jurnal prof

ATVB In FocusMetabolic Syndrome and Atherosclerosis

Series Editor: Marja-Riitta Taskinen

Preview Brief Reviews in this Series:• Grundy, SM. Metabolic syndrome pandemic. Arterioscler Thromb Vasc Biol. 2008;28:629–636.• Barter PJ, Rye KA. Is there a role for fibrates in the management of dyslipidemia in the metabolic syndrome.

Arterioscler Thromb Vasc Biol. 2008;28:39 – 46.• Kotronen A, Yki-Jarvinen. Fatty liver: a novel component of the metabolic syndrome. Arterioscler Thromb

Vasc Biol. 2008;28:27–38.• Gustafson B, Hammarstedt A, Andersson CX, Smith U. Inflamed adipose tissue: a culprit underlying the meta-

bolic syndrome and atherosclerosis. Arterioscler Thromb Vasc Biol. 2007;27:2276 –2283.• Rigamonti E, Chinetti-Gbaguidi G, Staels B. Regulation of macrophage functions by PPAR-�, PPAR-�, and

LXRs in mice and men. Arterioscler Thromb Vasc Biol. 2008;28:1050 –1059.• Kockx M, Jessup W, Kritharides L. Regulation of endogenous apolipoprotein E secretion by macrophages.

Arterioscler Thromb Vasc Biol. 2008;28:1060 –1067.

Overproduction of Very Low–Density Lipoproteins Is theHallmark of the Dyslipidemia in the Metabolic Syndrome

Martin Adiels, Sven-Olof Olofsson, Marja-Riitta Taskinen, Jan Boren

Abstract—Insulin resistance is a key feature of the metabolic syndrome and often progresses to type 2 diabetes. Bothinsulin resistance and type 2 diabetes are characterized by dyslipidemia, which is an important and common risk factorfor cardiovascular disease. Diabetic dyslipidemia is a cluster of potentially atherogenic lipid and lipoproteinabnormalities that are metabolically interrelated. Recent evidence suggests that a fundamental defect is an overproduc-tion of large very low–density lipoprotein (VLDL) particles, which initiates a sequence of lipoprotein changes, resultingin higher levels of remnant particles, smaller LDL, and lower levels of high-density liporotein (HDL) cholesterol. Theseatherogenic lipid abnormalities precede the diagnosis of type 2 diabetes by several years, and it is thus important toelucidate the mechanisms involved in the overproduction of large VLDL particles. Here, we review the pathophysiologyof VLDL biosynthesis and metabolism in the metabolic syndrome. We also review recent research investigating therelation between hepatic accumulation of lipids and insulin resistance, and sources of fatty acids for liver fat and VLDLbiosynthesis. Finally, we briefly discuss current treatments for lipid management of dyslipidemia and potential futuretherapeutic targets. (Arterioscler Thromb Vasc Biol. 2008;28:1225-1236)

Key Words: apolipoprotein B � VLDL1 � insulin resistance � metabolic syndrome� nonalcoholic fatty liver disease � stable isotopes � kinetics

Insulin resistance is a key feature of the metabolic syn-drome and can lead to the development of type 2 diabe-

tes.1,2 These conditions are today increasingly common,primarily because of the increased prevalence of a sedentary

lifestyle and obesity.3 Insulin resistance and type 2 diabetesare characterized by dyslipidemia, which is a major riskfactor for cardiovascular disease (CVD). This dyslipidemia ischaracterized by the so-called lipid triad4—high levels of

Original received November 28, 2007; final version accepted April 1, 2008.From the Sahlgrenska Center for Cardiovascular and Metabolism Research, Wallenberg Laboratory for Cardiovascular Research and the Department

of Molecular and Clinical Medicine (M.A., S.-O.O., J.B.), The Sahlgrenska Academy at University of Gothenburg, Sweden; and the Division of Diabetes(M.-R.T.), University of Helsinki, Finland.

