Acute Hypoglycemia in Healthy Humans ImpairsInsulin-Stimulated Glucose Uptake and GlycogenSynthase in Skeletal Muscle: A Randomized Clinical StudyThomas S. Voss,1,2 Mikkel H. Vendelbo,3 Ulla Kampmann,2 Janne R. Hingst,4 Jørgen F.P. Wojtaszewski,4

Mads V. Svart,1,2 Niels Møller,1,2 and Niels Jessen5,6

Diabetes 2017;66:2483–2494 | https://doi.org/10.2337/db16-1559

Hypoglycemia is the leading limiting factor in glycemicmanagement of insulin-treated diabetes. Skeletal mus-cle is the predominant site of insulin-mediated glucosedisposal. Our study used a crossover design to test towhat extent insulin-induced hypoglycemia affects glucoseuptake in skeletal muscle and whether hypoglycemiacounterregulation modulates insulin and catecholaminesignaling and glycogen synthase activity in skeletal mus-cle. Nine healthy volunteers were examined on three ran-domized study days: 1) hyperinsulinemic hypoglycemia(bolus insulin), 2) hyperinsulinemic euglycemia (bolus in-sulin and glucose infusion), and 3) saline control withskeletal muscle biopsies taken just before, 30 min after,and 75 min after insulin/saline injection. During hypogly-cemia, glucose levels reached a nadir of ∼2.0 mmol/L,and epinephrine rose to ∼900 pg/mL. Hypoglycemia im-paired insulin-stimulated glucose disposal and glucoseclearance in skeletal muscle, whereas insulin signalingin glucose transport was unaffected by hypoglycemia.Insulin-stimulated glycogen synthase activity was com-pletely ablated during hyperinsulinemic hypoglycemia,and catecholamine signaling via cAMP-dependent proteinkinase and phosphorylation of inhibiting sites on glycogensynthase all increased.

Hypoglycemia is the most common side effect of insulin-treated diabetes and the main factor precluding optimalglucose management (1). Hypoglycemia is defined by theWhipple triad: symptoms related to hypoglycemia, low plasmaglucose measured at the time of symptoms, and relief of

symptoms when glucose levels are raised to normal. Further-more, hypoglycemia is differentiated based on the severity ofsymptoms (or lack thereof) and glucose levels: asymptomatichypoglycemia, which presents with no symptoms but plasmaglucose ,3.9 mmol/L (,70 mg/dL); symptomatic hypo-glycemia, which shows symptoms and plasma glucose ,3.9mmol/L (,70 mg/dL); and severe hypoglycemia, whichis an event requiring aid from another person and lowplasma glucose (2). Insulin-induced hypoglycemia occursrapidly (nadir plasma glucose within 30 min) (3). In healthysubjects, hypoglycemia leads to a decrease in insulin andincreases in circulating levels of the counterregulatoryhormones glucagon, norepinephrine, epinephrine, growthhormone (GH), and cortisol (4). Catecholamines and glu-cagon inhibit glycolysis and increase glycogenolysis aswell as gluconeogenesis via increased adenylyl cyclase andcAMP-dependent protein kinase (PKA) activity (5). Thecombined effects of these hormones effectively andrapidly increase endogenous glucose production duringhypoglycemia (6).

Insulin-stimulated glucose disposal is primarily governed byskeletal muscle (7). Intracellular signaling in this process isinitiated by insulin binding to its receptor and the subse-quent signaling through the insulin receptor substrate1/phosphoinositide 3-kinase signaling pathway. This acti-vates Akt and stimulates phosphorylation of AS160 andTBC1D1 (8). This stimulates translocation of GLUT4 tothe cell surface and facilitates transport of glucose intothe muscle. Once glucose is taken up it is metabolized byeither oxidative or nonoxidative pathways (6).

1Department of Clinical Medicine, Aarhus University, Aarhus, Denmark2Department of Endocrinology and Internal Medicine, Aarhus University Hospital,Aarhus, Denmark3Department of Nuclear Medicine and PET Centre, Aarhus University Hospital,Aarhus, Denmark.4Section of Molecular Physiology, Department of Nutrition, Exercise and Sports,Faculty of Science, University of Copenhagen, Copenhagen, Denmark5Department of Clinical Pharmacology, Aarhus University Hospital, Aarhus, Denmark6Department of Biomedicine, Aarhus University Hospital, Aarhus, Denmark

Corresponding author: Niels Jessen, niels.jessen@biomed.au.dk.

Received 15 December 2016 and accepted 1 June 2017.

Clinical trial reg. no. NCT01919788, clinicaltrials.gov.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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COMPLIC

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The effects of insulin are inhibited during hypoglycemia(9), plausibly because of the action of counterregulatoryhormones and a reduced glucose concentration gradient (10).Catecholamines stimulate G protein–coupled b-receptors,which are present on skeletal muscle, and thereby activatePKA (11). Increased PKA activity can inhibit insulin actionand may thereby suppress glucose uptake during hypogly-cemia (12). Epinephrine administration to isolated rat skel-etal muscle decreases insulin-stimulated glucose uptake bydecreasing glycogen synthase (GS) activity, leading to accu-mulation of glucose-6-phosphate (G6P) and subsequent in-hibition of hexokinase activity (13). In humans, epinephrineadministration in adrenalectomized subjects during exercisedecreases glucose disposal (14). However, catecholamine-mediated signaling in skeletal muscle during acute hypogly-cemia has not been determined. Similar to b-receptors, GHreceptors are expressed on skeletal muscle, and GH stimu-lation potently inhibits insulin-stimulated glucose transportafter latency through unknown mechanisms (15). The aimof this study was to investigate insulin action in skeletalmuscle during acute hypoglycemia under the hypothe-sis that intracellular insulin action is impaired duringthis condition.

RESEARCH DESIGN AND METHODS

The study protocol was approved by the local scientific ethicscommittee (1-10-72-113-13) and registered at clinicaltrials.gov (NCT01919788). The study was conducted in accor-dance with the Helsinki Declaration II, and all subjects gaveoral and written informed consent to participate.

