyperinsulinemia stimulates CHO oxidation. Following an oral glucose load (lg/kg) at
rest, the overall energy requirements of muscle did not change as reflected by a constant
oxygen uptake (V02) over time (80-min) while the respiratory exchange ratio (RER)
increased significantly [Oldland, 2000 #135]. An increased RER following oral glucose
ingestion was associated with an increase in the calculated rate of CHO oxidation in this
study (basal; 44 & 22 to 75-min; 173 + 15 mg/min) and a similar increase following a 100
g oral glucose Ioad (85). The predominant fate of glucose taken up by the muscle
following oral CHO ingestion may be oxidation in contrast to glycogen synthesis.
Insulin, released in response to a glucose Ioad, is the main regulator that prevents
hyperglycaernia by promoting glucose uptake and storage in insulin-dependent tissues,
and by suppressing hepatic glucose release. In normal individuds, the ingestion of an
oral glucose load (lgkg) dramatically increases insulin-stimulated dnsposal of glucose
while hepatic glucose production is suppressed by approximately 50% (22).
When a CHO load is ingested at rest, blood glucose is iransported into skdetal
muscle by specific glucose transporters (GLUT-1 and GLUT4), and is subsequently
phosphorylated to G-6-P by hexokinase (HK). Inside the muscle, G-6-P is a substrate for
glucose storage as glycogen, and for glycolysis followed by oxidative glucose
metabolism. The two main fates of CHO inside the muscle are contralled by the activity
of glycogen synthase (GS) and pyruvate dehydrogenase (PDHa), respectively (Figure 1).
Since a i l of these processes are involved in the disposai of an oral glucose load, an
alteration at one or more sites may be involved in a state of insulin imsensitivity, where
glucose disposal by the skeletal muscle is reduced.
Fiare 1, Potential sites of dietary adaptation leading to aitered glucose disposal by skeletal mriscle. 1) glucose uptake, 2) glucose phosphorylation, 3) glucose storage, and 4) glucose oxidation
Muscle ceiI
' + Glucose - G-6-P -* -+ GLYCOGEN
1 PYRWATE
1 Mitochondria ,...*-
6 PDK i ri
Regulation of the ma-ior sites involved in skeletal muscle glucose disposa1 at rest
Glucose transport
Circulating blood glucose is taken up into cells by a family of integral carrier
proteins, known as glucose transporters (64). The two transporters found in skeletal
muscle are GLUT-1 and GLUT-4. GLUT-1 is found in low levels at the muscle
membrane, and regulates basal glucose uptake to support basic muscle cell fünctions.
The predominant glucose transporter in skeletal muscle is GLUT-4. GLüT-4 is localized
in intracellular vesicles under basal conditions and is translocated to the muscie ce11
membrane in response to insulin (64). The translocation of GLUT-4 to the muscle
membrane is mediated through an insulin-signaling pathway involving
phosphatidylinositol-3-kinase (34). The rapid redistribution and fusion of GLUT-4 with
the membrane results in a 30 to 40-fold increase in the ce11 surface content of the
transporter and pIays a dominant role in the disposal of glucose by the muscle under
conditions of insulin-stimulation. Most studies have shown that insulin increases glucose
transport through an increase in the Vmax and this appears to be mainly due to an
increased number of transporter proteins in the ce11 membrane.
Glucose phosphorylation
The hexokinases catdyze the ATP-dependent phosphorylation of hexose to
hexose-6-phosphate. In skeletal muscle, hexokinase (HK) catalyzes the transformation of
glucose to G-6-P:
Glucose + ATP G-6-P + ADP + Pi
There are four isofonns of hexokinase in mammalian tissues (HKI to IV) that differ in
tissue distribution, regulation and catalytic properties. HKII is the predominant isoform
expressed in skeletal muscle, but HKI is also present (28). HKI and II have a Km for
glucose in the subrniliimolar range and are allosterically inhibited by G-6-P.
The calculated Km of HKII for glucose in human skeletal muscle, which shares 94
% sequence hornology with the rat muscle isoform, is 0.13 mmol/l(56). G-6-P is a
potent inhibitor of HKII in skeletal muscle (28). The knowledge of the functional
organization and possible structure of HKII has rnainly been established from studies of
other HK isoenzymes. Studies of rat HKI suggest that ATP and glucose bind to the
catalytic C-temiinal of HK inducing a conformational change that prevents binding of G-
6-P at its allostenc site in the regulatory N-terminai (105). Binding of G-6-P induces a
conformational change that prevents the binding of ATP at the substrate site of the C-
terminal haif, but does not prevent the binding of glucose. Studies of human HKII have
shown that both the N- and the C- terminal halves of the enzyme have cataiytic activity
and are inhibited by G-6-P (4).
In vivo, hyperinsulinernia for a period of 4 to 6-hrs induces the expression of
HKII mRNA in human and rat skeletal muscle (56,77). In human skeletal muscle, only
one group (56) demonstrated that HKII activity was increased after 4-hrs of
hyperinsulinemia, while a representative study by KeIIey et al. (43) shows that this has
not been confirmed by other groups of researchers. Therefore, although insulin regulates
HKXI expression in skeletal muscle, the tirnecourse of the associated increase in HKII
protein and activity remains unconfïrmed. A single bout of exercise followed by 3-hrs of
rest also increased HKII mRNA in rat skeletal muscle (63) and increased HKTI mRNA
and activity in humans (50). The evidence for rapid induction of HKII expression and
possibly HKII activity in skeletal muscle suggest that it may be an important regulatory
site in situations where CHO utilization is altered.
Glucose storage
Gl ycogen s ynthase (GS) catalyzes the formation of a- 1,4-gl ycosidic linkages b y
transferrïng glycosyl units from uridine diphosphate glucose (UDP-glucose) to an
existing oligosaccharide chah:
UDP-glucose + glycogen. - UDP + glycogen,~
GS exists in two distinct isoforms, muscle-type GS and Iiver-type GS. Human muscle
GS has only 69% sequence homology to the liver isoforrn (61), whereas human and
rabbit muscle GS share 97% identity (1 10). It should be noted that other isoforms of GS
may exist. Two forms of glycogen exist in human skeletal muscle (1). MacrogIycogen is
recognized as the classic glycogen molecule, with a low protein and high CHO content.
Proglycogen exists as a stable intermediate form of glycogen and has a high protein
content. It has been proposed that the biosynthesis of proglycogen is mediated by a
separate proglycogen synthase, however so far this has not been well substantiated
biochemicaily (P.J. Roach, personai communication).
The initial stsp in glycogen biosynthesis involves the covalent attachrnent of
glucose to the priming protein glycogenin. Glycogenin has the ability to self-glucosylate
and catalyzes the sequential addition of glricosyl residues from UDP-glucose, in a
~ n ' + / ~ ~ * + - d e ~ e n d e n t reaction (76). The self-glucosylation results in the formation of a
chah of 8 to12 residues which is further elongated by GS when glycogenin and GS are
complexed together.
GS catalyzes the rate-limiting step in glycogen synthesis and is regulated by
reversible covalent phosphorylation and by Alostenc modification. The phosphorylated
forms of GS are inactive in skeletal muscle, while the dephosphorylated form of the
enzyme is active (60). The phosphorylation state of GS is determined by the activity of
regulatory protein kinases and a phosphatase enzyme (Figure 2). Traditionally, there are
at least three protein kinase enzymes that are known to phosphorylate and inactivate GS.
These are cyclic AMP-dependent protein kinase (also known as glycogen synthase
kinase-1 or GSK- l), phosphorylase kinase (dso known as GSK-2), and GSK-3 (60).
GSK-3 activity is specific for GS, while the other two participate in the coordinated
regulation of GS and glycogen phosphorylase, so that glycogen synthesis is inhibited in
situations where glycogenolysis predominates and vice versa (Figure 2). In vitro, a
number of additional protein kinases have been s h o ~ to phosphorylate GS (P.J. Roach,
personal communication), however the in vivo significance of the additional kinases has
not been determined. The dephosphorylation and activation of GS is achieved by a single
phosphatase enzyme, protein phosphatase- 1 (PP 1).
There are three important regulators that determine the relative rates of glycogen
synthesis and breakdown in the muscle (Figure 2). Ca* and CAMP (secondary to
elevated epinephrine) directiy and indirectly stimulate phosphorylase kinase to increase
glycogenolysis and decrease GS activity simultaneously (60). More importantly at rest,
insulin is thought to activate GS by increasing the activity of PP1 (19) or by decreasing
the activity of protein kinases, both of which would simultaneously decrease
glycogenolysis. Studies of rabbit muscle indicate that phosphate is released mainly fiom
three sites (3a, 3b and 3c) in response to insulin (67). The most important regulatory site,
3b, is specifically phosphorylated by GSK-3 (87). In addition, insulin was also shown to
promote dephosphorylation of site 2, which strongly inactivates GS and c m be
phosphorylated by a number of protein kinases in vitro (14) including phosphorylase
kinase. Therefore, insulin may activate GS by inhibiting a number of protein kinases or
by stimulating PPl, or both.
At physiological concentrations of G-6-P (0.1-0.5 mM), phosphorylated forms of
GS are much less active than the dephosphorylated form. G-6-P can allosterically
activate the phosphorylated form of GS (49), however G-6-P does not change drastically
in most aerobic situations in skeletal muscle (60). Therefore, the activity o f GS is rnainly
dependent on the overall phosphorylation state of the enzyme. Glycogen concentration
also plays a critical role in regulating GS activity through a negative feedback mechanism
(59).
Fiare 2. Regulation of glycogen synthase (GS) and glycogen phosphorylase. GSK- 3(glycogen synthuse kinasej), PK (CAMP-dependent protein kinase), PHOS-K (phosphoryl~se kinase), PPI (protein phosphatuse- 1).
Glycogen Phosphorylase (INACTIIE)
INSULIN PP I
(CAMP) : (Ca", INS ULZ-W
PK PHOS-K
Glycogen Phosphorylase - P (ACTIVE)
G-6-
~ 1 + 5 PHOS-K
GS-P GS - (INACTIVE) (ACTIVE)
Glycogen Phosphorylase - P (ACTIVE)
Glucose oxidation
Pymvate dehydrogenase (PDH) is a multienzyme complex that catalyzes the
conversion of pyruvate to acetyl-CoA:
Pyruvate + CoA + NADC ---+ Acetyl-CoA + NADH + COz
Since this is the first irreversible step in the oxidation of CHO, PDH controls the flux of
CHO-derived carbon into the TCA cycle. The complex contains multiple copies of three
enzymes; pyruvate dehdrogenase (El), dihydrolipoarnide transacetylase (E2), and
dihydrolipoamide dehydrogenase (E3). In addition to these catalytic components, PDH
contains a binding protein (protein X), which Links E3 to the complex, and two inbinsic
regulatory enzymes, a kinase and a phosphatase. Pyruvate dehydrogenase kinase (PDK)
phosphorylates the El subunit of PDH, resulting in inactivation of the complex, while
PDH phosphatase (PDP) dephosphorylates and activates PDH. The relative activities of
PDK and PDP determine the amount of complex in its active form, PDHa (8 1,94). In
contrast to GS, there is no direct allosteric regulation on the PDH complex itself, and the
metabolic effectors that regulate PDHa work through increasing and decreasing the
activities of PDK and/or PDP.
Acutely, PDK is regulated by intramitochondrial metabolic effectors (Figure 3).
Specificdy, a high NADHMAD+, acetyl-CoNfree CoA, and ATP/ADP ratio activate
PDIC, whereas pyruvate decreases PDK activity. At rest, PDK activity is high due to low
pyruvate, and high rnitochondrial effector ratios. PDH phosphatase (PDP) requires Mg+
and is activated by Ca+. During exercise, the increase in intraceliïlar Caf activates PDP
and the increase in pyruvate, as well as the decrease in ATP/ADP inactivate PDK.
Insulin has been shown to increase PDHa in rats (21) and in humans (57). In rats, the
acute effect of insulin to increase PDHa in alloxan diabetic rats was not mediated through
decreased PDK activity, suggesting that PDH is acutely activated by insulin through
direct PDP activation (21).
In the long-term, stable increases in PDK activity have been observed in
association with nutritional conditions (starvation, high fat feeding) or disease States
(alloxan diabetes). The mechanisms involved in the long-term regulation of PDK have
not been conclusively defined. However, possible rnechanisms may include a stable
upregulation of PDK specific activity or an increase in protein synthesis. Recent
evidence has indicated the existence of four isoenzymes in mammalian tissues (PDKl to
PDK4) that differ in tissue distribution, specific activity, and sensitivity to effectors (1 1,
29, 86). PDKl to 4 are present in human skeletal muscle with low Ievels of PDKl and 3
expression, while rat muscle expresses only the PDKl, 2 and 4 isoenzymes.
The kinetic properties and abundant expression of PDK2 in most tissues are
consistent with the idea that it is the isoenzyme mostly responsible for rnetabolic
regulation of PDH activity. Starvation and alloxan diabetes are common models used to
study the long-term regulation of PDK secondary to CHO restriction and elevated FFA.