Correspondence to Martin Adiels Wallenberg Laboratory Sahlgrenska University Hospital 41345 Gothenburg, Sweden. E-mail [email protected]© 2008 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.107.160192

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http://atvb.ahajournals.org/

plasma triglycerides, low levels of high-density lipoprotein(HDL) cholesterol, and the appearance of small, dense, low-density lipoproteins (sdLDL)—and excessive postprandiallipemia.1,2,5–7 Diabetic dyslipidemia frequently precedes type2 diabetes by several years, indicating that the disturbance oflipid metabolism is an early event in the development ofcardiovascular complications of type 2 diabetes.1,2 Indeed,patients with insulin resistance both with and without type 2diabetes display qualitatively similar lipid abnormalities.2

It is now recognized that the different components ofdiabetic dyslipidemia are not isolated abnormalities but areclosely linked to each other metabolically,1,2,5 and are mainlyinitiated by the hepatic overproduction of large triglyceride-rich very low–density lipoproteins (VLDL1).1,5 It is thus ofkey importance to elucidate the mechanisms involved in theoverproduction of VLDL1 in diabetic dyslipidemia. Here, wereview the pathophysiology of VLDL metabolism in themetabolic syndrome and discuss how increased liver fatinduces overproduction of VLDL1.

Formation and Metabolism of VLDLProduction of Triglyceride-Poor VLDL in the LiverThe assembly of VLDL involves a stepwise lipidation of thestructural protein apolipoprotein B100 (apoB100) in the liver(Figure 1).8,9 The initiating step is lipidation of apoB bymicrosomal triglyceride transfer protein (MTP) in the roughendoplasmic reticulum.10,11 This results in the formation of aprimordial pre-VLDL lipoprotein particle,12 which is convertedto a triglyceride-poor VLDL particle by additional lipidation.13

Maturation of VLDL in the LiverThe triglyceride-poor VLDL particle can either be secretedfrom the cell as VLDL2 or further lipidated to form a mature,triglyceride-rich VLDL (ie, VLDL1).13,14 The lipidation isdependent on the small GTP-binding protein ADP-ribosylation factor 1 (ARF-1).15 This protein plays a centralrole in membrane trafficking between the ER and the Golgi

apparatus, which is consistent with recent results showingthat the late steps of VLDL formation occur in the Golgiapparatus.14,16–18 However, there are data to suggest that theER is the site of maturation.19 The conversion of triglycer-ide-poor to triglyceride-rich VLDL requires a bulk additionof triglycerides and thus differs from the stepwise lipidationof apoB to form pre-VLDL.13 VLDL formation is highlydependent on the accumulation of triglycerides in the cytosol,and several authors have demonstrated that the fatty acidsused for the biosynthesis of VLDL-triglycerides are derivedfrom triglycerides stored in cytosolic lipid droplets.20–22

Delipidation of VLDLVLDL, intermediate-density lipoprotein (IDL), and LDL allcontain 1 apoB100 per particle and are linked in a delipida-tion cascade. Triglyceride-rich VLDL is released from theliver and converted to IDL by lipoprotein lipase (LPL)-catalyzed hydrolyzation of the lipids. LPL can be inhibited byapoCIII.23 IDL can be further hydrolyzed by hepatic lipase tocholesterol-rich LDL, which is catabolized mainly by hepaticuptake of LDL through LDL receptors.24 Results from kineticstudies suggest that the liver also secretes smaller particlessuch as IDL and LDL25,26 and that IDL can be catabolizedthough LDL receptor-mediated uptake.27,28 During theseprocesses, apoB100 remains with the particle.

Dyslipidemia in Insulin Resistance and Type2 Diabetes

Abnormal concentrations of lipids and apolipoproteins canresult from changes in the production, conversion, or catab-olism of lipoprotein particles. Thus, although static measure-ments are important, they do not reveal the underlyingmechanisms involved in the dysregulation of lipid disorders.To infer this information, it is necessary to perform in vivotracer/tracee studies in which the rates of synthesis orcatabolism of a particular lipoprotein or apolipoprotein can bedetermined.