The design is a randomized crossover study of 3 days’duration, previously described in detail (16). Briefly, eachsubject was allocated to 1) control (CTR) (bolus, 2 mL NaCli.v.); 2) hyperinsulinemic hypoglycemia (HH) (bolus insulin,0.1 unit Actrapid/kg in 2 mL NaCl i.v.); and 3) hyperinsu-linemic euglycemia (HE) (bolus insulin, 0.1 unit Actrapid/kgin 2 mL NaCl i.v. and i.v. glucose infusion 20% [varyingamounts]). The study days were separated by at least21 days; they commenced at 9.00 A.M. (t = 0 min) and endedat 10.45 A.M. (t = 105 min).

Our primary outcome for this study was forearm glucosedisposal. Secondary outcomes were glucose oxidation rates,insulin signaling in glucose transport, glycogen synthesis inskeletal muscle, and energy expenditure (EE).

Muscle biopsies from the vastus lateralis of the quadri-ceps muscle were obtained under sterile conditions 15 minafter local anesthesia (lidocaine 1%, 10 mL) was applied.Biopsies were obtained from the right leg (separated by.10 cm) just before t = 0 min and at t = 75 min, and fromthe left leg at t = 30 min. Biopsies were immediately snap-frozen in liquid nitrogen and subsequently stored at 280°Cuntil analyzed. All samples were homogenized twice for 30 sat 5,000 rpm in a buffer containing 50 mmol/L HEPES,20 mmol/L NaF, 2 mmol/L NaOV, 5 mmol/L EDTA,5 mmol/L nicotinamide, 137 mmol/L NaCl, 10 mmol/LNa4P2O7, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 0.01 mmol/L

trichostatin A, Halt Protease Inhibitor Cocktail (100X), 1%NP-40, and 10% glycerol in a Precellys Homogenizer (BertinInstruments, Montigny-le-Bretonneux, France). Then sampleswere rotated at 4°C for 30 min before being centrifuged at14,000g for 20 min. The resulting supernatant was collected.

Western blotting was performed using 4–15% CriterionXT Bis-Tris gels (Bio-Rad, Hercules, CA). The following pri-mary antibodies were used AKT, pAKTser473, pAKTthr308,AS160, pAS160ser642, pAS160ser588, GS, pGSser641 (site3a), glycogen synthase kinase-3 (GSK-3), pGSK-3a+b, mam-malian target of rapamycin (mTOR), pmTORser2448,AMPK, pAMPKthr172, 4EBP1, nonphosphorylated thr464EBP1, PKA phospho-substrate (catalog nos. 4691, 9271,9275, 5676, 4288, 8730, 3886, 3891, 5676, 9331, 2972,5536, 2532, 2531, 9644, 4923, 9624, respectively; all fromCell Signaling Technology); acetyl-CoA carboxylase (ACC),pACCser79, GLUT4 (streptavidin; Upstate 07-303,07-1404) (all from EMD Millipore, Darmstadt, Germany);and GSK-3b (610202; BD Biosciences). Antibodies detectingphosphorylated GS (pGS) ser7+10 (site2+2a) were raisedagainst peptide PLSRSL(Sx)MS(Sx)LPGLED (residues 1–16of rat GS), and pGS ser697 (site1a) and pGS ser710 (site1b)were raised against the peptides CEWPRRASpSCTSSTG(692–703 residues of human GS) and CSGSKRNSpVDTATS(704–716 residues of human GS); the specificity of theseantibodies have been reported previously (17,18). Phos-phorylation levels are expressed as a ratio with the targetedprotein, measured on the same membrane. Stain-free pro-tein technology was used to control for equal loading (19).GLUT4 and PKA phospho-substrate levels are expressed asa ratio with the protein content quantified by stain-freeprotein technology. PKA phospho-substrate antibody wasused as a measure of PKA activation through the detectionof PKA-phosphorylation on all detectable proteins .15 kDa.This antibody detects proteins containing a phospho-serine/threonine residue with arginine at the23 position, which isa bona fide PKA motif (20). Glycogen phosphorylase (GP) aand b antibodies were used as previously described (21).Proteins were visualized and quantified by enhanced chem-iluminescence after incubation with horseradish peroxidase–conjugated antirabbit/antimouse secondary antibodies usingImage Lab 5.0 software (Bio-Rad). Protein measurementsare expressed as the ratio with the mean CTR value att = 0 min relative units (RU).

Muscle glycogen concentration was determined as glycosylunits after acid hydrolysis of muscle lysate using thefluorometric method (22). GS activity was measured usinga modified version (18) of the method described by Thomaset al. (23). The activity was measured at 37°C, and themixture contained uridine diphospho-glucose at a concentra-tion of 1.7 mmol/L and G6P at concentrations of 0.02mmol/L(zero), 0.167 mmol/L (low), and 8.0 mmol/L (high). GS activityat a G6P concentration of (Zero/High) 3 100 is termedI-Form, whereas activity at a G6P concentration of (Low/High) 3 100 is termed fractional velocity (FV).

Biochemical parameters were measured using variousmethods: plasma glucose was measured with the oxidase

2484 Hypoglycemia and Insulin in Skeletal Muscle Diabetes Volume 66, September 2017

method (YSI 2300 STAT Plus; YSI Life Sciences, YellowSprings, OH); serum insulin, with ELISA (Dako DenmarkA/S, Glostrup, Denmark); plasma glucagon, with a radioim-munoassay kit (EMD Millipore); plasma epinephrine andnorepinephrine, with electrochemical detection after high-performance liquid chromatography (9); serum GH, withchemiluminescence technology (IDS-iSYS Multi-DisciplineAutomated System; Immunodiagnostic Systems Nordic S/A,Copenhagen, Denmark); and C-peptide, with ELISA (ALPCO,Salem, NH).

Indirect calorimetry (Deltatrac monitor; Dantes Instru-mentarium, Helsinki, Finland) was used from t = 90 to 105 minin order to determine total EE, the respiratory quotient,and glucose oxidation rates (24). Protein oxidation wasset at 12% of EE (25).