A representative study by Feldhoff et al. (21) showed that PDK increased and PDHa was
decreased in 48-hr alloxan diabetic rats. Since P D M expression increases several-fold in
rat heart and skeletal muscle during starvation (72,95, 107), it is thought that this
isoenzyme may be important in the long-term adaptation of PDK. Adding to this idea is
the observation that PDK4 has a relatively high specific activity and is much less
sensitive to inhibition by dichloroacetate, the synthetic analogue of pyruvate (1 1).
Therefore, an increased expression of PDK4 would be consistent with an increase in total
PDK activity and a decrease in the ability of pyruvate to reactivate PDH, seen in rat
cardiac rnyocytes and skeletal muslce after 48-hrs of starvation (78,95). In addition,
Bowker-Kinley et al. (1 1) determined that PDK4 does not respond to NADH in
combination with acetyl-CoA. Therefore, if PDK4 is overexpressed in long-term
adaptation, the total kinase activation by these effectors rnay be blunted. This rnay be
important in situations where fat oxidation is elevated, leading to increâses in the
concentration of NADH and acetyl-CoA. In humans, a high fat diet caused a stable
increase in skeletal muscle PDK activity as quickly as 3-days, with a hrther increase at
6-days (71). A subsequent study (70) showed that PDK4 protein and mRNA increased
dunng the first 3-days of the high fat diet. The expression of PDK4 rnay be important in
the stable adaptation of PDK activity in human skeletal muscle, as suggested in rats.
There are a number of potentiai mediators that rnay play a role in the stable
increase in PDK activity. Elevated FFA and ketones contribute to the increase in PDK in
starvation and diabetes secondary to increased oxidation of these substrates which
increases acetyl-CoAKoA and NADWNAD'. There is also evidence ro suggest that
insulin rnay be directly involved in the stable adaptation of PDK. In 48-hr starved rats,
the rapid decrease in circulating insulin levels correlated with a stable increase in cardiac
PDK activity (65). The effects of insulin rnay be due to an indirect effect of circulating
FFA levels, however evidence from culture experiments shows that insulin rnay also have
a direct effect on PDK (65,97). In cultured cardiomyocytes, insulin opposed the effects
of n-octanoate and dibutyrl-CAMP to increase cardiac PDK activity (65), suggesting that
a decrease in insulin levels or a decrease in insulin sensitivity rnay be obIigatory for
stable increases in PDK (97). Insulin increased PDHa activity in diabetic rats within 1-
hr, while 72-hrs of low dose insulin (2 units/kg/day) or 48-hours of high dose insulin (40
unitskg/day), was required to decrease PDK activity to control values (21)- This
indicated that the long-term effects of insulin may directly invoIve PDK whereas the
acute effects of insuiin on PDHa may be PDK-independent.
In humans, Majer et al. (53) showed that the expression of the PDK2 and PDK4
isoforms in skeletal muscle fiom Pima hdians correlated with indicators of the severity
of insuLin resistance. PDK2 and PDK4 mRNAs wem positively correlated with fasting
plasma insulin, 2-hr plasma insulin in response to oral glucose and percentage body fat,
and were negatively correlated with insulin-mediated glucose uptake rates. Dunng a
100-min euglycemic hyperinsulinernic clamp, the Ievels of PDK2 and PDK4 rnRNA
decreased in response to insulin. These results demonstrated that insulin had a direct
effect on the expression of two PDK isoforms, and that an increase in PDK activity in
insulin-resistant subjects could be linked to a deficiency in insulin-stimulated
downregulation of PDK expression. In this case, decreased glucose oxidation would be a
consequence of insu!in resistance.
Fimire 3. Acute regdation of pyruvate dehydrogenase (PDH). Pymvate dehydrogenase kinase (PDK) phosphorylates and inactivates PDH. Pyruvate dehydrogenase phosphatase (PDP) dephosphorylates and activates the complex (PDHa). PDK and PDP are regulated by concentrations of intramitochondnd effectors.
CoA + NAD' NADH + COz
PYRUVATE PDHa
ACETYL-COA
Insulin Resistance
Physiology
The main actions of insulin following an oral glucose load include decreasing
blood glucose levels by promoting the utilization and storage of glucose in peripheral
tissues. Both an increase in the blood glucose Ievel at a given insulin concentration
andor an increase in the plasma insulin Ievel at a given glucose concentration indicate
that the tissues have become less sensitive to insulin (5). Specifically, insulin resistance
can be defined as a state in which insulin produces a subnormal biological response (41).
There are several methods used to measure insulin sensitivity clinically and
experimentally. The administration of an oral glucose load, followed by measurement of
blood glucose at timed intervals is called an oral glucose tolerance test (OGTT). Another
approach, the euglycemic hyperinsulinemic clamp, is often used experimentaily to study
insulin resistance. This method is performed under nonphysiological steady state
conditions, where a defined concentration of insulin is infused intravenorisly and variable
amounts of glucose are infùsed sirnultaneously to maintain a constant Ievel of blood
glucose. Since glucose and insulin are administered intravenously, physiological
hormonal responses and the kinetics of glucose absorption and insulin secretion are lost
(5).
Belfiore et al. (5) developed an insulin sensitivity index (ISIgi,) to quantify insulin
sensitivity using the glycemic and insuhnemic results of OGTT: ISIgly= 2/[(INSp X
GLYp)tl], where PNSp and GLYp are insulinernic and glycemic areas recorded during
O G n . Zt should be noted that discrepancy has been observed when comparing results
of OG?T and euglycemic hyperinsulinemic clamps. It is thought that the consistent
hyperinsulinemic suppression of FFA during the clamp technique may alter the short-
t e m effects of elevated starting FFA and remove an important causal component of
insulin resistance in certain populations.
Since the pnmary physiological indication of decreased insulin sensitivity is the
elevation of glucose andor insulin levels, Belfiore et al. (5) concluded that OGTT is a
simple procedure that can be used to determine whole-body insuLin sensitivity under
physio1ogical conditions.
Mechanisms of insuiin resistance
Altered insulin-stirnulated glucose disposal by skeletal muscle occurs as a result
of elevated fat availability andor CHO restriction, and may be due to adaptive changes at
a number of sites in CHO metabolism. These inciude: 1) glucose transport into skeletal
muscle (GLUT-1 and GLUT-4 transporters), 2) the subsequent phosphorylation of
glucose to G-6-P (HK), 3) the storage of glucose as glycogen (GS), and 4) the oxidation
of glucose (PDKIPDHa) (Figure 1). It is unlikely that experimentally induced insulin
resistance secondary to elevated fat availability over the short-term would alter these
mechanisms to the sarne degree as that of chronic insulin-resistant disease sthtes such as
NIDDM. Kowever, experimental data in normal subjects may provide some insight into
the development of these conditions. A number of models have been used to study the
interaction between insulin-stimulated CHO disposal and substrate availability. It is
important to separate models that induce short-term regulatory changes (minutes to 6-hrs)
vs. models that cause moderate (>6-hrs to 6-days) and long-term (> 6-days) adaptations
in insulin-stimulated substrate utilization. Different sites rnay be involved in
downregulating glucose disposal at different stages of skeletal muscle adaptation to
substrate availability in the development of insulin resistance.
Thecourse of skeletal muscle adaptations to altered substrate availability that may downregutate insulin-stimulated gIucose disposal
Short-term adaptations (min to 6-hrs)
The idea that dtering substrate availability changes the handIing of fat and CHO
by skeletal muscle was originally proposed by Randle et al. in 1963 (38). The reciprocal
interaction between fat and CHO rnetabolism was formally termed the "glucose fatty acid
cycle" (GFA), and was suggested as a possible mechanism for altered CHO metabolism
in diabetes, where FFA are elevated. The GFA was based on observations of the acute
effects of FFA (minutes) to decrease the uptake and oxidation of glucose in contracting
rat heart and resting diaphragm muscle. The central mechanism for the GFA was
proposed to be an increase in the mitochondrial acetyl-CoA and citrate content,
secondary to the increased delivery and oxidation of FFA. The increase in acetyl-CoA
was believed to inhibit the activity of PDH by increasing PDK activity, and the increase
in mitochondrial citrate was believed to increase transport of citrate into die cytoplasm
where it would inhibit the glycolytic enzyme phosphofructokinase (PFK). The secondary
accumulation of G-6-P was proposed as an indirect mechanism leading to the inhibition
of HK and ultimately glucose uptake. These mechanisms were supported in classical
studies by in vitro inhibition of PDH, PFK and HK by acetyl-CoA, and by high levels of
citrate and G-6-P, respectively (16,74,79,99). Re-examination of PFK regulation using
physiological enzyme concentrations reveded less potent effects of citrate than originally
proposed (108). It was subsequently shown that the most potent inhibition of PFK is
present at resting citrate concentrations (73), and it was speculated that additional
increases in citrate achieved by experimental elevation of FFA would not lead to further
increases in PFK inhibition.
In addition to the ciassic indirect role of HK in rnediating glucose disposal,
evidence for rapid adaptation at the level of HK is also based on changes in HKII rnRNA,
and possibly in HKII activity, during hyperinsulinernia (4 to 6-hrs) and 3-hrs following a
single bout of exercise, as discussed previously. The rapid alteration of HKII rnRNA
rnay be important in regulating glucose disposal since total HK protein is related to the
total activity.
In vivo, the short-term (hrs) inhibitory effects of FFA on insulin-stimulated
glucose disposal and oxidation have been repeatedly shown by indirect cdorimetry in
conjunction with euglycemic hyperinsulinernic clamps. These studies will be discussed
in a subsequent section. In sumrnary, fat infusion during euglycernic hyperinsulinernic
clamps in rats and humans rapidly decreases glucose oxidation within 2-hrs, and glucose
disposal within 3 to 4-hrs, although most of these studies do not measure glucose
transport, HK, PDHa and PDK or metabolite effectors directiy. A decrease in non-
oxidative glucose disposal ar;d GS activity occurs after 4 to 6-hrs. This would be
expected in a situation where glucose is continuously administered while glucose
oxidation is suppressed by artificidly elevated FFA, leading to extra glycogen
accumulation within the muscle.
OveralI, there is evidence to suggest that many sites of CHO metabolism may
adapt rapidly to elevated fat availability. In an acute situation, this may be due to altered
levels of metabolic regulators in skeletal muscle. More direct rneasurements in skeletd
muscle are required to determine the mechanisms underlying the reduction in insulin-
stimulated glucose disposal in short-term manipulations of fat availability.
Moderate-term adaptations (>6-hrs to 6-days)
Although studies that alter substrate availability over a moderate timecourse are
lirnited, the literature does suggest that the acute effects of increasing FFA availability
rnay be supplemented with longer-term mechanisms that downreguiate CHO utilization
over a period of hrs to days. The main models used to study this timecourse are
starvation, chemically induced diabetes (alloxan diabetes) and high fat/low CHO diets.
In contrast to FFA infusion studies where FFA leveis are elevated, in starvation, diabetes,
and CHO restriction, eievated FFA as well as low CHO and insulin levels (or decreased
insulin sensitivity) may be important in the adaptation of the skeletal muscle.
In general, early diet studies showed that 5-days of a high fatflow CHO diet
decreased glucose tolerance in human subjects (30). Recently, a study in Our laboratory
(7 1) used a high fat/low CHO diet mode1 in humans to directly examine the adaptation of
skeletal muscle to altered dietvy substrate availability at the level of PDK activity over a
sirnilar timecourse. The administration of a high fatnow CHO diet for 3 and 6-days
resulted in a stable increase in PDK activity (3 to 5-fold). This may be an important site
of downregülation of CHO utilization that could be involved in the altered insulin-
stirnulated glucose disposal following a high fatAow CHO diet. The stable adaptation of
PDK in humans was consistent with PDK findings in rats following 24 to 48-hrs of
starvation, a state where substrate availability is altered such that FFA and ketones are
elevated. Rodent studies have s h o w that starvation increased the activity of PDK 2 to 3-
fold in extracts of heart (65), skeletal muscle (90) or liver (93) rnitochondria. Alloxan
diabetes (48-hrs) aiso resufted in an increased PDK and decreased PDHa (21). These
studies provided evidence for a longer-tenn mechanism for the stable activation of PDK
that persisted through rigorous rnitochondrial extraction procedures, and incubation of the
rnitochondna with an uncoupler to convert PDH to PDHa. As discussed earlier, some
evidence suggests that decreased insulin levels associated with these conditions rnay
potentiate the stable increase in PDK resulting in decreased CHO metabolism.
In summary, the stable adaptation of PDK described above indicates that this is
one important site that rnay be involved in the shift in glucose handling by skeletal
muscle secondary to an elevated supply of FFA and ketones and/or to a decrease in CHO
availability. With respect to the other possible sites of adaptation in resting skeletal
muscle, adaptations in glucose transport, HK and GS secondary to altered substrate
availability have not been exarnined over this tirnecourse. Since some of these sites can
adapt to altered substrate availability over min to hrs, it would seem possible that
adaptations at these sites may also be extended into the short-term to days).
Long-term adaptations (>6-days)
Studies examinhg skeletal muscle adaptations to substrate avaiIability over the
Iong-term often involve human subjects with non-insulin-dependent diabetes mellitus
(NTDDM), since this condition is associated with chronic muscle insulin resistance.