Figure 1. Assembly and secretion of apoB100-containing lipoproteins. ApoB is synthesized andtranslocated into the lumen of the endoplasmicreticulum (ER) (1). The growing apoB molecule iscotranslationally lipidated by MTP to form the pri-mordial VLDL particle (pre-VLDL) (2). Alternatively,apoB fails to be lipidated and is incorrectly folded(3) and sorted to proteasomal degradation (4). Latein the ER compartment, the pre-VLDL particle isconverted to a triglyceride-poor VLDL particle (5).Triglyceride-poor VLDL exits the ER by Sar1/CopIIvesicles that bud off (6) from specific sites on theER membrane.16 The vesicles fuse to form the ERGolgi intermediate compartment (ERGIC) (7), whichthen fuses with the cis-Golgi (8). The triglyceride-poor particles are either transported through thesecretory pathway and then secreted (9), or furtherlipidated (10) to form triglyceride-rich VLDL (VLDL1)particles, which are then secreted (11). The forma-tion of triglyceride-rich VLDL is highly dependenton the accumulation of triglycerides in cytosoliclipid droplets. These lipid droplets are formed assmall primordial droplets from microsomal mem-branes (12) and increase in size by fusion (13). The

triglycerides within the droplets undergo lipolysis and are re-esterified (14) before they lipidate the triglyceride-poor VLDL to form tri-glyceride-rich VLDL.

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Kinetic StudiesToday, the majority of in vivo lipoprotein kinetic studies areperformed using infusion of stable isotopes. The metabolismof lipoprotein particles can be followed by injecting aminoacids labeled with stable isotopes that are then incorporatedinto proteins such as apoB100.29 The triglyceride content canbe followed by infusion of labeled glycerol or free fattyacids.30 Several multicompartmental models have been pro-posed over the years to provide estimates of protein secretionand catabolism,31 and have been designed to analyze eitherthe VLDL-apoB32,33 or the VLDL-triglycerides.34,35 Indeed,in recent years, much has been learned about the metabolicchanges that contribute to dyslipidemia (Figure 2). To en-hance the understanding of the pathways leading to VLDLsubpopulations, we developed a combined multicompartmen-tal model that allows the kinetics of triglyceride and apoB100in VLDL1 and VLDL2 to be simultaneously assessed (Figure3).36 Previous models combined infusion of [3H]glycerol andVLDL exogenously labeled with iodine,37–40 or assessed theapoB and triglyceride kinetics independently.41

Increased Levels of VLDLThe dyslipidemia seen in insulin resistance and type 2diabetes is associated with excess hepatic production ofVLDL.42–45 We have observed that this increase is in theVLDL1 fraction.46,47 By contrast, hepatic secretion of VLDL2

is comparable in insulin-resistant and insulin-sensitive sub-jects.5,46,48,49 In subjects with type 2 diabetes, hepatic uptakeof VLDL, IDL, and LDL is decreased, resulting in increasedplasma residence time of these lipoproteins,50–52 which fur-ther contributes to the increased accumulation. There are alsoreports of increased production of IDL and LDL in insulin-resistant women without diabetes,53 and in men with mild butnot severe diabetes.54

Formation of sdLDLThe formation of sdLDL is closely associated with insulinresistance and hypertriglyceridemia,6 and the VLDL1-triglyc-eride level is the major predictor of LDL size in individuals

with or without type 2 diabetes.1,7,49,55 The mechanism thatleads to the formation of sdLDL is well elaborated, and bothcholesteryl ester transfer protein (CETP) and hepatic lipaseare involved: (1) CETP facilitates the transfer of triglyceridesfrom VLDL1 to LDL; (2) the resulting triglyceride-rich LDLis a preferred substrate for hepatic lipase; and (3) increasedlipolysis of triglyceride-rich LDL results in the formation ofsdLDL.1,6 Thus, it seems that the presence of large triglyceride-rich VLDL1 particles is a prerequisite for sdLDL formation, andsuch correlations have been observed.7,49,55 However, sdLDL arealso observed in patients with type 2 diabetes and insulinresistance with close to normal triglyceride levels.56 This mightbe explained by increased hepatic lipase activity.