Forearm blood flow (FBF) was measured using strain-gauge plethysmography (26) and forearm glucose disposalwas assessed using the product of FBF and arteriovenousdifferences in glucose concentrations. Arterialization of ve-nous blood was obtained as described previously (16). Whenlooking at differences in glucose disposal using arteriove-nous differences 3 FBF, possible differences in glucose dis-posal due to differences in glucose gradient across the cellmembrane (glucose concentration) are not accounted for.To account for these differences in glucose levels, plasma glu-cose clearance in mL plasma $ 100 mL forearm21 $ min21

was calculated as follows: glucose disposal (mmol $ 100 mLforearm21 $ min21) O arterial glucose concentration(mmol/L) 3 1,000.

Statistical analyses were performed using a repeated-measures mixed model with visit order, visit number, time,intervention, and the interaction between intervention andtime as factors (Stata 13.0; StataCorp, College Station, TX).When only one daily measure was applied, a repeated-measures mixed model was used, with visit order, visitnumber, and intervention as factors. Unequal correlationsand SEs, when detected, were accounted for in the analysis.The model was validated by inspecting quantile-quantileplots of the residuals and scatterplots of the predictedversus the fitted values. If residuals were not normallydistributed, logarithmic transformation was performed.P , 0.05 was considered statistically significant. Data ingraphs show geometric means with SEMs. Numerical dataare geometric means with 95% CIs.

RESULTS

Subject CharacteristicsInformation about the study subjects has in part beenpublished previously in a report of a study describing lipidmetabolism and adipose tissue signaling during acuteinsulin-induced hypoglycemia (16). The subject characteris-tics are summarized in Table 1.

Glucose Disposal in Skeletal MusclePlasma glucose and insulin levels have been publishedpreviously in detail (16). Comparable peak insulin levels(;900 pmol/L) were reached during both HH and HE

(P = 0.87) at t = 15 min. Insulin levels remained slightlyelevated during HE throughout the rest of the study (over-all P , 0.001) (Fig. 1A). This was likely because of a mildoverdose of glucose during HE, leading to increased endog-enous insulin production, which was confirmed by overallC-peptide levels ;2.7 times higher during HE than duringHH (P , 0.001) and ;1.3 times higher than the CTR (P ,0.001).

FBF showed no difference among interventions at any timepoint. Forearm glucose disposal wasmarkedly larger (;2.5 timesoverall) during HE than during HH (overall P, 0.001) (Fig.1C). These differences were already present at t = 20 min.

Plasma glucose clearance (Fig. 1E) was reduced ;35%during HH compared with HE (overall P , 0.01). Differ-ences were evident at t = 40 min (P = 0.01), t = 60 min (P =0.04), and t = 80 min (P , 0.01), thus confirming reducedinsulin-stimulated glucose uptake in skeletal muscle duringhypoglycemia.

Insulin Signaling in GLUT4 Translocation and ProteinSynthesisInsulin increased phosphorylation of Aktser473 (Fig. 2A)and Aktthr308 (Fig. 2B) 30 min after injection by ;10-foldcompared with the saline control. No differences werefound between the hypoglycemic and euglycemic insulin-stimulated conditions. These findings were associatedwith increased signaling at the downstream target AS160at thr642 (Fig. 2C) and ser588 (Fig. 2D) at the same time

Table 1—Baseline characteristics of the nine subjects

Variable Value

Age (years) 22 (18–27)*

BMI (kg/m2) 23.6 (21.7–26.1)*

Smoking (cigarettes/day) 0

Alcohol (units/week) 3.7 (0–7.4)

Systolic blood pressure (mmHg) 124 (117–131)

Diastolic blood pressure (mmHg) 75 (71–80)

Heart rate (bpm) 61 (53–68)

Exercise (h/week) 4.6 (3.0–6.1)

Glucose (mmol/L) 5.0 (4.6–5.5)

HbA1c (mmol/mol) 30 (28–32)

HbA1c (%) 4.9 (4.7–5.1)

C-peptide (nmol/L) 0.63 (0.46–0.80)

TSH (1023 IU/L) 1.93 (1.42–2.43)

CRP (mg/L) 0.8 (0.5–1.0)

Creatinine (mmol/L) 87 (77–96)

Hemoglobin (mmol/L) 9.3 (9.0–9.6)

HOMA (%S) 152 (105–200)

HOMA (%B) 80 (56–103)

Data are mean (95% CI) or median (range), indicated by *. Most ofthese data have been published previously (16). HOMA estimatessteady-state b-cell function (%B) and insulin sensitivity (%S), aspercentages of a normal reference population. CRP, C-reactiveprotein; TSH, thyrotropin.

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point during both HE and HH. Akt phosphorylationsat ser473 and thr308 were still elevated, but only aboutfourfold, 75 min after insulin injection during HH andHE conditions compared with the saline control. Phosp-horylation on AS160ser588 remained elevated duringboth HH and HE, but thr642 phosphorylation was in-creased during HE only. We did not detect any differ-ences in Akt, AS160, or GLUT4 protein levels among allinterventions.

Insulin stimulates protein synthesis through activationof the mTOR pathway (27). Insulin increased phosphoryla-tion of mTOR at ser2448 ;2.5-fold 30 min after injectionduring both HE and HH compared with CTR (both P ,0.001) (Fig. 2E) but remained stable throughout CTR. At t =75 min, the increased mTOR phosphorylation during HHwas ;75% higher than that of the CTR (P , 0.001) and;35% higher than that during HE (P , 0.001). Similarregulation by insulin was evident at the downstream targetprotein 4EBP1 (Fig. 2F).

Counterregulatory Hormones and SignalingHypoglycemia profoundly increased circulating levels ofepinephrine, norepinephrine, GH, and glucagon. For clarity,epinephrine and glucagon, which have been publishedpreviously (16), are described here.

Epinephrine levels were low and comparable between HEand CTR at all time points (P = 0.14) (Fig. 3A). During HH,epinephrine levels rose rapidly to a peak level of 891 pg/mL(560–1,221 pg/mL) at t = 30 min. Overall comparisonrevealed differences between both HH and CTR (P ,0.001) as well as between HH and HE (P , 0.001).