Also, animai models use chronic high-fat feeding (typically 28-days) to induce skeletai
muscle insulin resistance and to study the possible underlying mechanisms.
Euglycemic hyperinsulinemic clamp s tudies in NIDDM subjects have s ho wn bo th
a decrease in skeletal muscle G-6-P (84) as well as an increase in intracellular free
glucose and G-6-P concentrations (55). These results suggest defects in glucose transport
or phosphorylation, and defects in a step distal to the G-6-P pool respectively, as possible
rate limiting steps in CHO metabolism in NIDDM. Vestergaard et al. (103) found
decreased HKII mRNA, protein and activity in NIDDM patients, however Kelley et al.
(43) did not observe a difference in total or HMT activity in the vastus IateraIis of
NIDDM subjects vs. non-diabetic controls. Glucose transport is a critical step in the
regulation of glucose metabolism and changes in the concentration of GLUT-4 and/or the
response of GLUT-4 to insulin may be involved in insulin resistance. Total GLUT-4
protein and gene expression is normal in NIDDM patients (69), however the translocation
of GLUT-4 to the plasma membrane in response to insulin may be impaired (1 11).
Long-term regulatory alterations leading to insulin resistance may involve chronic
adaptations in insulin-stirnulated GS activation, as seen in full-blown NIDDM. The
major defect in insulin stimulated muscle glucose metabolism in NIDDM is reported to
be decreased storage of glucose as glycogen, calculated based on indirect calorimetry
(100). Basal md insulin-stimulated GS mRNA expression were found to be reduced in
subjects with NIDDM (102, 104). It is interesting to note that decreased insulin
activation of GS Fractional velocity (GSn) has been observed in NIDDM patients during
euglycemic hyperinsulinernic clamps (40), but not during hyperglycemic
hyperinsulinemic clamps (55). Since defects in the GS gene itself have not been found,
aiterations in the pathway of insulin-stimulated GS activation may be important in insulin
resistance.
Long- term alteration of insulin sensitivity may involve an increase in PDK
mRNA and protein, or specific activity. Similar to starvation (48-hrs), high fat feeding
(28-days) in rats Ieads to a stable increase in PDK activity (65). Altered expression of
PDK isoforms has more recently been proposed as a mechanism invotved in the
adaptation of PDK activity and kinetics in response to disease or nutritional
manipulation, as discussed previously. In insulin-resistant Pima Indians with elevated
PDK rnRNA (53), a 100-min eugykemic hypennsulinemic clamp decreased PDK2 and
PDK4 mRNA to normal levels, indicating a direct effect of insulin on PDK expression.
This recent evidence suggests that altered expression of PDK isoforms and insufficient
insulin-mediated downregulation of PDK mEWA may be a cause of defects in insulin-
mediated glucose disposal in chronic insulin-resistant States. Further support for this
mechanism was observed in cardiac mitochondria of 28-day high fat fed rats (96) and in
alloxan diabetic rats (21), where acute insulin administration (1 to 6-hrs) failed to
suppress elevated PDK activity, in cornparison to control rats.
In sumrnary, evidence from subjects with NIDDM and from long-term dietary
manipulation of fat and CHO availability suggests that glucose transport/phosphorylation,
glucose storage and glucose oxidation may al1 be sites of skeletal muscle adaptation
involved in a state of insulin resistance.
Models used to study the metabolic adaptation to altered substrate availabilitv
The adaptation of skeletal muscle to altered substrate availability and the
subsequent changes in substrate handling and insulin sensitivity have been studied
extensively in animais including the hurnan. It has been shown repeatedly that elevating
the supply of FFA andor restricting CHO levels over different timecourses (min to days)
causes adaptations in skeletal muscle that inhibit glucose disposal, oxidation and storage.
Fat rnetabolism is also reciprocally downregulated when CHO supply is abundant (94).
However, the mechanisms underlying the adaptations of skeletai muscle in these
situations have not been conclusively defined. This review will focus on examining the
metabolic adaptations that occur in skeletal muscle from the perspective of alterations in
CHO metabolisrn in the presence of elevated fat availability and/or decreased CHO
availability. In addition, only studies that examine these interactions and mechanisrns in
normal subjects will be included, in contrast to those that examine adaptations secondary
to chronic disease States.
Two cornmon models used to experimentally decrease insulin sensitivity and alter
the metabolic handling of CHO are euglycemic hyperinsuhernic clamps with intralipid
infusion (vs. saline infusion), and a high fat and/or low CHO diet (vs. normal dietary
intake or a high CHO diet). A srnall number of studies have used the OGTT as a means
of studying aiterations in metabolism during the elevation of FFA. These models will be
reviewed in both animals and humans, where data is available.
Euglycemic hyperinsulinemic clamp + intralipid infusion
In humans and rodents, an increase in fat availability has been achieved by
infusing lipidheparin during euglycemic hypennsulinernic clamps to prevent the nomal
insulin-mediated suppression of FFA. In humans, continuous indirect calorimetry is used
to detennine rates of whole-body glucose oxidation during the clamp. The idmion of 3-
3~-glucose or 6,6-Dz-glucose coupled with measurements of specific activity in blood
sarnples is most often used to measure overall glucose turnover and is also used in
conjunction with calorimetry data to calculate nonoxidative glucose disposal. In rats,
insulin-stimulated whole body glucose utilization, glycolysis, and glycogen synthesis are
estimated based on tracer concentrations in the plasma and in the muscle glycogen pool.
Studies that attempt to directiy examine the mechanisms underlying altered substrate
handling using this mode1 usudly measure GS activity and G-6-P, and occasionaily
measure PDHa in skeletal muscle biopsies. However, in humans one study (83) used
13c-~MR spectroscopy to quanti@ the rate of muscle glycogen synthesis, and 3 L ~ - ~ ~ ~
spectroscopy to measure changes in G-6-P concentration in the muscle dunng clamp
experiments.
G-6-P is the end result of glucose transport and subsequent phosphoryiation by
HK in skeletal muscle, and is the initial substrate in the glycogen synthesis pathway. For
this reason, the concentration of G-6-P in the muscle is often measured dunng the clamp
protocols as an indicator of which of these mechanisms are altered to cause reduced
glucose disposd under experimental conditions of insulin resistance. It should be
recognized that G-6-P is also a source of pyruvate for PDHa. If the reduction in glucose
disposal is due to inhibition of insulin-stimuiated GS activity or decreased oxidative
glucose disposal, we would expect to see an accumulation of G-6-P in the muscle. If
glucose transport andor phosphorylation is irnpaired, we would expect to see a decrease
in the G-6-P pool.
Animal Studies
In rats, lipidhzparin infusion during the euglycemic hyperinsulinemic clamp
procedure does not consistently lead to the development of impaired insulin action at the
whole body Ievel during the first 2-hrs, however glucose disposal decreases with
iipidheparin infusion in the 3d hr, and thereafier. Consistent with human data, the effect
of the 2-hr clamp with elevated FFA on GS activity and glycogen rnass is variable.
When the lipidheparin infusion was extended to 5-hrs, Chalkiey et ai. (13) observed the
development of insulin resistance after 2 to 3-hrs and found that 3 to 5-hrs of FFA
elevation significantly impaired GS activity in rat skeletal muscle. Park et al. (66) also
found that glucose disposal during euglycernic hyperinsulinemia was only significantly
lower after 3 to 5-hrs of lipid elevation, and that GS activity significantly decreased at
this time. In the latter study, whole body glycoIysis was calculated to be lower
throughout the clamp with lipid/heparin infusion, consistent with a rapid downregulation
of glucose oxidation in the presence of elevated FFA. During the initial 3-hrs, this was
compensated by an increase in calculated whole body glycogen synthesis and an increase
in the accumulation of radioactivity in muscle glycogen. Also, in the initia1 hours of the
clamp, muscle G-6-P was elevated, suggesting that the FFA-induced inhibition of
glycolysis (PFK, PDH) resulted in the accumulation of G-6-P, which would then
stimulate GS activity. Another study (47) found that decreased glucose transport was
secondary to decreased glycolysis and the accumulation of G-6-P during a 3-hr intralipid
clamp.
In a more chronic elevation of FFA (24-hr inaalipid infusion) that examined
glucose transport as a possible limiting mechanism, whole body glucose disposal
decreased in lipid perfüsed rats (52). The amount of total GLUT4 protein was not
different, suggesting that penpheral insulin resistance after the elevation of plasma FFA
may have been due to a defect in translocation of the transporters to the plasma
membrane or transporter intrinsic activity.
Human Studies
The clamp model in normal human subjects has consistentiy resulted in impaired
insulin action at the whole body level, suggesting a state of decreased insulin sensitivity.
While oxidative glucose metabolism decreases rapidly under these conditions, a decrease
in non-oxidative glucose disposal only occurs after 4 to 6-hrs of intralipid infusion (8-10,
44). A representative study by Yri-Jarvinen et al. (109) is typical of some studies that
have not shown a decrease in insulin-stimulated GS activity during a 2-hr clamp. It is
accepted that this discrepancy is due to the time dependence of the fatty acid inhibition of
GS in the euglycemic hyperinsulinemic clamp situation (10).
As suggested earlier, muscle G-6-P levels rnay provide insight into the
mechanisms underlying altered skeletal muscle glucose disposal in this experimentaI
model. Roden et al. (83) measured a decrease in muscle G-6-P concentration starting at
1.5 hours of a euglycemic hyperinsulinemic clamp with elevated plasma FFA. After 3-
hrs, a decrease in glucose disposal was observed accompanied by a reduced rate of
glucose oxidation and glycogen synthesis, suggesting that initially, glucose
transport/phosphorylation was inhibited followed by a reduction in oxidative and non-
oxidative glucose disposal. Boden et al. (9) also found that decreased insulin-stimulated
glucose disposal only occurred after 3 to 4-hrs of fat infusion, despite decreased glucose
oxidation and an elevated acetyl-CoAfCoA ratio within the first hour. The decrease in
glucose disposal was associated with a decrease in GS activity, although no change in G-
6-P was rneasured. The elevated acetyl-CoNCoA was consistent with an inhibition of
PDHa in skeletal muscle secondary to FFA infusion, as measured by Kelley et al. (44). In
a subsequent study (8), the effects of FFA on decreasing glucose disposai were shown to
be dose dependent. After 4 to 6-hrs of fat infusion in normal subjects, high FFA
concentrations (750uM) were associated with impaired GS activity and an increase in G-
6-P concentration. This high FFA concentration is similar to that seen in NIDDM, where
non-oxidative glucose metabolism is thought to be the main defect responsible for
decreased glucose disposai. In contrast, Iower FFA concentrations (550uM) were
associated with a decrease in G-6-P and a speculated reduction in glucose
transport/phosphorylation. In normal subjects, at the lower FFA concentrations,
decreased CHO oxidation and glycogen synthesis contnbuted equaily to the overd1
decrease in glucose disposal.
Whereas most of the studies discussed above are. based on systemic measurements
of glucose metabolism, Kelley et al. (44) used the leg artenovenous balance technique to
more directly assess the effect of fat infusion on skeletai muscle glucose metabolism in
normal subjects. Maintenance of FFA for 4-hrs dunng hyperinsulinernia resulted in a 36
% decrease in the rate of glucose uptake. The calculated rate of glucose storage
accounted for 23 % of the glucose disposal during intralipid infusion (vs. 5 1 % in the
control clamp), and this was accompanied by a decreased GS activity at the end of the
clamp. The contribution of glucose to leg muscle oxidation was Iower when FFA were
rnaintained during the clamp (53% vs. 76 %), and PDHa activity was decreased
accordingly. The results cited above (44) were calculated based on data collected from
210 to 240-min and enzyme activities were measured only at 240-min, therefore it is
difficult to obtain a sense of the components contributing to muscle glucose disposal in
the early stages of the clamp, which may be physiologically relevant.
It should also be noted that although muscle glucose transporters are important in
insulin-mediated glucose homeostasis, studies investigating transport as a rate-Iimiting
step directly in humans are lirnited. Glucose transport is often combined with the
phosphorylation step and these are based on G-6-P levels as mentioned above. Both
gIucose transporters and HK should be examined directly in human skeletai muscle under
experimental conditions of insulin resistance.
Overall, euglycemic hyperinsulinemic clamp studies show that fat infusion
decreases CHO oxidation rapidly, probably by PDK-mediated inhibition of PDHa, and
that elevated FFA may also decrease glucose transport or phosphorylation. Fat infusion
decreases insulin-stimulated glycogen synthesis in a time and dose dependent manner. It
is suggested that the accumulation of glycogen within the initial hours of a euglycemic
hyperinsulinemic clamp may later inhibit GS activity. In addition, decreased levels of G-
6-P secondary to decreased glucose &ansport and phosphorylation may also lead to
decreased glycogen synthesis even when insulin-stimulation of GS is not impaired.
It is clear fiom studies using this mode1 that direct measurements of glucose
transport and glucose phosphorylation by HK are lacking and that the contribution of
these mechanisms to insulin resistance in this situation is not defined.
It is important to examine differences between these results and those measured
under more physiological conditions. The clamp studies examine the effect of elevated
FFA on the disposal of glucose without the physiological increases and fluctuations in
glucose and insulin concentrations that occur following the normal ingestion of CHO.