Several studies have shown that the presence of sdLDLparticles is associated with increased cardiovascular risk.57–60

However, it is still under debate whether sdLDL levels addindependent information on risk assessment over standardrisk factors.61

Decreased Levels of HDLIncreased levels of VLDL1 also alter the composition of HDLthrough the actions of CETP and hepatic lipase, leading to theformation of small dense HDL and increased catabolism ofthese particles.62 Thus, there is an inverse correlation betweenHDL and liver fat.49

Postprandial LipemiaIntestinal-derived apoB48-containing chylomicrons contributeto the large triglyceride-rich lipoproteins in the postprandialstate. Although approximately 80% of the increase in triglycer-ides after a fat load meal comes from apoB48-containinglipoproteins,63 approximately 80% of the increase in particlecount is from apoB100.64,65 Moreover, the area under the curve(AUC) for apoB100 is 10-fold higher than that of apoB48,66 andthe production rate of apoB100 is 15 to 20 times higher than thatof apoB48.67,68 ApoB48 and apoB100-containing particles are

Figure 2. Changes in lipoprotein metabolism in type 2 diabetesand the metabolic syndrome. Subjects diagnosed with the met-abolic syndrome display, most noticeably, an increased produc-tion of VLDL (1), and there is a reduction in the catabolic rate ofapoB-containing lipoproteins, in particular IDL and LDL (2).50–52

Together, these result in increased concentrations of apoB-containing lipoproteins.50–52 The catabolism of apoA1, the mainapolipoprotein of HDL, is increased by 48% but apoA1 produc-tion is increased by 25%, probably because of some compen-satory effect (3).52 This results in a 16% reduction in the con-centration of HDL-apoA1.52

Figure 3. Compartment model of VLDL-apoB and VLDL-triglyc-erides. The combined model allows for simultaneous determina-tion of the plasma kinetics of apoB and triglycerides in VLDL1

and VLDL2 after a bolus injection of 2H3-leucine and 2H5-glycerol.36 The model consists of 2 subsystems for the freetracers, a glycerol-to-triglyceride conversion subsystem, and asubsystem for the assembly and secretion of the lipoproteinparticles. Secreted particles are either cleared by the system ortransferred down the delipidation chain, before being convertedto IDL.

Adiels et al Overproduction of VLDL in the Metabolic Syndrome 1227



cleared from the circulation by a common pathway and thereforecompete for clearance.69 Thus, the major contribution to anatherogenic lipoprotein profile from chylomicrons is likely theirinterference with apoB100 catabolism.

The Lipid TriadCollectively, the key components of the diabetic dyslipidemiacan be attributed to increased accumulation of VLDL particles,predominantly caused by overproduction of VLDL1. A similardyslipidemia is also present in other syndromes, such as familialcombined hyperlipidemia,70 hyperapobetalipoproteinemia,71 fa-milial dyslipidemic hypertension,72 LDL subclass pattern B,73

and the Reaven syndrome,74 which are all characterized byincreased hepatic apoB overproduction and insulin resistance.