Norepinephrine quickly rose during HH to a peak levelof 387 pg/mL (349–425 pg/mL) at t = 30 min, and wasclearly increased compared with both HE (P , 0.001) andCTR (P , 0.001) (Fig. 3B). No overall difference was de-tected between CTR and HE (P = 0.07).

Glucagon levels increased during HH to a peak levelof 133 pg/mL (119–147 pg/mL) at t = 45 min and wererestored to CTR levels at t = 90 and 105 min (P . 0.05)

Figure 1—Glucose disposal in skeletal muscle during CTR (bolus, 2 mL NaCl i.v.) (white squares), HH (bolus insulin, 0.1 unit Actrapid/kg) (blackcircles), and HE (bolus insulin, 0.1 unit Actrapid/kg and glucose 20%) (black triangles). A: Insulin concentrations in picomoles per liter (publishedpreviously in [16]). B: Glucose infusion rate (GIR) (mL/h) during HE. C: Forearm (FA) glucose disposal (GD) (mmol/100 mL forearm/min). D:Arteriovenous (A-V) glucose differences (mmol/L). E: Glucose clearance (mL/100 mL forearm/min). *P< 0.05, HH vs. HE; †P< 0.05, HH vs. CTR;‡P < 0.05, HE vs. CTR; §P < 0.05 for all.

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(Fig. 3C). During HE, glucagon levels were suppressed com-pared with those of the CTR (P , 0.001). Differences be-tween these two study days were evident from t = 30 min.Glucagon levels were stable during CTR, with an overallmean of 52 pg/mL (47–56 pg/mL).

GH was comparable between HE and CTR at all times(Fig. 3D). During HH, GH levels increased from t = 45 minand were elevated throughout the study day, with a peaklevel of 15.6 mg/L (6.3–38.7 mg/L) at t = 60 min. Overall

comparison between interventions revealed increased GHlevels during HH, HH and CTR (P = 0.001), and HH and HE(P , 0.001).

Activity of phospho-PKA substrate, as a surrogate marker ofcatecholamine stimulation, increasedby;75% at t = 30min du-ring HH compared with both CTR (P , 0.001) and HE (P ,0.001) (Fig. 3E). At t = 75 min, PKA substrate phosphory-lation decreased to only ;10% more than that during CTR(not significant), and to ;30% above HE levels (P , 0.01).

Figure 2—Insulin signaling in glucose transport and protein synthesis during CTR (bolus, 2 mL NaCl i.v.) (white squares), HH (bolus insulin, 0.1unit Actrapid/kg) (black circles), and HE (bolus insulin, 0.1 unit Actrapid/kg and glucose 20%) (black triangles). Phosphorylation of proteins at t =0, 30, and 75 min. Values are shown relative to those of the CTR at t = 0 min. A and B: Ratios of ser473 (A) and thr308 (B) phospho-Akt to Aktprotein level. C and D: Ratios of thr642 (C) and ser588 (D) phospho-AS160 to AS160 protein level. E: Ratio of ser2448 phospho-mTOR to mTORprotein level. F: Ratio of nonphosphorylated thr46 4EBP1 to 4EBP1 protein level. *P < 0.05, HH vs. HE; †P < 0.05, HH vs. CTR; ‡P< 0.05, HEvs. CTR; §P < 0.05 for all.

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No differences in AMPKthr172 phosphorylationwere detected between the interventions (Fig. 3F),nor did we detect any effect of the interventions on phos-phorylation of the downstream target ACC (data notshown).

Regulation of Glycogen Synthesis and Breakdown, TotalGlycogen, Glucose Oxidation, and EE

GS ActivityInsulin potently increased GS FV by ;35% during HEat both t = 30 and 75 min compared with CTR values

Figure 3—Plasma levels of counterregulatory hormones and PKA signaling in skeletal muscle during CTR (bolus, 2 mL NaCl i.v.) (white squares),HH (bolus insulin, 0.1 unit Actrapid/kg) (black circles), and HE (bolus insulin, 0.1 unit Actrapid/kg and glucose 20%) (black triangles). A–D:Counterregulatory hormone concentrations in plasma: epinephrine (A), norepinephrine (B), glucagon (C), and GH (D). E: Phosphorylation of arange of proteins >15 kDa at t = 0, 30, and 75 min, using an antibody directed against phosphorylated PKA-motif peptide sequences (PKA-phos.). Values are shown relative to the CTR level at t = 0 min. F: Ratio of thr172 phospho-AMPK to AMPK protein level (Western blot shownbelow). G: Representative image of Western blot for phospho-PKA substrate >15 kDa. *P < 0.05, HH vs. HE; †P < 0.05, HH vs. CTR; ‡P <0.05, HE vs. CTR; §P < 0.05 for all.

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(P , 0.001) (Fig. 4A). Hypoglycemia totally abolished theeffect of insulin on GS activity, and thus no differenceswere detected when comparing HH with CTR at t = 30 or75 min.I-Form. The same pattern was seen with I-Form, which wasincreased by;75% at t = 30 min (P, 0.001) and by;65%at t = 75 min (P , 0.001) compared with CTR (Fig. 4B).Again, hypoglycemia completely impaired GS activity whenI-Form was used, and no differences were detected whencomparing HH with CTR.

These findings confirm that storage of glucose asglycogen in muscle tissue was inhibited by hypoglycemia.To unfold possible explanatory mechanisms for this

decrease in GS activity, we also investigated knownregulatory phosphorylation sites on GS and the up-stream GSK-3.

GS PhosphorylationsSite 3a (ser641). Insulin induced the activation of de-phosphorylation on site 3a, leading to;30% reduced phos-phorylation during HE (P , 0.001) (Fig. 4C), whereas thecombined effects of insulin and hypoglycemia during HHdecreased phosphorylation on site 3a by ;13% (not signif-icant) compared with CTR at t = 30 min. At t = 75 min, theinsulin effects during HE were a little smaller, and phos-phorylation on site 3a was decreased ;25% (P , 0.01)

Figure 4—GS activity and phosphorylation during CTR (bolus, 2 mL NaCl i.v.) (white squares), HH (bolus insulin, 0.1 unit Actrapid/kg) (blackcircles), and HE (bolus insulin, 0.1 unit Actrapid/kg and glucose 20%) (black triangles). GS activity expressed as FV (percentage) (A) and I-Form(percentage) (B). C–F: Phosphorylation of GS sites 3a, 2+2a, 1a, and 1b relative to total GS protein at t = 0, 30, and 75 min. Values are shownrelative to the CTR level at t = 0 min (representative Western blots are shown below the graphs). *P < 0.05, HH vs. HE; †P< 0.05, HH vs. CTR;‡P < 0.05, HE vs. CTR; §P < 0.05 for all.