OGTT + intralipid infusion
The mechanisms involved in lipid-induced insulin resistance during euglycernic
hyperinsulinemia do not necessarily define the mechanisms that alter glucose metabolism
in the postprandial state of insulin-resistant subjects, where plasma glucose levels are
elevated. Although euglycemic clamps offer the advantage of precise control ofglucose
and insulin levels in experimental situations, the effects of elevated lipids may be more
relevant if evaiuated under the physiological conditions of OG?T
Euglycemic hyperinsulinemic clamp studies provide evidence for decreased
oxidation and storage of glucose during infusions of triglyceride (TG) emulsions,
however the same TG infusions in nomal subjects did not result in decreased glucose
storage during the acute hyperglycaemic conditions of an OGïT (85). More recently
(82), OGïT using double-labeled glucose with lipid infusion in normal subjects resulted
in an increase in non-oxidative glucose disposal.
It should be noted that glucose oxidation was decreased during OG'IT with
intralipid infusion (82, 85) and that this was accompanied by a moderate, although
significant glucose intolerance (85). An earlier study (26) also demonstrated that glucose
tolerance decreased despite higher serurn insulin levels in normal subjects during OGTT
(100 g glucose) with intralipid infusion. Indirect calorimetry in this study indicated that
glucose oxidation was lower during OGTT.
Although studies using OGTT are limited, they consistentiy show that glucose
oxidation is decreased in response to a glucose load when FFA are elevated during
OGTT, whereas non-oxidative disposal is not inhibited in this short-term insulin resistant
state. The non-oxidative results are in contrast to the results of clamp studies where a
constant infusion of glucose and insulin during the artificial elevation of FFA over a
period of hours leads to a decrease in the storage of glucose.
High fat/low CHO diet
Animal studies
In animals, high. fat feeding has been used as a mode1 to study the metaboiic
interactions and insulin resistance associated with elevated FFA and CHO restriction.
Chronic high fat feeding (2 to 4-wks) has been shown consistently to induce insulin
resistance in rats (45,46, 51,92), measured using the euglycernic hyperinsulinemic
clamp. The diets studied included those high in saturated fat (66.5 % fat as calories,
mainly shortening) (45,46) and (59% fat as calories, edible tallow and safflower oil)
(92), those high in polyunsaturated fat (59 % fat as calories, mainly saffiower oil) (51,
92), and those high in monounsaturated fat (59 % fat as calories, mainly olive oil).
Results of the various studies indicated that high fat feeding impaired insuIin-stimulated
glucose metabolism at the level of glucose transport, glycolysis, and glycogen synthesis.
The effects of high fat feeding in rats has been shown to be dependent on the type of fat
ingested (25,92). Diets high in saturated fat decreased muscle insulin sensitivity,
whereas diets substituted with n-3 polyunsaturated FFA reversed this effect (92). In one
study (25), the effects of a 28-day high fat diet (47 % fat by energy; 43 % lard and 4 %
corn oil) to increase PDK activity were completely suppressed with the dietary
substitution of 7 % n-3 polyunsaturated FFA (40 % lard and 7 % n-3) within 24-hrs.
In a recent study (43, rats were fed a high saturated fat (mainly shortening) diet
(high fat; 66.5 % fat, 12.5 % CHO, 21 % protein by calories) for 3-wks (vs. control; 12.5
% fat, 66.5 % CHO, 21 % protein by calories). Glucose metabolism was measured using
data from a 2-hr euglycernic hyperinsulinemic clamp, and directly by measuring enzyme
activities and protein levels. Foliowing the 3-wk high fat diet, plasma FFA
concentrations were elevated compared to the control diet. In contrast to the euglycemic
hyperinsulinemic clampAipid infusion studies, where FFA levels were artificially
elevated, FFA levels decreased sirnilarly in both groups during hypennsulinemia- The
high fat diet decreased insulin-stimulated glucose uptake. Results also showed that G-6-
P was increased, accornpanied by a decrease in GS activity and a reduced accumulation
of 3~-glycogen in skeletal muscle. There was no change in total GLUT-4 protein content
or in HKII activity. It was concluded that high fat feecling induced insulin resistance at
sites distal to the G-6-P pool, indicating that impaired glycolysis and glycogen synthesis
were mostly responsible for the decrease in glucose uptake. Also, the possibility of
decreased insulin-stimulated translocation of GLUT-4 cannot be mled out, despite the
unaltered levels of total GLUT-4 protein. It should be noted that in the study by Kim et
al. (43, glucose oxidation was not measured. We would expect that the oxidation of
glucose rnay also have been reduced since high fat feeding in rats increased PDK activity
(25) and subsequentiy decreased PDHa activity . Decreased oxidative disposal of
glucose rnay also have been contributing significantiy to the downregulation of glucose
disposal and the accumulation of G-6-P.
Other studies have also shown that high fat feeding is associated with insulin
resistance and a decreased insulin-stimulated glycolysis and glycogen synthesis (5 1, 9 1,
92). It is interesting to consider that rat studies have suggested that decreased insulin-
stirnulated GS activity rnay not be the primary mechanism responsible for insulin
resistance foliowing high fat feeding. In one study (46), insulin-stimulated GS activity
was increased during the initial days of high fat feeding and then decreased to control
values after 2-wks of the diet. Insulin-stimulated glycolysis decreased rapidly, dunng the
initial Zdays of high fat feeding. Impaired insulin-stimulated GS activity rnay not be
involved as a mechanism underlying insulin resistance until2 to 3-wks of high fat
feeding in rats (45,46). Therefore, glucose oxidation rnay be an important primary event
leading to decreased glucose disposal under the conditions of a high fat diet. Chronic
high fat feeding (28-days) is known to induce a stable increase in PDK activity in rat
skeletal muscle (25), suggesting that this rnay be an important mechanism involved in the
adaptation of skeletal muscle to a high fat diet.
High fat diets have been shown to decrease insulin-stimulated glucose transport
without changing the total protein content of the transporters (31, 32,45, 112). The
decreased glucose transport is presumably due to a decrease in the translocation of
GLUT-4 to the plasma membrane in response to insulin, as confirmed by Zierath et al.
(1 12) in high fat fed mice. More proIonged exposure to a high fat diet (>IO-wks) resulted
in a decrease in GLUT-4 mRNA in rat soleus muscle (48). Nthough it is not measured
as ofien as the glucose transport step, post-transport phosphorylation of the glucose
seems to be unaffected in rodents following high fat feeding (1 12), as indicated by
unaltered HKII activity &ter a high fat diet.
Overail, chronic high fat diet studies in animal models seem to indicate that the
elevation of FFA induces insulin resistance and that a number of mechanisms may be
involved. Altered transport of glucose may contribute to the insulin resistance due to
decreased insulin-stimulated translocation of GLUT-4, whereas the major defects in
glucose disposal are distal to G-6-P, including glucose oxidation and glycogen synthesis.
Human studies
Studies using high fat andlor low CHO diets to examine the interaction between
elevated FFA and insulin resistance are lirnited. In an early study, Gordon et al. observed
a rise in FFA in normaI subjects foLlowing dietary restriction of CHO (27). Hales et al.
(30) measured plasma glucose, FFA and insulin dunng OGTT (100 g glucose) in normal
subjects following a 5-day low CHO diet (less than 50 g CHO per day, fat and protein
unlimited). The dietary alteration led to a reduction in glucose tolerance as evidenced by
higher gIucose concentrations at 30 and 60-min during OGTT, and maintename of
elevated plasma insulin at 60 and 150-min during O G T . FFA were elevated following
the low CHO diet, and were suppressed during OGTï in the control and diet condition.
The suppression occurred rapidly in the control condition, but was delayed following the
low CHO diet- Another study (3) showed that increasing the fat content of the diet from
43 to 65 % and decreasing the CHO content fiorn 40 to 20 %, resulted in a marked
deterioration of glucose tolerance in normal subjects. In a different study (88), a 3-day
high fat/low CHO diet (65% fat by energy) with normal daily energy intake resulted in an
enhanced piasma insulin response during OGTT (1.5g glucosekg). Therefore, subjects
required a greater level of insulin after LCD to achieve a similar rate of forearm glucose
uptake and to maintain simiiar blood glucose concentrations.
Anderson et ai. (2) demonstrated that a 5-day low CHO diet (57 g CHO/day) did
not impair glucose tolerance in normal men if the fat content remained sirnilar to their
normal dietary fat intake (145 dday). However, when the fat content of their diet was
increased to 16 % higher than normal (168 g/day), the low CHO diet caused impaired
glucose toIerance. The glucose levels measured during OGTT were higher between 30
and 150-min after the 16 % increase in fat content of the diet. These results suggest that
the increased fat content of high fat diet models is important for the development of
glucose intolerance.
Decreased CHO oxidation may be involved in the downregulation of glucose
disposa1 seen after a high fat diet. The direct effects of a high fat diet on PDHa are not
well known. A significant reduction in resting RER has been observed following 3 and
5-days of a high fat diet (65 % and 72 % fat, respectively), suggesting that CHO
oxidation was downregulated by the dietary alteration (39, 88). Putman et al. examined
PDHa directly following exhzustive exercise and a 3-day high fat/low CHO diet (3 %
CHO, 51 % fat, 46 % protein) and found that resting skeletd muscle PDHa was
decreased vs. a high CHO diet (80). Cutler et al. (17) examined the effects of longer-
term (21-days) ingestion of a high fatnow CHO diet (8% CHO, 75% fat, 17% protein) on
glucose metabohm and insulin sensitivity. Indirect calonmeûy data indicated that
glucose oxidation was decreased during a euglycemic hyperinsulinemic clamp, and this
was accompanied by decreased PDHa activity measured in muscle biopsies.
Overall, diet studies in rodents and in humans indicate that elevating the dietary
intake of fat and/or restricting the intake of CHO is associated with a decrease in insulin-
stimulated glucose disposal. Results from Limited hurnan studies indicate that glucose
oxidation is downregulated whereas rodent studies suggest a nurnber of possible sites of
adaptation in CHO metabolism including transport, oxidation and glycogen synthesis. In
general, the mechanisms underlying the decreased insulin-stimulated glucose disposal
following a high fat and/or low CHO diet remain undefined, and the majority of the
studies in normal human subjects have not directly measured enzymes, proteins and
metabolites in the muscle. Therefore, although a decrease in insulin sensitivity following
a high fat/low CHO diet is not a novel finding, a comprehensive approach to examining
the possible sites of adaptation underlying the insulin resistance in humans is lacking in
the current literature.
More recently, in o u lab, enzymatic adaptation to a high fatnow CHO diet was
studied directly in muscle biopsies h m normal subjects. The diet (5 % CHO, 63 % fat,
33 % protein) resulted in a dramatic and rapid increase in PDK activity at 3-days (0.35 +
0.09 vs. 0.10 + 0.02 min-'), with a further increase at 6-days (0.49 + 0.06 min-'). Resting
PDHa activity was decreased accordingly (0.17 + 0.04 vs. 0.63 + 0.17 mm01 acetyl-
~ o ~ . m i n - ' - k ~ - ' ) after 6-days of the high fatllow CHO diet. In conjunction with elevated
blood FFA, glycerol and B-hydroxybutyrate levels following the diet, the decreased
PDHa strongly indicated that the diet shifted fuel metabolism by increasing the utilization
of fat and ketone bodies, and reducing CHO metabolism. A subsequent pilot study in Our
Iaboratory showed that PDK activity increased afier only 2-days of a high fatnow CHO
diet (10 % CHO, 63 % fat, 27 % protein) in normal subjects. In addition, blood glucose
increased to a higher peak and remained elevated throughout a 3-hr OGïT (lgkg)
following the high fat/low CHO diet when compared to pre-diet OGïT results. This
suggested that penpheral glucose uptake and utilization may be decreased as early as 2-
days after a high fat/low CHO diet, resulting in decreased glucose tolerance in normal
subjects.
STATEMENT OF THE PROBLEM
As discussed throughout this review, there are many possible sites of regulation
that may decrease the disposd of a CHO load when FFA are elevated and CHO is
restricted during LCD. Some of these sites include decreased glucose uptake (GLUT-
1,4), inhibition of glycolytic enzymes (PFK, HK), inhibition of glucose oxidation
(decreased PDHa, mediated by PDK), and decreased glucose storage (mediated by GS).
In addition, insulin receptor binding and insulin signaling events that precede these
glucose disposd mechanisms may also be irnpaired, however these were not discussed
within the scope of this review. Adaptations at any number of these sites may alter the
disposal of glucose by skeletal muscle.
A short-term (56-hr) high faülow CHO diet (LCD) mode1 was used to examine
adaptations in whole-body glucose disposal and insufin sensitivity following dietary
alteration in normal individuds. The first purpose of this study was to c o d m that the
short-term LCD altered whole body glucose disposai and insulin sensitivity in normal
subjects. The second purpose was to identify potential sites in the muscle where the
handling of CHO was altered. Specifically, the effect of LCD on total GLUT4 protein,
and on GS, total HK and PDWPDHa activity was examined.