Regulators of VLDL AssemblyFatty Acids Increase VLDL FormationAn increased delivery of fatty acids increases the secretion ofVLDL-triglycerides and apoB100 from human liver and fromhepatocytes and HepG2 cells.75,76 Our turnover studies invivo have demonstrated the importance of hepatic triglycer-ides for the assembly and secretion of VLDL.49 These studieshave also confirmed the stepwise lipidation of VLDL1 anddemonstrated that the secretion of VLDL1-apoB100 increaseswith increasing concentrations of liver lipids.5,49 Importantly,the relationship between triglyceride and apoB productionrates for VLDL1 showed that subjects with type 2 diabetessecrete more—not larger—VLDL1 particles than nondiabeticcontrols.5,36,49 Thus, the amount of lipid added to an individ-ual VLDL2 particle to produce VLDL1 is equal in subjectswith type 2 diabetes and nondiabetic controls, but the rate ofconversion is increased in subjects with type 2 diabetes.

Insulin Shifts the Balance From VLDL1 to VLDL2Only a few studies have investigated the acute effect ofinsulin on VLDL kinetics in humans in vivo.75,77–80 Althoughthe modeling approaches differ, they all show decreasedsecretion of VLDL-triglycerides and VLDL-apoB. Further-more, insulin infusion has a greater effect on the secretion ofVLDL-triglycerides than VLDL-apoB,75,78,79 and it has beenshown to suppress mainly VLDL1-apoB production, withlittle effect on VLDL2-apoB100 production.77,80 Thus, insulinnot only reduces the number of overall VLDL particles, butalso shifts the balance between VLDL2 and VLDL1 to reducethe relative proportion of VLDL1 particles. However, theacute effect of insulin on lipolysis of circulating lipoproteinsstill remains to be fully elucidated.

Insulin has been shown to decrease VLDL formation by atleast 2 mechanisms: (1) by regulating the amount of fattyacids in the circulation79; and (2) by direct suppression of theproduction of VLDL1 in the liver, independent of the avail-ability of fatty acids.78 The molecular mechanisms involvedin the direct suppression of VLDL1 are elusive, and severalmechanisms have been proposed. Sparks and coworkers haveshown that activation of phosphatidylinositol 3-kinase(PI3-K) is necessary for the insulin-stimulated decrease inapoB secretion from rat hepatocytes.81–83 In addition, insulindownregulates MTP expression via activation of the mitogen-activated protein kinase (MAPK) pathway.84,85 Insulin may

also decrease VLDL secretion by inhibiting the activity of thetranscription factor Foxa2.86 Recent studies in ob/ob micehave shown that Foxa2 and its coactivator peroxisomeproliferator-activated receptor gamma (PPAR�) coactivator �(Pgc-1�) promote fatty acid oxidation and stimulation ofMTP in livers, resulting in increased VLDL secretion.87,88

However, these results require further investigation as it issurprising to observe the combination of increased fatty acidoxidation and VLDL secretion.

PPAR� Agonists DecreaseVLDL-Triglyceride SecretionFibrates activate PPAR�, which plays a key role in the regula-tion of energy homeostasis and inflammation.89 Fibrates havebeen used since the 1970s for their lipid-modifying properties,which include reducing plasma triglycerides and VLDL andincreasing HDL cholesterol.89

Experiments in rat liver cells have shown that the triglycer-ide-lowering effect of fibrates is partly explained by increasedoxidation of free fatty acids,90 diverting them away fromtriglyceride synthesis and thus reducing the hepatic synthesis oftriglyceride-rich lipoproteins.91,92 However, in humans, data ondirect effects of fibrates on VLDL production rate are inconclu-sive. The effect on lowering plasma triglycerides seems to becaused by an increased clearance rate,52,93,94 which is supportedby the observations that PPAR� induces expression of LPL95

and inhibits the synthesis of apoCIII.96

Brain Glucose Controls VLDL SecretionRossetti and coworkers have recently shown that hypotha-lamic glucose-sensing mechanisms regulate liver, but notintestinal, VLDL-triglyceride production, and that this regu-lation is lost in diet-induced obesity.97,98 The cross-talkbetween the brain and the liver couples carbohydrate sensingto lipoprotein secretion by curtailing the activity of stearoyl-coenzyme A (CoA) desaturase-1 (SCD1) in the liver and byinterfering with a late step in the hepatic assembly andsecretion of VLDL particles. These findings are consistentwith a homeostatic loop in which the increased availability ofcarbohydrates limits the endogenous output of lipids into thecirculation. This mechanism would, together with insulin,acutely downregulate VLDL secretion after a meal. Glucose-induced hyperglycemia-hyperinsulinemia lowers VLDL-tri-glyceride secretion by 50%,99 but the effects of insulin andglucose cannot be separated from each other.