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compared with CTR. The effects of insulin were totallyabrogated by hypoglycemia at t = 75 min when comparingHH with CTR.Sites 2+2a (ser7+ser10). Insulin decreased phosphorylationon site 2+2a by;20% (not significant) at t = 30 min and by;45% at t = 75 min (P = 0.001) during HE compared withCTR (Fig. 4D). The effects of hypoglycemia totally abolishedthe effects of insulin on GS site 2+2a, and thus increasedphosphorylation by;90% (P, 0.001) at t = 30 min and by;60% (P , 0.01) at t = 75 min when comparing HH withCTR. Comparison of HH and HE revealed a ;2.5-fold in-crease in phosphorylations on site2+2a during HH at botht = 30 and 75 min (both P , 0.001).

Sites 1a and 1b. Phosphorylation on sites 1a and 1brevealed no statistically significant interaction (time 3 in-tervention; P = 0.09 and 0.06, respectively) (Fig. 4E and F).Thus the results were analyzed accordingly. A main effect ofthe intervention between HH and CTR was found regardingboth sites 1a and 1b, with ;25% increased site 1a phos-phorylation (P = 0.01) and ;15% increased site 1b phos-phorylation (P , 0.05) during HH.GSK-3 Phosphorylations (a and b). Insulin increasedphosphorylation of GSK-3a ;40% (P , 0.001) and ofGSK-3b ;25% (P , 0.01) during both HE and HH com-pared with CTR at t = 30 min (Fig. 5A and B). No statisti-cally significant differences in either GSK-3a or GSK-3b

Figure 5—Regulators of GS phosphorylation and glycogen breakdown and total glycogen during CTR (bolus, 2 mL NaCl i.v.) (white squares), HH(bolus insulin, 0.1 unit Actrapid/kg) (black circles), and HE (bolus insulin, 0.1 unit Actrapid/kg and glucose 20%) (black triangles). A: pGSK-3arelative to GSK-3a. B: pGSK-3b relative to GSK-3b. C and D: GPa and GPb. E: Glycogen. Values are shown relative to the CTR level at t = 0 min(representative Western blots are shown below the graphs). †P < 0.05, HH vs. CTR; ‡P < 0.05, HE vs. CTR.

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phosphorylation were detected when comparing HE withHH at t = 30 or 75 min. At t = 75 min, GSK-3a phosphor-ylation had normalized during HH, whereas GSK-3b phos-phorylation was increased;20% compared with that of theCTR (P = 0.04). No statistically significant differences inGSK-3b phosphorylation were detected when comparingHE with both HH and CTR.

GPa and GPbGP is present in two forms, GPa and GPb (Fig. 5C and D).They differ in phosphorylation, and GPb is converted intoGPa by phosphorylation at two sites (28).GPa. No statistical significant interaction (time 3 inter-vention; P = 0.3) or main effect of any intervention (P .0.05) was observed.GPb. At t = 30 min, GPb decreased during CTR and in-creased during HH, revealing an ;75% increase inGPb levels during HH compared with CTR (P = 0.003).Levels were stable during HE and not statistically sig-nificantly different from those during either the CTR orHH at t = 30 and 75 min (P . 0.05). Total glycogen inskeletal muscle biopsies was not statistically significantlydifferent between any time points among the interventions(Fig. 5E).

Glucose oxidation was determined using indirect calo-rimetry. Oxidation rates were at their lowest during CTR(669 kcal/day) and highest during HE (1,278 kcal/day) (P,0,001). During HH (924 kcal/day), glucose oxidation werebetween that of CTR (not significant) and HE (P = 0.07). EEincreased during hypoglycemia to 1,826 kcal/day, comparedwith 1,717 kcal/day during HE (P , 0.01). EE during CTRwas 1,762 kcal/day; this was not statistically different fromEE during HE or HH.

DISCUSSION

The key finding of this study is that insulin action inskeletal muscle is profoundly inhibited during acute symp-tomatic hypoglycemia in humans. This was determinedusing a design that reflects a clinically relevant situation.Our study therefore allows for direct comparisons betweenacute insulin action during euglycemia and hypoglycemia.By contrast, previous investigations of hypoglycemia usedinsulin-clamp conditions (9,29–32) under which glucose lev-els are reduced for longer periods. The clamp conditionsallow for steady-state estimates of glucose disposal, butthe condition is far from the acute hypoglycemia experi-enced by insulin-treated patients. A previous investigationcomparing hypoglycemia induced by either a 10-min or a12-h insulin infusion found a substantial reduction of glu-cose disposal during prolonged, but not acute, hypoglycemia(33). This is in contrast to the findings in the current study,but several differences in the models could explain thediscrepancies. Most important, the level of hypoglycemiawas more pronounced in this study, in which glucoselevels reached a nadir of ;2.0 mmol/L, compared with;2.8 mmol/L in the previous study. The lower glucose levelsin the current study were associated with a substantially

higher epinephrine response at ;900 pg/mL, comparedwith a peak ,400 pg/mL in the previous investigation. Asimilar but less pronounced pattern was observed whencomparing norepinephrine levels between the two studies.Furthermore, it is interesting that the increase in adrenalineconcentration was much larger than the limited increase innoradrenaline, which indicates an adrenal effect rather thanregulation by the sympathetic nervous system. This mayimplicate a role of b2 adrenergic receptors for counterregu-lation in muscles.

Because of the above-mentioned differences, our resultsdo not necessarily conflict with existing data, but ratherdemonstrate the mechanisms involved during a more pro-nounced counterregulatory response.