HYPOTEESES
The overail hypothesis was that the short-term (56-hr) LCD wouid decrease
whole body glucose disposal and that this would be related to an increase in skeletal
muscle PDK activity and a decrease in PDHa.
Specific hypotheses
1. Whole body glucose disposal would be decreased following LCD.
2. Resting PDK activity wouId increase, and resting PDHa would decrease after
LCD. It was hypothesized that PDHa would be iower after the administration of
oral glucose in LCD vs. CON.
3. Total GLUT-4 protein would not be altered by LCD.
4. Total HK activity would not be different after LCD vs. CON.
5. Basai and insulin-stimulated GS activity would not be altered by the short-term
LCD.
METHODS
Six heaithy, male university students volunteered for this study, mean age and weight
(22.8 + 1.0 yr and 74.1 + 12.9 kg). Al1 of the subjects were aerobically active on a
regular basis (3 to 6 times per wk). The mean relative maximum 0 2 consumption
(VOzma) was 55.4 + 8.3 ml-rnin-L-kg-l (range 43.6 to 67.6 ml.min-'-kg-'). Subjects were
informed of the study protocol and associated nsks before giving their written informed
consent. The study was approved by the Ethics Cornmittees of McMaster University and
of the University of Guelph.
Pre-experimentalprotocol. Prior to the expenment, VOzm was determined using a
continuous incremental protocol on a cycle ergorneter (Excalibur, Quinton Instruments,
Seattle, WA) with a metabolic cart (SensorMedics Mode1 2900, Yorba Linda, CA).
Subjects also completed 3-day dietary records. Dietary records were analyzed using
Nutripro Diet Analysis Software (West Publishing, Salem, OR) to determine normal
dietary intake. This software was then used to design experimental diets. If necessary,
slight adjustments were made to the normal diet to achieve a standardized pre-diet (CON;
5 1 % CHO, 29 % fat, 20 % protein). A 56-hr LCD was designed for each individuai
subject (LCD; 5 % CHO, 73 % fat, 22 % protein). CON and LCD were eucaloric with
the normal diet of the subjects. The dietary composition (% CHO, % fat, % protein) is
reported as the mean of the six individual diets.
ExperimentaiprotocoL During the expenment, subjects reported to the laboratory on
two separate occasions (Figure 4). For 3-days prior to their first visit, subjects consumed
the standardized pre-diet, CON. Physical activity was restricted for 24-hrs before the
CON trial. On their first visit to the laboratory (DAY O), subjects arrived after an
overnight fast (10 to 12-hrs). A catheter was inserted into an antecubital vein of one
forearm, and a resting blood sample (-30-min) was obtained. Patency was maintained
with a sterile isotonic saline solution. One leg was prepared for muscle biopsies taken
from the vastus lateralis under local anesthetic, as previously described (7). A second
resting blood sample was taken followed by two resting muscle biopsies (O-min). A
portion of the frst biopsy was dissected free of blood and connective tissue, and
mitochondria were extracted on the fresh muscle for determination of PDK activity. An
oral glucose load (1 @kg) (Tmtol75, Custom Laboratories Inc., Baltimore, ML) was then
administered and blood samples were taken at 30-min intervals over a period of three
consecutive hours. Seventy-five min after the administration of the oral glucose, a third
muscle biopsy was taken from the vastus lateralis (75-min). An additional biopsy was
taken at this time if the amount of muscle was inadequate for the necessary analysis.
After completion of the 3-hr OG'M', subjects irnrnediately began their LCD. Detailed
dietary guidelines were provided for each subject in addition to al1 of the required food
for the 56-hr period. Physical activity was restncted to activities of daily living during
LCD, and alcohol was prohibited.
Following LCD, subjects fasted ovemight (10 to 12-hrs) and retumed to the
laboratory in the moming (DAY 3). The above protocol was repeated in the LCD
condition. Muscle biopsies were taken from the altemate leg at this time.
Fimire 4. Schematic of experimental protocol for both the CON and LCD trial.
Following the CON trial, subjects began LCD for 56-hrs followed by an overnight fast.
Subjects then returned to the laboratory for the LCD trial.
CONLCD caîheter lgkg glucose
-30 O 30 60 90 120 150 180 (minutes)
t
Blood t f t t f t
Fasted (IO-12h)
Muscle biopsy Oû fIU (75-min)
overnight 1 LEG PREP OGTT
Blood analyses. BIood samples (3 to 4 ml) were dram from the indwelling catheter.
One portion of wholz blood (200 pl) was added to 0.6 N perchloric acid (800 pl),
vortexed and centrifuged at 10,000 rpm for 1-min. The supernatant was removed for
analysis of glucose, phydroxybutyrate (B-OH), lactate and glycerol(6). A second
portion of whole blood was cenerifuged and 400 pl of plasma were removed into 5 M
NaCl (100 pl) and incubated for 30-min at 56OC to inhibit lipoprotein Lipase activity.
Plasma free fatty acids were measured in the plasma using a Wako NEFA C test kit
(Wako Chernicals, Richmond, VA). The remaining aliquot of plasma was analyzed for
insulin using a Coat-a-Count Insulin test kit (Diagnostics Products, Los Angeles, CA).
Muscle analyses. A portion of fresh muscle (-50 to 60 mg) was separated from the first
resting muscle biopsy and processed for the extraction of intact mitochondria to measure
PDK and citrate synthase (CS) activities. A second portion of the biopsy (-20 mg) was
frozen immediately in liquid nitrogen (&) for analysis of total homogenate CS. CS
activities were used to calculate mitochondnal recovery, and the quality of the
rnitochondrial extraction (discussed later). Extra muscle from the first biopsy was frozen
separately in N2 and was used for the measurement of muscle glycogen.
The second resting biopsy as well as the 75-min biopsies were frozen immediately
in N2 and stored for Iater analysis, including PDHa, HK, GS and muscle metabolites.
GLUT-4 protein was measured in the 75-min biopsy samples only.
PDK act ive . Intact mitochondria were extracted from the muscle homogenate using
differential centrifugation, as previously descnbed (38,54). The final rnitochonckial
suspension was incubated for 20-min at 30' C in a buffer containing 10 p M carbonyl
cyanide rn-chlorophenyl-hydrazone, 20 mM Tris-HCI, 120 mM KC1,2 rnM EGTA, and 5
mM potassium phosphate (monobasic), pH 7.4. This incubation decreases ATP
concentration to zero thereby causing complete conversion of PDH to the active form,
PDHa (20). Mitochondria were pelleted (7,000 g, 10-min) and stored in Nz for later
analysis of PDK activity.
PDK activity was measured following the protocol previously outlined in our lab
by Peters et al. (71). Briefly, the rnitochondnal pellet was resuspended in a phosphate
buffer, pH 7.0, and freeze-thawed twice to break al1 of the mitochondria. The suspension
was warmed to 30' C and two aliquots were removed into a sodium fluoride,
dichloroacetate buffer, pH 7.8 (dilution 1: 1) to lock PDHa activity through inhibition of
the phosphatase and kinase. These samples represent "zero-tirne" or "total P D H
Magnesium-ATP (final concentration 3 mM) was added to the remaining suspension and
timed samples were removed from the original suspension every 30-sec for 2-min, and
then every min up to 5-min. The samples were placed immediately into the sodium
fluonde, dichioroacetate buffer and stored on ice for analysis of PDHa by the
radioisotopic measurement of acetyl-CoA production as previously described (15,80).
PDK activity is reported as the apparent first-order rate constant of the inactivation of
PDH (min-') or as the slope of ln[%(PDHa activity with AïJ? addition)/(total PDH
without ATP addition)] vs. time (20, 101). The slope was deterrnined by regression
analysis.
Mirochondrial and total homogenate CS activity. CS activity was measured
spectophotometrically using an enzymatic method linked to a colored product, as
previously described (89). CS activities in the total muscle hornogenate (CShm), and in
mitochondrial suspensions were used to calculate the recovery and quality of the
rnitochondrial preparations (71). In the rnitochondrial suspension, extrarnitochondrial CS
(CS,,) was measured, and total CS (CS,) was measured after freeze-thawing to break the
mitochondria:
Fractional recovery = (CSts - CSed/CSh,
% Intact mitochondria = 100 X (CSts - CSem)/CSts
PDNa, A srnaII piece of frozen wet muscle (approxirnately 10 mg) was removed from
each of the biopsies under Nz for the determination of PDHa using tAe methods of
Constanin-Teodosiu et al. (15) as modified by Putman et al. (80). Briefly, muscle
samples were homogenized in a homogenizing buffer, pH 7.8 (30 p1:l mg), containing
NaF and dichloroacetate to inhibit PDH phosphatase and kinase respectively. PDHa
activity was deterrnined at 37OC by adding muscle hornogenate (30 pl) to a reagent
mixture, containing the necessary coenzymes (CoA, NADH and thiamine
pyrophosphate), as previously outlined (80). The reaction was initiated using pyruvate
foliowed by the removai of aliquots (200 pl) at precisely tirned intervals (1,2 and 3-min)
into 0.5 M PCA (40 pl) to stop the reaction. Samples were neutralized using 1.0 M
K2C03. The neutralized extracts were srored at -80°C for subsequent radioisotopic
analysis of acetyl-CoA (12). Linear regression of plots of acetyl-CoA vs. time was
performed to determine reaction rates. Total creatine content was measured in
neutralized PCA extracts of PDHa homogenates, using the method of Bergrneyer et al.
(6). PDHa activity was corrected to the highest creatine concentration in a set of biopsies
from a given subject. This allowed for compensation for the presence of blood and
connective tissue in the muscle biopsy samples. PDHa activity was calculated as mm01
acetyl-CoA per kg wet muscle per minute.
GZycogen synthase. A second piece of frozen wet muscle (6 to 10 mg) was removed from
the biopsies under N2 for the determination of GS activity. GS activity was calculated as
nmol of UDP-glucose incorporated into glycogen per minute per mg of protein, and was
used to calculate GS fractional velocity (GSfv). GSrv is defined as the activity of GS at 0.1
rnM G-6-P (active GS) divided by the activity at 10.0 rnM G-6-P (total GS). G& is a
sensitive indicator of in vivo GS activity (49) and the ratio increases in response to insulin
infusion in humans (56,57).
GS was measured fluorometrically as described by H e ~ k s s o n et al. (35) with
modifications. The muscIe samples were homogenized by hand in buffer (50 pl: I mg)
containing 50 mM Tris-HCI, 5 mM EDTA, 20 mM NaF, and 5 mM DT, pH 7.2-7.4.
Homogenates were centrifuged (7000 g / 8300 rpm, + 4 O C ) for 5-min and aliquots of the
cytosolic fraction (supernatant) were incubated for the determination of active and total
GS activity. The incubation media consisted of 50 mM Tris-HC1,2 mM EDTA, 10 rnM
NaF, 10 mM glycogen, 0.5 mM Dm, 0.02% BS A, and either 0.1 rnM or 10.0 mM G-6-
P, pH 7.2 to 7.4. Muscle homogenate (100 p1) was incubated with 450 pl each of 0.1
mM and 10.0 mM incubation media for 45411 at 37OC. The reaction was started with
the addition of 50 pl of UDP-glucose (final concentration 8 rnM), and was stopped by
heating at 90° C for 2.5-min. Samples were centrifuged and the supernatant was removed
for fluoromettic assay of UDP using the reactions catalyzed by pyruvate kinase (PK)
(PEP + UDP + Pynivate + UTP) and lactate dehyrogenase (LDH) (Pyruvate + NADH
i Lactate + NAD?. Bnefly, samples, blanks and standards (UDP, 25 pM to 150 pM)
were added to an assay reagent containing 20 mM Tris-HCl, 30 mM KC1,4 mM MgC12,
0.4 mM phosphoenolpyruvate, 20 p M NADH, 0.4 U/rnl LDH, pH 7.6. Pyruvate kinase
was added (3.0 U/ml) to stitrt the reaction. The samples were incubated for 15-min at
room temperature followed by fluorometric determination of NADH.
Note: In order to carry out fluorometric measurements after a 15-min incubation
period, a series of initial experirnental assays were run. These determined the endpoint of
the assay reaction and confirrned that any drift inherent to the assay over time was
consistent between blanks and standards as well as samples. In the initial assays, samples
were read fluorometrically followed by the addition of PK to start the reaction. Samples
were read at 1 and 10-min, and then every 5-min for a total of 40-min. Blanks, standards
and samples continued to drift between 1 and 10-min, however after 10-min the
fluorometric readings leveled out (ie. the reaction was completed) and were constant up
to 40-Mn with no indication of further dnfting. Therefore, in the final experimental
assays, an initial reading was taken followed by a 15-min incubation with PK at room
temperature and then a final reading.
Hexokinase. Maximat HK activity was measured at room temperature as origindly
described by Henriksson et al. (35) and modified by Phillips et al. (75), using a small
piece of wet muscle (3 to 5 mg). HK activity was determined in muscle homogenates by
measuring the fluorescence of NADPH (linked to [Gd-PI) against a range of G-6-P
standards (1.67 to 10.0 mM). HK activity was calculated as mol D-glucose incorporated
into G-6-P per hour per kg protein.