Further Evidence for Independent Regulation ofVLDL1 and VLDL2

In addition to independent regulation of VLDL1 and VLDL2

production by insulin and PPAR� agonists (as describedabove), there is evidence to indicate a primary effect ofethanol on the stimulation of production of VLDL1 particlesin humans.100 In addition, endogenous cholesterol synthesiscorrelates with VLDL2-apoB but not VLDL1-apoB produc-tion.101 This finding provides further support for independentregulation of VLDL1 and VLDL2, and may explain whyVLDL2 but not VLDL1 is increased in patients with increasedplasma cholesterol, as in moderate hypercholesterolemia26

and familial hypercholesterolemia.28




Which Factors Predict Overproductionof VLDL1?

We recently analyzed which features of type 2 diabetes andinsulin resistance correlate with VLDL1 production, andrevealed strong correlations with plasma glucose that are notapparent in the normal range of plasma glucose.46 By extend-ing our study to monitor liver fat, intra-abdominal fat,subcutaneous fat, and adiponectin, we showed that fastinginsulin, plasma glucose, intra-abdominal fat, and liver fat andHOMA-IR are predictors of VLDL1-apoB and VLDL1-tri-glyceride production.49 However, in a multiple regressionanalysis, only liver fat and plasma glucose remain signifi-cant.49 Moreover, the key predictors of liver fat are intraab-dominal fat, adiponectin, and plasma glucose.49

Liver Fat Correlates with Reduced Effect of InsulinNonalcoholic fatty liver disease (NAFLD) is defined as fataccumulation in the liver that exceeds 5% to 10% of liver weightin individuals who do not consume significant amounts ofalcohol.102 Recent data show that NAFLD strongly associateswith type 2 diabetes, obesity, and hyperlipidemia.103–110

We tested the relationship between liver fat and VLDL1

suppression in subjects with a broad range of liver fatcontent.80 This study confirmed that liver fat predicts baselineVLDL1 production,49 and it also showed that liver fat isassociated with lack of VLDL1 suppression in response toinsulin: insulin downregulates VLDL1 secretion in subjectswith low liver fat but fails to suppress VLDL1 secretion insubjects with high liver fat, resulting in overproduction ofVLDL1.80 The reason for this lack of effect is not known.Insulin suppresses the nonesterified fatty acid (NEFA) pool toa similar extent regardless of liver fat level,80 and thus thehigh VLDL1 production in individuals with high liver fatmust be facilitated either by using a greater portion ofsystemic NEFA or recruiting other sources of triglycerides,such as from hepatic stores.

Does Liver Fat Cause Insulin Resistance?Is the observed association between high liver fat and reducedsuppression of VLDL1 by insulin the result of fatty liver, or isfatty liver merely a consequence of hepatic insulin resistance?The existence of a causal relationship between liver fat andhepatic insulin resistance is controversial with conflictingresults: (1) Fatty liver and insulin resistance may be separatemanifestations of metabolic derangements, and hepatic insu-lin resistance may reflect inflammation rather than lipidaccumulation in this tissue.111 (2) Some studies suggest thatinsulin resistance is an essential requirement for the accumu-lation of hepatocellular fat.107,112,113 It is indeed possible thathepatic steatosis results from insulin resistance because theinsulin signaling pathways that drive fatty acid biosynthesis inthe liver are relatively sensitive to the high levels of portalinsulin flux to the liver that accompany systemic insulinresistance. (3) Several lipid intermediates (eg, ceramides,GM3 ganglioside, and diacylglycerol) appear capable ofinactivating components of the insulin signaling path-way.114,115 The levels of such metabolites are elevated inindividuals with insulin resistance,116,117 and it is also likelythat yet to be identified lipids or lipid-derived metabolites

affect insulin sensitivity. However, the existence of a caus-ative association between accumulation of specific lipidspecies and insulin resistance remains controversial.