The threshold for autonomic symptoms to hypoglyce-mia is ;3.2 mmol/L; for neuroglycopenic symptoms it is;2.8 mmol/L (4). Population-based data demonstratethat 30–40% of individuals with type 1 diabetes mellitusexperience between one and three episodes of severe (ac-quiring assistance from another person) hypoglycemiaeach year (34), indicating that, in a clinical setting, glucoselevels ,2.8 mmol/L (and probably even much lower) oftenoccur in type 1 diabetes mellitus. Our data therefore dem-onstrate that decreased glucose disposal in skeletal muscle,in addition to increased endogenous glucose production,can be an important factor contributing to glucose counter-regulation during symptomatic hypoglycemia.

When circulating glucose levels are within the physio-logical range and metabolism is normally regulated, trans-port across the cell membrane is the limiting factor forglucose uptake in skeletal muscle (35). During insulin stim-ulation, glucose is primarily disposed in skeletal muscle byfacilitated transport through GLUT4 (36). Under these con-ditions, the amount of GLUT4 in the cell membrane and thegradient of glucose into the cell determine uptake. There-fore the decreased glucose gradient during HH contributessignificantly to the decreased forearm glucose disposal ob-served in this and other studies (10). Km for GLUT4 is;4.3 mmol/L (37), and glucose clearance, defined as theratio between glucose disposal and circulating glucose levels,can be assumed to be constant (33). In this study, glucoseclearance was significantly reduced during hypoglycemia,and this could indicate reduced GLUT4 content in the cellmembrane. We were not able to measure GLUT4 transloca-tion in skeletal muscle biopsies in this study, but asexpected, the interventions did not affect the total amountof GLUT4 protein. Nor did we observe impaired activationof signaling intermediates regulating GLUT4 trafficking30 min after insulin stimulation, when glucose disposalpeaked. In fact, phosphorylation of Akt and AS160 tendedto be higher during HH than during HE, although thesedifferences did not reach statistical significance. At t =75 min, when glucose levels were nearly normalized, phos-phorylation of AS160 at thr642 was lower during HH thanduring HE. This phosphorylation site on AS160 has consis-tently been shown to regulate glucose uptake (38), but thelower phosphorylation level at this time point is unlikely to

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explain differences in glucose disposal during acute hypo-glycemia. It may instead be caused by the slightly elevatedinsulin levels during HE. Therefore, a lower glucose gradientthrough the plasma membrane during HH decreases glu-cose transport during these conditions. In addition, reducedGLUT4 content in the cell membrane cannot be excluded asa mechanism for decreased glucose clearance during symp-tomatic hypoglycemia, but it is not a consequence of re-duced insulin signaling in GLUT4 translocation.

Under nonphysiological conditions in mouse models,glucose metabolism, and not transport across the cellmembrane, can become the limiting factor for glucoseuptake in skeletal muscle (39). Similar mechanisms mayreduce glucose clearance during hypoglycemia in situationswhere insulin-stimulated GLUT4 translocation to the cellmembrane is normal. Unlike the subtle effects of hypogly-cemia on insulin signaling in glucose transport, we foundprofound effects of acute hypoglycemia on important reg-ulators of glycogen synthesis. GS is the key enzyme respon-sible for glycogen synthesis in muscle tissue, and underphysiological conditions, GS activity is stimulated by insulin(40). Reduced glycogen synthesis can impair glucose uptakethrough mechanisms originally described by Randle et al.(41) and may contribute to reduced glucose clearance dur-ing hypoglycemia. Impaired glycogen synthesis could becaused by the observed effects of hypoglycemia on severalregulating phosphorylation sites on GS. Insulin signals toGS via activation of Akt and subsequent phosphorylation ofGSK-3. This reduces the activity of GSK-3 and inhibitsphosphorylation, especially of the inactivating site 3a onGS (42,43). Hypoglycemia did not affect insulin-stimulatedAkt and GSK-3 phosphorylation; instead, we found evidenceof direct inhibition of GS activity by catecholamine signal-ing. These findings are supported by the data from a pre-vious study investigating the effects of insulin andadrenaline on Akt, GSK-3, GS activity, and phosphorylation(44). PKA directly inhibits GS activity via phosphorylationof sites 1a, 1b, and 2 (45). In addition, PKA possibly in-directly increases phosphorylation on site 3a via inhibitionof the GM protein phosphatase 1 complex (12). The in-creased GS phosphorylations on sites 3a and 2+2a duringhypoglycemia were therefore likely caused by catecholamine-stimulated PKA activity. This was further supported by thetrends toward inhibition of phosphorylation at the sites 1aand 1b, although these differences failed to achieve statisti-cal significance (P = 0.09 and 0.06, respectively).

Hypoglycemia not only decreased GS activity, it alsodecreased glucose oxidation despite an increase in total EE.In agreement with the reduced glucose oxidation, we didnot find evidence of measurable glycogen degradation, andGPa, the active form of GP, was not increased duringhypoglycemia (46). We were not able to measure all impor-tant regulators of glycogen metabolism, including proteinphosphatase I complexes and G6P, in our biopsy material(46). However, our data suggest that glycogenolysis alonewas not able to compensate for the reduced glucose uptakeat the time point when the biopsy was obtained. This

indicates that lipids become the primary metabolic substrateduring hypoglycemia, which is supported by our previouslypublished observations of the same subjects (16). Underphysiological conditions, insulin reduces free fatty acid(FFA) levels. During hypoglycemia, counterregulatory hor-mones abrogate insulin inhibition, and lipolysis in adiposetissue is potently stimulated, leading to increased lipid avail-ability (16). An increase in circulating FFAs induces insulinresistance in skeletal muscle in a dose-dependent manner,but this occurs only hours after FFA levels increase (47).Activation of lipolysis in adipose tissue by catecholamineshas been suggested to have protein-sparing/anabolic ef-fects in skeletal muscle (11). In rat skeletal muscle, thishas been shown to be associated with catecholamine-induced Akt and mTOR activation (11). Our data extendthese findings to human skeletal muscle and indicate thatcatecholamines may affect protein synthesis via mTOR sig-naling. It is therefore likely that the increase in lipid oxida-tion affects protein synthesis, but lipid-induced insulinresistance is unlikely to be involved in the acute responseto hypoglycemia.