Protein. The protein content for GS and HK activity was rneasured using a BCA reagent
kit containing BS A standard (2 rng/rnl) and reagents (Pierce, Rockford, L).
Total GLUT-4protein. GLUT-4 protein content was deterrnined in collaboration with
Dr. Bonen at the University of Waterloo (Department of Kinesiology). Western blotting
procedures were used to determine the total protein, as previously described by this group
(58,75). A portion of frozen muscle (50 mg) fiom the 75-min biopsy samples was
homogenized and total membranes were isolated by centrifugation (250,000 g). Samples,
containing 30 pg protein were mixed with 62.5 pl of L a e d sarnple buffer containing
2.5 % dithiothreitol and brought to 125 pl with buffer containing 25 mM Tris (pH 8.3),
0.19 mM glycine, and 1 % sodium dodecyl sulfate (SDS). Sarnples were separated by
SDS-polyacrylarnide electrophoresis on a 12 % resolving gel and transferred to an
irnrnobilon polyvinylidene difluoride membrane (Millipore, Malton, ON) by
elecromembrane transfer (i 10 V, constant voltage, 90-min). GLUT-4 protein was
detected using a polyclonal immuno-A purified GLUT-4 antibody (1:750, East Acres
Biologicals) followed by incubation with a horseradish peroxidase-labeled second
antibody (15000, anti-rabbit imrnunoglobuIin G; Arnersharn). GLUT-4 protein was
visualized using an enhanced cherniluminescence detection system (Amersharn) and the
blots were quantified using a Macintosh LC cornputer with an Abaton scanner and
appropriate software (Scan Analysis, Biosoft, Cambridge, UK). GLUT-4 protein was
measured as the intensity of a fixed area and expressed in relative absorbante units.
Musck metabolites. The remainder of the frozen muscle was f'reeze-dried, powdered and
dissected of ail visible blood, connective tissue and fat. The muscle metabolites were
measured using standard enzymatic methods that link the concentration of the metabolite
to a product that absorbs light or fluoresces at a given wavelength, or to a labeled product
that can be measured radioisotopically. Glycogen content was measured in the resting (0-
min) samples (2 to 3 mg), as descnbed by Hanis et al. (33). The rest of the metabolites
were measured in resting and 75-min biopsies on neutralized PCA extracts. Creatine,
phosphocreatine (PCr), ATP, lactate and G-6-P were deterrnined by spectrophotometric
andysis (6,33), pyruvate was measured fluorometrically (68), and acetyl-CoA and
acetyl-camitine were determined by radioisotopic measurement (12). Al1 of the
metabolites were corrected to the highest total creatine concentration from a set of
biopsies to compensate for the presence of blood and connective tissue.
Calculations. A 90-min OGTï area under the curve (AUC) for blood-[glucose] and
plasma-[insulin] was calculated for each subject as follows; resting glucose and insulin
concentrations (O-min) were used as the baseline value and the total area of deviation
from the baseline was calculated between O and 90-min. Values above and below the
baseline were designated as positive and negative respectively.
An insulin sensitivity index for glycemia (ISI& was calculated to quantifi
whole body insulin sensitivity based on OGTï results, as descnbed by Belfiore et aL(5);
ISI(gly)= 2/[mSp x GLYp) + 11, where INSp and GLYp are 2-hr insulinernic and
glycemic areas during O G n . The change in ISI@ly) for each individual subject
following dietary aiteration was calculated by normalizing LCD results to a subject's
normal OGTT response (CON).
Resting or basal blood (glucose, P-hydroxybutyrate, glycerol and lactate) and
plasma (insulin and FFA) concentrations were calculated by averaging the -30 and O-min
concentrations.
Stutistical analyses. Paired student's t-tests were used to compare GLUT-4 protein
content, glycogen concentration, and PDK activity in muscle samples (CON vs. LCD).
AUC's for blood-[glucose] and piasma-[insulin] were cornpared using a paired student's
t-test. Basal concentrations of blood and plasma parameters were directly compared
between CON and LCD using a paired student's t-test. Blood parameters, enzyme
activities (PDHa, GS, HK), and muscle metabolite concentrations over time during
OG?T were analyzed using a two-way repeated rneasures ANOVA (time x diet) with a
Tukey post-hoc test for al1 painvise multiple comparisons. Al1 blood and muscle data are
presented as means t SE. Siopificance was accepted at pc0.05.
RESULTS
Diet analysis
Dietary compliance during CON and LCD was monitored and subjects completed
check-lists outlining specific quantities of foods as well as preparation instructions. If
subjects required slight modifications to the diet during the LCD, these were precisely
recorded after verbal approval, and then adjusted in a final dietary analysis. The subjects
consumed 51 + 0.5 % CHO, 29 + 0.5 % fat, 20 + 0.8 % protein in the CON diet (range
50.0 to 53.a %, 27.0 to 30.0 %, 17.8 to 23.0 %) and 5 + 0.2 % CHO, 73 + 0.6 % fat, 22 +
0.5 % protein (range 4.0 to 5.0 %, 71.2 to 75.0 %, 20.0 to 23.7 %) in the LCD diet. The
total energy intake was eucaloric with the subjects normal dietary intake. The total
caloric intake and breakdown of the diets is presented in Table 1.
Blood Resuits
Glucose and piasma insulin
Basal blood-[glucose] and plasma-[insulin] were lower following LCD vs. CON
(3.39 + 0.19 vs. 4.22 + 0.19 mM and 5.64 I 0.3 1 vs. 8.54 + 0.67 pIU/rnl) (p<0.05).
During OGTT, the blood-&lucosel decreased to the baseline level in CON and
decreased aimost to baseline in LCD at 90-min (Figure 5). The plasma-[icsulin]
decreased towards baseline values at 90-min in both conditions and then leveled out at
this concentration with slight decreases toward baseline in the final hour (Figure 5). A
90-min area under the curve (AUC) was calculated to compare the response to the oral
glucose load between conditions, since the majority of the glucose and insulin response
occurred within this time. The calculated 90-min AUC blood-[glucose] was 2-fold
higher and the AUC plasma-[insulin] was 1.25-fold higher following LCD vs. CON
(pcO.05). It should be noted that the 120 and 180-min AUC for blood-[glucose] were
significantly higher in LCD vs CON (pc0.05). However, the data was disproportionate
to the acfual diet effects on glucose disposal since blood glucose levels were well below
baseline after 90-min in CON, but not in LCD. The 120 and 180-min AUC for plasma-
[insulin] were higher after LCD vs. CON (p~0.05 and p=0.055 respectively).
In addition, an insulin sensitivity index for glycemia (ISI~gi,l) was calculated to
quantify whole body insulin sensitivity based on OG?T results (descnbed in Methods)
(4). There was a signif~cant decrease in insulin seiisitivity following LCD vs.CON
(ISIgly = 0.32 + 0.07 vs. 1) ( ~ ~ 0 . 0 5 ) .
Blood lactate, glycerol, p-hydroxybutyrate and plasma free fat@ acids
Basal blood [%hydroxybutyrate] (B-OH) and plasma F A ] were significantly
elevated after LCD. B-OH increased from O. 1 I + 0.02 to 0.29 t 0.05 rnmoVl and FFA
increased from 0.3 1 + 0.05 to 0.48 + 0.05 rnmol/l following LCD (p<0.05). FFA, B-OH
and glycerol were suppressed significantly vs. basal levels (O-min) during OGTï in both
conditions (pe0.05) (Figure 6). Blood lactate was lower from O to 120-min during OG'IT
following LCD (Figure 6) (NS). (See appendix for individual blood results).
Table 1. Dietary analysis for CON and LCD. CHO (carbohydrate), PR (protein), g (grams),Tot FAT (total fat), sat (saturated fat), mono (monounsaturated fat), poly (polyunsaturated fat). SE are presented in brackets-
CON LCD
kcal
Yo CHO
Yo FAT
?4o PR
g CHO
g PR
g Tot FAT
g sat
g mono
9 PO~Y
Figure 5. Blood-[giucose] and plasma-bulin] over time during OGTT. During OGTT, blood-[glucose] decreased more slowly and plasma-[insulin] reached a higher peak following LCD. "Significant ciifference within a given time point (LCD vs. CON), pcO.05.
Tlme (min)
+CON
-Ci- LCD
+CON
-iï- LCD
-30 O 30 60 90 120 150 180
T h e (mM)
Figure 6. Plasma FFA, and blood B-OH, glycerol and lactate during OGTT. " Significant difference within a given time point (LCD vs. CON) (pc0.05). Significant difference vs. basal concentrations (-30 and O-min) in both CON and LCD (pc0-05)
-
b b b b
Fiare 6. Plasma FFA, and blood B-OH, glycerol and lactate during OGTT. a Significant difference within a given time point (LCD vs. CON) (p<0.05). Significant difference vs. basai concentrations (-30 and O-min) in both CON and LCD @<O.OS)
+CON
4 LCD
-30 O 30 60 90 120 150 180
lime (min)
-o- CON
-a- LCD
-30 O 30 60 90 120 150 180
Tirn e (m in)
Muscle b io~sv results
GLUT-4 protein. Total GLUT-4 protein was unaltered by LCD (Figure 7). The
intensity of a fixed area for GLUT-4 was normalized to CON. GLUT-4 protein was 1.13
+ 0.06 (LCD) vs. 1 (CON). -
Enzyme activities
Hexokinase. Maximal HK activity was not altered by LCD (CON; 0.42 + 0.04 vs. LCD;
0.36 + 0.04 mol-kg%fL). There was also no change in maximal HK dunng OGTT in
either condition, CON; 0.43 + 0.05 and LCD; 0.40 + 0.05 motkg-'*hfl).
Glycogen synthase. At rest, GS activity (in the presence of 0.1 mM G-6-P) was sirnilar
in CON vs. LCD (1.66 2 0.36 vs. 1.43 + 0.18 nm~l*rnin'~m~-~). OGTï significantly
increased the active GS to 3.04 + 0.46 and to 2.69 + 0.53 nrnol.min-'mg-' in the CON and
LCD condition, respectively. Total GS activity (in the presence of 10.0rnM G-6-P) was
unchanged throughout the experiment. Therefore the GS fractional velocity (GSf,,)
increased significantly in response to insulin in CON and LCD (Figure 8).
It shouId be noted that both HK and GS activities are reported here based on
normalization to muscie protein ievels. However, HK and GS were aIso corrected to total
creatine concentration in the samples and compared with the protein-normalized data,
since it is standard practice to correct to total creatine in some laboratones. The creatine
correction did not alter the outcome of the HK or GS results. Therefore the data is
reported based on protein norrnalization.
PDK. The rnitochondrial recovery for this study was 18.9 + 4.4% of the total
mitochondria, and the quality of the extraction (percentage of intact mitochondria) was
87 t 4%. Resting PDK activity was significandy increased by LCD (0.19 0.05 vs. 0.08
+ 0.02 min-') (pc0.05) (Figure 9). -
PDHa, Resting PDHa activity was significantly lower after LCD vs. CON (0.38 + 0.08
vs. 0.79 + 0.10 mm01 acetyl-CoA/kg/min). During OGïT, PDHa increased significantly
in both conditions, but remained lower after LCD (0.60 f: 0.1 1 vs. 1-04 + 0.09 mm01
acetyl-CoA/kg/min) (Figure 9).
Muscle fuels and metabolites
Muscle glycogen
Mean muscle glycogen, measured in the resting biopsy sarnple was not different
between triais (CON; 346.1 4 22.3 and LCD; 320.0 + 14.7 mmolkg dm).
Muscle metabolites
Resting muscle ATP and G-1-P were lower following LCD vs. CON (Table 2).
Muscle acetyl-CoA and acetyl-carnitine decreased significantly dunng OGTT in both
conditions.
Fimire 7. Western blot of total GLUT-4 protein for CON and LCD. Representative western blot of GLUT-4 protein for CON and LCD in three subjects (1 to 3). The band representing GLUT-4 protein is indicated by the black arrows. Total GLUT-4 protein was not significantly different in LCD vs. CON.
CON 1 LCD I CON 2 LCD 2 CON 3 LCD 3
Fimire 8. Basal (O-min) and insulin-shulated (OGTT, 75-min) glycogen synthase fractional velocity (GSfv). GSfv represents active (0.1 mM)/total (10.0 mM) GS activity . *Significantly different from O-min (pc0.05).
CON O C O N 7 5 LCDO L C D 7 5
Fimre 9. PDK and PDHa activities. PDK (top panel) was measured in intact mitochondria at rest. PDHa (bottom panel) was measured at rest (O-min) and during OGTI' (75-min). The delta increase in PDHa fiom O to 75-min was not different between trials. 'Significanly dflerent from CON, bsignificantly different fiom O-min (p<0.05)
CON O-min LCD O-min
O 7 5 O 7 5 CON LCD
TabIe 2. Muscle metabolites. Metabolites are reported as mmolkg dm and *pmoVkg dm. SE is reported in brackets. aSignificantly different from CON, b~ign5cantly different from O-min, (p<O.OS).