Hepatic Insulin Resistance Induces DyslipidemiaWithout Fatty LiverLiver insulin receptor knockout (LIRKO) mice develophyperinsulinemia but their livers do not respond to it, and thusthey can be used to investigate the effects of pure hepaticinsulin resistance.118 These mice develop a proatherogeniclipid profile (low HDL cholesterol and VLDL particlesenriched in cholesterol) but not fatty liver.118 Normally,insulin promotes apoB degradation,82 but the insulin-resistantLIRKO mice over secrete apoB100.118 In contrast to insulin-resistant humans who over secrete both apoB and triglycer-ides as VLDL,29 LIRKO mice secrete less VLDL-triglyceridethan control mice.118 This is probably attributable to lowexpression of SREBP-1c and SREBP-2 and their targets, andsuggests that apoB is secreted as denser particles in LIRKOmice. These data suggest that pure insulin resistance driveschanges in cholesterol homeostasis whereas other factors,such as hyperinsulinemia, drive increased production oftriglycerides and the development of fatty liver. The signifi-cance of these results for humans with the metabolic syn-drome remains to be determined.

Models of Fatty Liver With and WithoutInsulin ResistanceShulman and coworkers have presented data supporting thehypothesis that hepatic steatosis leads to hepatic insulinresistance (see review119). In a rat model of NAFLD, lipidaccumulation stimulates gluconeogenesis and activates pro-tein kinase C epsilon (PKC-�) and c-Jun N-terminal kinase-1(JNK1), which may interfere with tyrosine phosphorylationof insulin receptor substrate-1 (IRS-1) and IRS-2 and impairthe activation of glycogen synthase by insulin.120 Furthermore,recent data from this rat model show that antisense oligonucle-otide (ASO)-mediated inhibition of PKC-� reverses hepaticinsulin resistance.121 PKC-� has also been shown to be activatedin the liver in patients with type 2 diabetes.122

Further work in transgenic animals has also directly testedand confirmed the hypothesis that hepatic steatosis can leadto insulin resistance: (1) Overexpression of glyceraldehyde-3-phosphate acyl transferase (GPAT) in rat liver results inmajor accumulation of triglycerides and in insulin resis-tance.123 (2) Expression of LPL in mouse liver causes hepaticsteatosis and hepatic insulin resistance as manifested byincreased hepatic glucose output.124 (3) Expression ofmalonyl-CoA decarboxylase in liver of rats with diet-inducedinsulin resistance resolves hepatic steatosis and improveswhole-animal, liver, and muscle insulin sensitivities.125

Choi et al showed that ASO-mediated inactivation of acylCoA diacylglycerol acyltransferase 2 (DGAT2) reverses diet-induced hepatic steatosis and insulin resistance by loweringhepatic diacylglycerol content and PKC-� activation.126 How-ever, Yu et al showed that ASO-mediated inhibition of DGAT2reduces liver triglyceride content but does not improve insulin orglucose tolerance in mice fed a high-fat diet.127 Futhermore,Monetti et al showed that DGAT2-mediated lipid accumula-




tion in the liver does not cause insulin resistance, indicatingthat hepatic steatosis can occur independently of insulin resis-tance.128 It is known that hepatic fat does not always associatewith insulin resistance in humans: for example, liver steatosiswithout hepatic insulin resistance is observed in patients withfamilial heterozygous hypobetalipoproteinemia.129