This study examined the effects of hypoglycemia inhealthy volunteers, and this ensured homogeneous counter-regulatory hormone responses. Unlike healthy individuals,subjects with diabetes treated with insulin often haveimpaired glucagon responses to hypoglycemia. In such cases,the catecholamine response is crucial in attempts to restorecirculating glucose levels (48). The pronounced increase incatecholamine signaling to GS and the associated inhibitionof glycogen synthesis observed in this study therefore dem-onstrate mechanisms that are likely intact among patientswith impaired glucagon responses. This could indicate thattreatment with b-receptor antagonists may dampen coun-terregulation in skeletal muscle, in addition to the well-known shift of glycemic thresholds for symptoms towardlower plasma glucose concentrations (49). The preservedglucagon response in healthy subjects is expected to in-crease endogenous glucose production, but it is not expectedto directly target skeletal muscle because of the absence ofglucagon receptors in this tissue (50). It is therefore likelythat our data regarding insulin action in skeletal musclefrom healthy volunteers can be extrapolated to patientswith diabetes—even those with long disease duration andimpaired glucagon response.

In conclusion, we found that acute symptomatic hypo-glycemia decreases insulin-stimulated glucose disposal inskeletal muscle. These findings are not associated withdecreased insulin signaling in glucose transport. Instead,insulin-stimulated glycogen synthesis is potently inhibited.This is explained, at least in part, by increased counter-regulatory catecholamine levels leading to increased PKAsignaling and to subsequent GS inactivation.

Acknowledgments. The authors thank A. Mengel, K. Nyborg Rasmussen,E. Søgaard Hornemann, and K. Mathiassen, of Medical Research Laboratories;H. Zibrandtsen of the Research Laboratories for Biochemical Pathology; L. Pedersen

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of the Department of Endocrinology and Internal Medicine, Aarhus University Hospital,Aarhus, Denmark. The authors also thank B. Bolmgren and J.B. Birk of the Section ofMolecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Sci-ence, University of Copenhagen, for their excellent technical assistance.Funding. GPa and GPb antibodies were raised in sheep and donated byYu-Chiang Lai, MRC Protein Phosphorylation and Ubiquitylation Unit, University ofDundee. This study was supported by Aarhus University, the Novo NordiskFoundation, the Beckett Foundation, the KETO Study Group/Danish Agency forScience Technology and Innovation (grant no. 0603–00479 [to N.M.]), and theDanish Medical Research Council.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. T.S.V. recruited the participants, conducted the trial,and performed the statistical analyses. T.S.V., M.H.V., U.K., N.M., and N.J. designedthe study. T.S.V., J.R.H., J.F.P.W., M.V.S., N.M., and N.J. collected the data. T.S.V.and N.J. wrote the manuscript. M.H.V., U.K., J.R.H., J.F.P.W., M.V.S., and N.M.reviewed and edited the manuscript. All authors approved the final version of themanuscript. N.J. is the guarantor of this work and, as such, takes responsibility forthe integrity of the data and the accuracy of the data analysis.

References1. Cryer PE. Hypoglycemia: still the limiting factor in the glycemic management ofdiabetes. Endocr Pract 2008;14:750–7562. Workgroup on Hypoglycemia, American Diabetes Association. Defining andreporting hypoglycemia in diabetes: a report from the American Diabetes AssociationWorkgroup on Hypoglycemia. Diabetes Care 2005;28:1245–12493. Ajala O, Lockett H, Twine G, Flanagan DE. Depth and duration of hypoglycaemiaachieved during the insulin tolerance test. Eur J Endocrinol 2012;167:59–654. Mitrakou A, Ryan C, Veneman T, et al. Hierarchy of glycemic thresholds forcounterregulatory hormone secretion, symptoms, and cerebral dysfunction. Am JPhysiol 1991;260:E67–E745. Rui L. Energy metabolism in the liver. Compr Physiol 2014;4:177–1976. Sprague JE, Arbeláez AM. Glucose counterregulatory responses to hypogly-cemia. Pediatr Endocrinol Rev 2011;9:463–473; quiz 474–4757. DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP. Theeffect of insulin on the disposal of intravenous glucose. Results from indirect calo-rimetry and hepatic and femoral venous catheterization. Diabetes 1981;30:1000–10078. Leto D, Saltiel AR. Regulation of glucose transport by insulin: traffic control ofGLUT4. Nat Rev Mol Cell Biol 2012;13:383–3969. Orskov L, Bak JF, Abildgård, et al. Inhibition of muscle glycogen synthaseactivity and non-oxidative glucose disposal during hypoglycaemia in normal man.Diabetologia 1996;39:226–23410. Capaldo B, Napoli R, Guida R, et al. Forearm muscle insulin resistance duringhypoglycemia: role of adrenergic mechanisms and hypoglycemia per se. Am JPhysiol 1995;268:E248–E25411. Lynch GS, Ryall JG. Role of beta-adrenoceptor signaling in skeletal muscle:implications for muscle wasting and disease. Physiol Rev 2008;88:729–76712. Walker KS, Watt PW, Cohen P. Phosphorylation of the skeletal muscle glycogen-targetting subunit of protein phosphatase 1 in response to adrenaline in vivo. FEBSLett 2000;466:121–12413. Lee AD, Hansen PA, Schluter J, Gulve EA, Gao J, Holloszy JO. Effects of epi-nephrine on insulin-stimulated glucose uptake and GLUT-4 phosphorylation inmuscle. Am J Physiol 1997;273:C1082–C108714. Howlett K, Galbo H, Lorentsen J, et al. Effect of adrenaline on glucose kineticsduring exercise in adrenalectomised humans. J Physiol 1999;519:911–92115. Møller N, Jørgensen JO. Effects of growth hormone on glucose, lipid, andprotein metabolism in human subjects. Endocr Rev 2009;30:152–17716. Voss TS, Vendelbo MH, Kampmann U, et al. Effects of insulin-inducedhypoglycaemia on lipolysis rate, lipid oxidation and adipose tissue signallingin human volunteers: a randomised clinical study. Diabetologia 2017;60:152–152