CON O CON 75 LCD O LCD 75
ATP
PCr
G-6-P
G-1-P
AC-COA*
Ac-carn
Lactate
Pyruvate
Glucose
DISCUSSION
An important finding of this study was that a 56-hr LCD (5 + 0.2 % CHO, 73 2
0.6 % fat, 22 + 0.5 % protein) was an effective mode1 to reduce whole-body insulin
sensitivity in normal human subjects. In the muscle, the activity of resting PDK was
increased following LCD and this resulted in a decreased PDHa at rest and following the
administration of oral glucose. GS activity was unaltered by LCD. These resuIts suggest
that decreased skeletal muscle glucose disposal after a LCD is related to decreases in
oxidative CHO disposal and is not related to decreases in glycogen storage. Although the
total GLUT-4 protein did not change following LCD, the concentrations of glycolytic
intermediates in the muscle indicated that the uptake of the oral glucose load into the
muscle was decreased. GLUT-4 translocation may have been downregulated secondary
to LCD.
Insulin sensitivity
The AUC for blood-[glucose] and plasma-[insulin] during OGïT increased
folIowing LCD. Despite higher circulating plasma insulin, the subjects were unable to
dispose of an oral glucose load as efficiently after LCD, resulting in higher blood glucose
levels during OGTT. In addition, the calculated insulin sensitivity index (ISIgly) (5) was
lower after the diet.
The majority of an oral glucose load (71%) is taken up by skeletai muscle, as
evidenced by the direct measurement of k g glucose uptake using fernoral venous
cathetenzation (42). This predicts that the majority of the decrease in insulin sensitivity
following LCD would be accounted for by decreased insulin-stirnulated skeletal muscle
glucose disposal.
It is also important to bnefly consider the involvement of the liver in deterrnining
the blood glucose level during O G T . In normal subjects afier an overnight fast? hepatic
glucose uptake accounted for 2 5 8 of the disposal of an oral glucose load (42) and hepatic
glucose output was suppressed by 50% (22). Therefore, in addition to a decrease in
muscle glucose uptake, factors that may contribute to an elevated AUC for blood-
[glucose] following LCD, are a decrease in hepatic glucose uptake or an increase in
hepatic glucose output. The suppression of hepatic glucose output during OGïT was
impaired only in severe NIDDM subjects vs. mild NIDDM and control subjects, and was
associated with a deficient insulin response (23). In our normal subjects, it is unlikely
that the suppression of hepatic glucose output during OGïT would have been impaired
after LCD. Secondly, we would speculate that hepatic glucose uptake would have been
higher in the LCD condition vs. CON in Our study, since the liver glycogen stores would
have been depleted to a greater extent after fasting and LCD vs. an overnight fast in CON
(37). Overall, the net contribution of the b e r to blood glucose levels rnay have actually
been less in the present study after LCD and masked the severity of the decreased insulin
sensitivity. Therefore, the majority of the elevation in blood-[glucose] after LCD would
have been due to reduced insulin-stimulated disposal of oral glucose by the muscle.
Possible mediators of insulin insensitivitv
Elevated FFA induce a state of insulin insensitivity as evidenced by euglycemic
hypennsulinemic clamp studies that infuse intralipid during hyperinsulinemia (8,9,44,
53). In studies that use starvation or high fatllow CHO feeding as a model to study
metabolic adaptation, FFA and ketones are elevated and there is a shift towards reiiance
on these metabolic fiels. In addition, circulating insulin levels (or insulin sensitivity)
may be decreased in the latter models, and this could be an important signal involved in
altenng substrate utilization. In Our study, LCD resulted in elevated resting FFA and
ketone levels, and lower resting plasma insulin, as expected. A previous study (71)
demonstrated that circulating insulin levels in the fed state were also suppressed by LCD
(7.0 2 1.7 vs. 21 .O + 6.7 plU/d). Therefore, possible mediators of reduced insulin-
stimulated CHO utilization in the LCD condition include decreased circulating insulin,
and increased FFA and ketone levels.
In the present study we were unable to assess the actual mechanisms by which the
mediators of insulin insensitivity may exert their effects. The main purpose of the study
was to examine the potential sites of adaptation in skeletal muscle that may contribute to
decreased insulin-stimulated glucose disposal following LCD. The four major sites
investigated were glucose transport (GLUT-4), glucose phosphorylation (HK), glucose
storage (GS), and glucose oxidation (PDK and PDHa). These sites will be discussed in
the following section.
Sites of skeletal muscle adaptation to LCD
Glucose transport and phosphorylation
Total GLUT-4 protein levels were not altered by 56-hrs of LCD. However, it is
possible that insulin-stimulated GLUT-4 translocation rnay have been dowmegulated
following LCD and contnbuted to a reduced glucose uptake into skeletal muscle. In most
rodent studies, long-term high fat feeding (3 to 8-wks) has resulted in decreased insulin-
shu la ted glucose transport without changing the total protein content of GLUT-4 (3 1,
32,45, 1 12), suggesting a decrease in GLUT-4 translocation. Due to the arnount of
muscle required to measure GLUT-4 translocation in humans, we were unable to directly
investigate the effects of LCD on this mechanism. However, total GLUT-4 protein levels
had not been measured foilowing a short-term diet mode1 in human subjects pnor to this
study. Since GLUT-4 protein increases within 5-days of endurance training (79, it was
possible that the protein levels may have adapted over the timecourse of LCD (2.33-
days) .
Total HK activity was not different after LCD. Studies in rodents have shown
that HKII activity was unaltered after a long-term high fat diet (3 to 4-weeks) (45, 113).
Total HK activity had not been measured previously in humans following a short-terrn
LCD. This was an important site that rnay have altered glucose disposal over the short-
tenn, based on the capacity of HKII expression to adapt rapidly to insulin levels (56,77).
In addition to direct measurements of glucose transport and phosphorylation, G-6-
P is often used as an indicator of the activity at these sites. This and other glycolytic
intermediates, including G-1-P, lactate and pyruvate, are indicative of the relationship
between the uptake of glucose into the muscle (coupled with phosphorylation) and the
utilization of glucose for storage and oxidation. In the case where the uptake of glucose
into the muscle is in excess of the utilization, these intermediates would accumulate. The
decrease in PDHa suggested that the oxidative glucose disposal was reduced following
LCD. Since none of the glycolytic intermediates accumulated in the muscle during
OGTT in the LCD triai (vs. CON), this suggests that the transport of glucose into the
muscle was decreased accordingly. In this situation it seems that HK worked in concert
with GLUT-4 to phosphorylate the incoming glucose, since intracellular glucose did not
accumulate in the muscle during the OGTï in the LCD condition. In a situation where
glucose uptake is chronically decreased HK rnay eventually adapt at the level of protein
expression to alter its total activity, as seen in NIDDM (103).
Glucose storage
LCD did not change the basal activity of GS. During OGTT, GS activity was
increased sirnilarly at 75-min in both conditions to contribute to the disposal of the oral
glucose load by storing some of the glucose as glycogen in the muscle. The fractional
velocity of GS, GSfv, is used as an indicator of the arnount of GS in the active
(dephosphorylated) form, and was increased in response to insulin during OGTT (75-
min) in both trials, as expected. Our values for basal and insulin stirnulated GS activity
and GSfv were in accordance with the literature from human skeletal muscle (56). It is
important to consider that the results of this study with respect to GS activity during
OGTT are only for one point in time and may not reflect GS activity over the duration of
the 90-min glucose disposa1 penod.
Euglycemic hyperinsulinemic clamp studies have demonstrated that in an induced
state of insulin resistance secondary to elevated FFA, glucose disposal is decreased after
3 to Chrs accompanied by a decrease in insulin-stimulated GS activity (8, 10,44). In
these studies, blood glucose and high insulin levels are constantly maintained while FFA
levels are artificially elevated over a penod of hours. It would be expected that glycogen
levels should be increasing in the muscle due to the inhibition of glucose oxidation by
FFA and the stimulation of GS activity by insulin, and that this could start to inhibit GS
activity. In contrast, in the current study, under physiological conditions of
hyperglycemia and elevated insulin for a shorter duration, it is unlikely that glycogen
accumulated sufficiently in the muscle to inhibit GS activity. The starting muscle
glycogen concentration was equivalent in CON vs. LCD in the present study. Based on
calculations for an average 75 kg subject, if ail of the oral glucose load was stored as
glycogen in the muscle (assuming 40% of rnass as muscle), there was the potential for a
59.7 rnrnol glucosyl units/kg dm increase that could occur in the muscle. Assuming that
only -80 % of the glucose was absorbed and -70 % of the absorbed glucose was taken up
into the muscle, less than 35.8 rnmol/kg dm would have accumulated in the muscle at 75-
min of OGïT. In addition, a portion of the oral glucose load is oxidized within the
muscle. Based on these calculations, it was not surprising that 75-min after the
administration of oral glucose, insulin-stimulated GS activity was not inhibited following
LCD.
Insulin-stimulated GS activity is decreased in chronic States of insulin resistance
(MDDM). A recent study in rodents (45) demonstrated decreased GS activity and a
decreased accumulation of muscle glycogen during a 2-hr euglycemic hypennsulinemic
clamp following a 3-wk high fat diet. This group aIso found that a decrease in insulin-
stimulated GS activity did not occur after 2-wks of a high fat diet, suggesting that it is not
an initial adaptation in the development of insulin resistance (46). Measurements of GS
activity in human skeletal muscle following a short-term LCD are lacking. Our results
indicate that insulin-stimulated GS activity is not inhibited during OGTT (75-min)
following 56-hrs of LCD. However, 4 of the 6 subjects had decreased GS activity during
OGTï after LCD vs. CON (subject I to 4, see appendix). The mean decrease in insulin-
stimulated GS foIIowing LCD was not statistically signifîcant. Therefore, the ability of
insulin to stimulate GS activity may be dowmegulated in some subjects by 56-hrs of
LCD. Longer dietary alteration may lead to an adaptation in the ability of insulin to
stimulate GS, as seen in rodents (45). Also, more frequent biopsy sampling during
OGTT would provide insight into the insulin-stimulated disposal of a glucose load by GS
following dietary alteration.
Glucose oxidation
An important finding in this study was the rapid and stable increase in resting
PDK activity following a 56-hr LCD. The PDK-mediated inhibition of PDHa may be
one mechanism involved in the decreased disposal of an oral glucose load following
LCD. We have previously measured a dramatic increase in PDK activity after 3-days of
LCD (7 l), and a pilot study with two subjects indicated that the increase rnay be
O C C U ~ ~ ~ as early as Zdays into the diet. Our current results confirm that PDK increases
rapidly, within 56-hrs of LCD.
Accompanying the increase in resting PDK activity was a corresponding decrease
in resting PDHa. A decrease in PDHa was measured in our previous study after 6-days
of LCD (7 L), however PDHa activity was not measured after 3-days when PDK was
found to be increased. When the oral glucose load was adrninistered, PDHa activity
increased in both CON and LCD, but remained lower in LCD. Assurning that PDHa is
representative of PDHa flux and that the 75-min biopsy sample is representative of the
entire 90-min period, the decreased PDHa would have contributed to the overall decrease
in the disposal of glucose folIowing LCD.
The increase in resting PDK after LCD was a stable increase that persisted
through a rigorous mitochondrial extraction. At rest (O-min) there were no major changes
in the known acute regulators of PDK activity, indicating that the increase in resting PDK
was not a function of acute regulation. As discussed earlier, a stable increase in PDK
activity may involve the altered expression of PDK isoforms following LCD. Recently,
it was shown that increased PDK activity during LCD was accompanied by an increase in
PDK4 protein and mRNA in human skeletal muscle, while PDK2 protein was unchanged
by the diet (70).
During OGTT, PDHa increased in both conditions. Acetyl-CoA decreased
sirnilarly in both conditions while pyxuvate and ATP were unchanged. We would also
expect that [ C a r would have been the same in CON and LCD. The decrease in acetyl-
CoA may have been involved in the increase in PDHa during OGTT by decreasing PDK
activity acutely. However, the decrease in acetyl-CoA observed in the present study mziy
not have been physiologically significant. A previous study (62) in Our lab measured an
increase in PDHa without a change in acetyl-CoA dunng a standard OGTT . PDHa
activation dunng OGïT in the present study may have been independent of acute
mediation of PDK. Increased insulin levels during OGTï may have directly increased
PDH phosphatase activity, as suggested in rat studies (21).
The downregulation of PDHa by PDK was shown to be an important site involved
in the initial adaptation of skeletal muscle to LCD. It seems that this is the major
mechanism that changes in concert with glucose uptake to decrease the ability of the
muscle to dispose of an oral glucose load at this stage of adaptation. For this reason, we
will bnefly discuss the possible regulators involved in the stable adaptation of PDK to
LCD. These mainly include an increase in FFA and a decrease in circulating insulin (or a
decrease in insulin sensitivity) secondary to the diet.
With prolonged reliance on fat metabolism in starvation, diabetes and high fat
feeding, PDK activity has been shown to increase in a stable adaptive manner in order to
downregulate CHO oxidation. In rat heart and skeletal muscle, the temporal increase in
circulating FFA in these models correlates with an increase in PDK activity and
subsequent decrease in PDHa (36,69). In cell culture experiments, 24-hr culture with n-
octanoate , dibutyryl-CAMP, or a combination of these mediators resulted in an increase
in PDK sirnilar to that seen in starved rats (65,90). However, the stable upregulation of
PDK with high-fat feeding is mediated through a CAMP-independent mechanism (96).