17. Højlund K, Staehr P, Hansen BF, et al. Increased phosphorylation of skeletalmuscle glycogen synthase at NH2-terminal sites during physiological hyper-insulinemia in type 2 diabetes. Diabetes 2003;52:1393–140218. Højlund K, Birk JB, Klein DK, et al. Dysregulation of glycogen synthase COOH-and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus.J Clin Endocrinol Metab 2009;94:4547–455619. Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loadingcontrol to b-actin for Western blots. Anal Biochem 2013;440:186–18820. Grønborg M, Kristiansen TZ, Stensballe A, et al. A mass spectrometry-basedproteomic approach for identification of serine/threonine-phosphorylated pro-teins by enrichment with phospho-specific antibodies: identification of a novelprotein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics 2002;1:517–52721. Bouskila M, Hirshman MF, Jensen J, Goodyear LJ, Sakamoto K. Insulin pro-motes glycogen synthesis in the absence of GSK3 phosphorylation in skeletalmuscle. Am J Physiol Endocrinol Metab 2008;294:E28–E3522. Lowry OH, Passonneau JV. A flexible system of enzymatic analysis. s NewYork, Academic Press, 197223. Thomas JA, Schlender KK, Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem 1968;25:486–49924. Meriläinen PT. Metabolic monitor. Int J Clin Monit Comput 1987;4:167–17725. Møller N, Jørgensen JO, Alberti KG, Flyvbjerg A, Schmitz O. Short-term effectsof growth hormone on fuel oxidation and regional substrate metabolism in normalman. J Clin Endocrinol Metab 1990;70:1179–118626. Whitney RJ. The measurement of volume changes in human limbs. J Physiol1953;121:1–2727. Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translationalcontrol. Nat Rev Mol Cell Biol 2009;10:307–31828. Cohen P. The hormonal control of glycogen metabolism in mammalian muscleby multivalent phosphorylation. Biochem Soc Trans 1979;7:459–48029. De Feo P, Perriello G, Torlone E, et al. Demonstration of a role for growthhormone in glucose counterregulation. Am J Physiol 1989;256:E835–E84330. Fanelli CG, De Feo P, Porcellati F, et al. Adrenergic mechanisms contribute tothe late phase of hypoglycemic glucose counterregulation in humans by stimulatinglipolysis. J Clin Invest 1992;89:2005–201331. Lucidi P, Rossetti P, Porcellati F, et al. Mechanisms of insulin resistance afterinsulin-induced hypoglycemia in humans: the role of lipolysis. Diabetes 2010;59:1349–135732. Meyer C, Saar P, Soydan N, et al. A potential important role of skeletal musclein human counterregulation of hypoglycemia. J Clin Endocrinol Metab 2005;90:6244–625033. De Feo P, Perriello G, De Cosmo S, et al. Comparison of glucose counter-regulation during short-term and prolonged hypoglycemia in normal humans. Di-abetes 1986;35:563–56934. International Hypoglycaemia Study Group. Minimizing hypoglycemia in diabetes.Diabetes Care 2015;38:1583–159135. Shepherd PR, Kahn BB. Glucose transporters and insulin action–implications forinsulin resistance and diabetes mellitus. N Engl J Med 1999;341:248–25736. Kim YB, Peroni OD, Aschenbach WG, et al. Muscle-specific deletion of the Glut4glucose transporter alters multiple regulatory steps in glycogen metabolism. Mol CellBiol 2005;25:9713–972337. Nishimura H, Pallardo FV, Seidner GA, Vannucci S, Simpson IA, Birnbaum MJ.Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes.J Biol Chem 1993;268:8514–852038. Sakamoto K, Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in theregulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 2008;295:E29–E3739. Fueger PT, Shearer J, Bracy DP, et al. Control of muscle glucose uptake: test ofthe rate-limiting step paradigm in conscious, unrestrained mice. J Physiol 2005;562:925–93540. Bogardus C, Lillioja S, Stone K, Mott D. Correlation between muscle glycogensynthase activity and in vivo insulin action in man. J Clin Invest 1984;73:1185–1190

diabetes.diabetesjournals.org Voss and Associates 2493

41. Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle.Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.Lancet 1963;1:785–78942. Skurat AV, Dietrich AD, Roach PJ. Glycogen synthase sensitivity to insulin andglucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylationsites. Diabetes 2000;49:1096–110043. Skurat AV, Roach PJ. Phosphorylation of sites 3a and 3b (Ser640 and Ser644)in the control of rabbit muscle glycogen synthase. J Biol Chem 1995;270:12491–1249744. Jensen J, Ruge T, Lai YC, Svensson MK, Eriksson JW. Effects of adrenaline onwhole-body glucose metabolism and insulin-mediated regulation of glycogen syn-thase and PKB phosphorylation in human skeletal muscle. Metabolism 2011;60:215–22645. Nielsen JN, Wojtaszewski JF. Regulation of glycogen synthase activity andphosphorylation by exercise. Proc Nutr Soc 2004;63:233–237

46. Roach PJ, Depaoli-Roach AA, Hurley TD, Tagliabracci VS. Glycogen and itsmetabolism: some new developments and old themes. Biochem J 2012;441:763–78747. Gormsen LC, Nielsen C, Jessen N, Jørgensen JO, Møller N. Time-course effectsof physiological free fatty acid surges on insulin sensitivity in humans. Acta Physiol(Oxf) 2011;201:349–35648. Bolli G, de Feo P, Compagnucci P, et al. Important role of adrenergic mech-anisms in acute glucose counterregulation following insulin-induced hypoglycemia intype I diabetes. Evidence for an effect mediated by beta-adrenoreceptors. Diabetes1982;31:641–64749. Hirsch IB, Boyle PJ, Craft S, Cryer PE. Higher glycemic thresholds for symptomsduring beta-adrenergic blockade in IDDM. Diabetes 1991;40:1177–118650. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am JPhysiol Endocrinol Metab 2003;284:E671–E678

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