The effect of F'FA to increase PDK in high-fat-fed rats has been shown to be dependent
on the type of fat ingested. Only 24-hrs of an n-3 poIyunsaturated diet reversed the
increase in PDK activity due to the standard 28-day high-saturated fat diet typically used
in the rat studies (25). The exact mechanism by which FFA alter the activity of PDK is
not known. Recent evidence suggests that peroxisornal proliferating activating receptors
(PPARs) may be a possible link between FFA and metabolic regulation. PPARs are
nuclear steroid-like receptors that act as trans-activators of many genes involved in lipid
metabolism. Exposure to a PPAR-a agonist (3-days) increased skeletal muscle PDK
activity and PDK4 mRNA and protein to levels observed in diabetes or 48-hr starved
animals, suggesting that PPARs may play a roie in downregulating CHO metabolism
(106).
In starvation and diabetes, the decrease in circulating insulin concentrations (or in
insulin sensitivity) correlates with the stable increase in PDK in rat skeletal muscle (65).
The effects of decreased insulin may be due to an indirect effect of elevating circulating
FFA due to a decrease in insulin-mediated suppression of lipolysis. However, in ceil
culture studies, insulin directly opposes the effects of FFA and CAMP to increase PDK
activity (65,97). It has been suggested that a decfine in insufin Ievels (or a decrease in
insulin sensitivity) may be obligatory for a stable increase in PDK. In alloxan diabetic
rats that have a stable increase in PDK, the long-term administration of insulin is required
to decrease PDK to control values (21). More recently, a study in Pima Indians showed
that the expression of PDK isoforms was correlated with the severity of a subject's
insulin resistance (53). These authors demonstrated that insulin had a direct effect on the
expression of PDK2 and 4, and suggest that in insulin-resistant individuals, there may be
insuffkient insulin-mediated downregulation of PDK. This would mean that decreased
insulin responsiveness may be a cause and not a consequence of increased fat utilization.
OGTT and acetyi-carnitine
Acetyl-carnitine decreased during OGTT in both trials. The reason for this
decrease is not known. We have previously observed a similar decrease in acetyl-
carnitine during a standard OGTT (62). One possibility is that there was insufficient
pyruvate to match the activation of PDHy even &ter LCD when PDHa was decreased. In
this case, the muscle may draw on the acetyl-camitine stores as a source of acetyl-CoA,
since the contribution of fat to the acetyl-CoA pool is minimal during OGTT due to the
insulin-mediated suppression of lipolysis. If pyruvate was insufficient, a decrease in
pyruvate would have been expected during OGTT, and this did not occur. However, G-
6-P does tend to decrease at 75-min in both conditions, suggesting that GS may compete
successfully for G-6-P, leaving less available pyruvate than could be handled by PDHa.
Conclusion
The initial adaptation of human skeletal muscle to the short-term (56-hr) dietary
elevation of fat and resmction of CHO involves a stable upregulation of PDK activity and
a subsequent decrease in PDHa. This data strongly suggests that the ability of the muscle
to dispose of an oral glucose oxidatively is decreased following LCD. If CHO flux
through PDH is in fact reduced by LCD, and insilin-stirnulated GS activity is unaltered
in response to an oral glucose load, the decrease in PDHa would necessitate a decrease in
glucose uptake. This is in accordance with the observed decreased in glucose disposa1
over 90-min of OGTï in the current study, and the lack of accumulation of glycolytic
intermediates at 75-min. Studies measuring GLUT-4 translocation directiy in humans are
limited due to the current techniques available.
Future directions
Since this was the first study that attempted to obtain a comprehensive picture of
skeletal muscle adaptation to a short-term LCD in humans by directly measuring muscle
proteins, enzyme activities and metabolites, it was important to maximize the information
that we could obtain from a lirnited quantity of muscle. We have now been able to
identify sites of adaptation thar require further study to determine the mechanisms
underlying decreased insulin-stimulated glucose disposal by the muscle following LCD.
Future studies should directly address the insuiin-stimulated translocation of
GLUT-4 following a short-term LCD. A longer-term (weeks) dietary stimulus should be
used to investigate adaptations in GLUT-4 protein levels, and in HK (activity, mRNA
and protein) and GS activity.
Measurements of CS and PDHa activity by repeated biopsy sarnpling over time
during insulin-stimulated glucose disposal should be performed in order to obtain more
conclusive evidence for the altered fate of glucose inside the muscle after LCD.
Further work is required to determine the mechanisms by which FFA and /or
insulin may be involved in inducing skeletai muscle insulin resistance. Measurements of
intermediates in the insulin-signaling pathway would be an important contribution to this
work. In addition, it will be important to study dBerent dietary fat compositions and the
possible differential effects on PPARs.
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APPENDIX
BLOOD RESULTS over time, during OGTT (O to 180-min)
1. Blood glucose, mmoVl PRE
1 2 3 4 5 6
MEAN Sem
LCD 1 2 3 4 5 6
MEAN Sem
2. Plasma Insulin, ulU/ml PRE
1 2 3 4 5 6
MEAN Sem
LCD 1 2 3 4 5 6
MEAN Sem
3. FFA, mmoUi PRE
1 2 3 4 5 6
MEAN Sem
LCD 1 2 3 4 5 6
MEAN Sem
PRE 1 2 3 4 5 6
MEAN sem
LCD 1 2 3 4 5 6
MEAN sern
PRE 1 2 3 4 5 6
MEAN Sem
LCD 1 2 3 4 5 6
MEAN Sem
5. Glycerol, mmoüi
1
6. Lactate, mmoüi
PRE 1 2 3 4 5 6
MEAN sem
LCD 1 2 3 4 5 6
MEAN Sem
MUSCLE RESULTS
1. GLUT-1 protein, intensity of fixed area
PRE LCD SET PRE =1
2. GLUT4 protein, intensity of fixed area
PRE LCD SET PRE =1
1 31056 48626 2 92226 118093 3 120502 84395 4 100198 119209 5 127339 115582 6 73497 82383
MEAN 90803 94715 sern 15718 12593
3a). HK activity, moükg proteinlhr
CON O 1 0.271 2 0.556 3 0.439 4 0.361 5 0.384 6 0.493
MEAN 0.417 Sem 0.041
CON 75 0.384 0.387 0.633 0 -274 0.397 0.520 0.432 0.051
LCD O 0.296 0.31 7 0.484 0.274 0.345 0.462 0.363 0.036
(O-rest, 75-during OGTT)
LCD 75 0.443 0.450 0.453 0.1 39 0 -476 0.407 0.395 0.052
PRE
1 1 1 1 1 1
1 O
PRE
1 1 1 1 1 1
1 O
LCD
0.67 0.90 2.20 0.88 0.99 1.51
1 .l9 0.25
LCD
1 -57 1 -28 0.70 1.19 0.91 1.12
1 . l3 0.06
3b). HK activity, mmoVg wet musclehin (corrected to total muscle Cr)
CON0 CON75 LCDO 1 4.550 4.51 0 4.1 10 2 3.860 2.860 2.690 3 3.760 4.1 20 3.090 4 2.480 2.390 1.990 5 4.710 5.1 70 3.71 O 6 2.460 3.1 40 4.01 0
MEAN 3.637 3.698 3.267 sem 0.399 0.437 0.339
4ai). GS activity, nmoUmin/mg cytosolic pr
CON (Omin)
0.1 mM 1 3.02 2 1.32 3 1.06 4 2.05 5 1.66 6 0.82
MEAN 1.66 Sem 0.36
CON (75min)
0.1 mM 2.39 3.12 4.5 4.03 2.22 1.98 3.04 0.46
4aii). GS fractional velocity (O.l/l OmM)
5. PDK activity, min -' CON0 LCDO
1 0.127 0.379 2 0.034 O -223 3 0.116 0.071 4 0.072 0.128 5 0.05 0.1 13 6 0.097 0.231
LCD 75 4.340 3.770 3.820 1.1 60 3-61 O 2.950 3.275 0.461
LCD (Omin)
0.1 mM 1-19 1 -84 1.98 0.96 1.27 1.32 1.43 0.1 8
LCD (75min)
0.1 mM 1-14 2.28 3.76 2.1 6 2.4 4.37 2.69 0.53
MEAN 0.083 0.1 91 sern 0.015 0.046
6. PDHa activity, mm01 acetyl-CoA'kg-'-min" (corrected to total muscle Cr)
CON0 CON75 LCDO 1 0.413 0.568 0.270 2 1.168 1 -220 0.1 97 3 0.663 0.743 0.723 4 0.752 0.828 0.462 5 0.847 1 .O22 0.448 6 0.908 1.298 0.1 92
MEAN 0.792 0.947 0.382 sem 0.103 0.1 16 0.084
7. Muscle gtycogen, mmoükg dw
CON LCD 1 315.29 303.61 2 377.23 366.56 3 428.84 314.33 4 303.76 265.59 5 367.53 351.68 6 284.20 318.44
8. Muscle metabolites
ATP
1
2
3
4
5
6
MEAN
Sem
PCr
1
2
3
4
5
6
MEAN
sem
CON O
29.092
25.973
25.468
27.31 7
27.994
23.978
26.637
0.830
CON O
91.309
95.1 13
89.098
87.252
88.102
85.866
89.457
1 -485
CON 75
25.467
25.601
29.487
27.599
26.1 25
24.1 1
26.398
0.844
CON 75
84.559
91.207
83.086
82.654
80.379
78.069
83.326
2.005
LCD O
23.27
23.003
21.733
24.1 08
26.1 35
23.621
23.645
0.652
LCD O
86.938
83.277
89.936
64.263
85.787
85.58
82.630
4.1 39
LCD 75 1.112 0.312 0.658 0.988 0.766 0.282
0.686 0.1 39
LCD 75 mmoikg dm
25.258
23.812
29.793
27.374
25.939
21 -737
25.652
1.250
LCD 75 mmoUkg dm
92.302
84.989
79.824
78.149
85.336
76.485
82.848
2.622
glucose
1
2
3
4
5
6
MEAN
sern
CON O
1.284
0.382
1 .O61
0.3
0.569
0.674
0.71 2
0.1 73
CON O
0.1 81
0.248
0.697
0.1 4
0.485
0.667
0.403
0.1 11
CON O
3.437
1 343
2.658
2.72
1.499
3.298
2.493
0.396
CON O
5.94
9 -38
10.79
13.89
6.32
5.88
8.700
1.456
CON 75
0.505
0.17
0.306
0.236
0.998
0-1 96
0.402
0.1 41
CON 75
0 .27
0.038
0-1 25
O
0.084
0.26
0.1 30
0.051
CON 75
19.1 98
1.481
1.593
1.897
2.1 97
17-41 6
7.297
3.824
CON 75
4.86
4.1 4
4.27
4.69
4.54
6.81
4.885
0.438
LCD O
1.4
0.265
0.486
0.721
1.153
0.299
0.721
0.209
LCD O
0.021
0-1 4
0.C66
0.1 13
0.049
0.288
0.113
0.043
LCD O
7.861
4.1 59
1.12
3.469
2.638
1.569
3.469
1 .O88
LCD O
9.1 6
7.65
7.32
8.642
10.09
8.99
8.642
0.457
LCD 75 mmoikg dm
0.596
0.71 5
0.378
0.244
0.738
0.63
0.550
0.088
LCD 75 mrnoflkg dm
0.02
0.048
0.095
0.1 4
0.108
0.092
0.084
0.01 9
LCD 75 mmoükg dm
4.627
1.595
9.764
6.891
1.989
5.355
5.037
1.374
LCD 75 umoükg dm
3.75
6.08
5.87
6.388
8.76
7.48
6.388
0.752
Lactate
1
2
3
4
5
6
MEAN
sem
Pyruvate
1
2
3
4
5
6
MEAN
sem
CON O
2.91
2.969
2.281
6.81
1 -75
5.607
3.721
0.901
CON O
6.481
2.203
2.01 5
3.647
2.637
6.223
3.868
0.898
CON O
0.1 42
0.061
0.043
0.067
0.1 26
0.379
0.1 36
0.056
CON 75
0.748
0.439
1.308
0.378
0.1 59
4.81 6
1.308
0.789
CON 75
8.597
3.679
5.43
2.594
2.1 35
10,145
5.430
1.473
CON 75
0.204
0.1 06
0.064
0.092
0.1 36
0.31 8
0.1 53
0.042
LCD O
4.592
2.809
2.049
2.482
3.607
6.931
3.745
0.806
LCD O
6.453
5.1 25
7.83
13.946
7.448
6.1 76
7.830
1.407
LCD O
0.065
0.086
0.C29
0.238
0.087
0.1 55
0.110
0.034
LCD 75 mmoükg dm
0.978
2.336
0.545
0.1 11
2.584 3-41 7
1.662
0.583
LCD 75 mmoükg dm
6.333
3.501
3.888
13.839
4.563
5.873
6.333
1 .il7
LCD 75 mmoükg dm
0.057
0.064
0.1 1 1
0.131
0.063
0.224
0.1 O8
0.029
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