How Could Storage of Neutral Lipid DropletsCause Insulin Resistance?The storage of lipids in mammalian cells was long consideredto be a relatively simple process in which excess fatty acidsare converted to neutral lipids and deposited in cytoplasmiclipid droplets. In recent years, the cell biology of the lipiddroplet has begun to be understood in more detail.130 Accu-mulation of lipids in cells is a balance between the formationof lipid droplets and the hydrolysis of lipids in these droplets,catalyzed by hormone-sensitive lipase (HSL)131 and adiposetriglyceride lipase (ATGL),132–134 and seems to be regulatedby droplet-associated PAT proteins.135–137 Combined knock-down of the PAT proteins adipose differentiation-relatedprotein (ADRP) and tail interacting protein of 47 kDa (TiP47)in cultured liver cells results in large lipid droplets with highturnover of triglycerides and insulin resistance.138 These datasupport an important structural role for ADRP and TiP47 assurfactant proteins at the surface of the lipid droplet, pack-aging lipid in smaller units, protecting lipid droplets againstendogenous lipases, and facilitating triglyceride formation.Furthermore, by promoting lipid packaging into lipid drop-lets, the proteins may protect against the development ofinsulin resistance in hepatic cells.

A number of other enzymes and signaling proteins havealso been shown to be associated with lipid droplets, andinclude fatty acid metabolic enzymes, eicosanoid-formingenzymes, specific kinases, and small GTPases.139–146 Thus,lipid droplets may integrate lipid metabolism, inflammatorymediator production, membrane trafficking, and intracellularsignaling,130,147 consistent with the hypothesis that a disturbedinterplay between these pathways may link liver fat andinsulin resistance. However, the mechanisms involved remainto be elucidated. Recent studies identifying the SNAREprotein SNAP23 as a molecular link between lipid dropletsand insulin resistance in muscle cells support the hypothesisthat it is not lipid droplets per se that promote the develop-ment of insulin resistance but other molecular mechanisms.148

Sources of Fatty Acids for Liver Fatand VLDL-Triglycerides

Potential sources of fatty acids for liver fat and VLDL-tri-glycerides include: (1) peripheral fats stored in adipose tissuethat flow to the liver via the plasma NEFA pool; (2) fattyacids synthesized within the liver through de novo lipogene-sis (DNL); (3) dietary fatty acids that are transported viachylomicrons from the intestine to the NEFA pool and then tothe liver; and (4) uptake of chylomicron remnants by the liver(Figure 4).149,150

Parks and coworkers have recently developed methodol-ogy to determine the fate of dietary fatty acids during thepostprandial state and combined this technique with liver biopsyto simultaneously measure all 4 pathways of fatty acid delivery

to the liver in patients suspected of having NAFLD.151 Thesequantitative metabolic data demonstrate that both elevatedperipheral fatty acid flux and DNL contribute to liver fat andlipoprotein-triglycerides in NAFLD. Furthermore, a signifi-cant similarity was observed between the contributions offatty acid sources for the liver triglyceride pools and theVLDL-triglyceride pool.

NEFA PoolThe hepatic uptake of fatty acids is not regulated and, as aresult, the plasma NEFA concentration is directly related tothe influx of fatty acids to the liver.152 Adipose tissuecontributes approximately 80% of fatty acid content to theplasma NEFA pool in the fasted state, and even in the fedstate it contributes approximately 60%.152 Thus, the mostlikely explanation for excess triglyceride accumulation inNAFLD is increased release of fatty acids from adipose tissue,which flow to the liver via the NEFA pool.149–151,153–155 Ininsulin-resistant states, insulin fails to suppress the activity ofHSL and results in enhanced lipolysis and flux of fatty acidsto the plasma NEFA pool.156

Visceral adiposity has been identified as an independentrisk factor for cardiovascular disease and the metabolicsyndrome, and the severity of NAFLD has been shown to bepositively related to the visceral fat accumulation regardlessof body mass index.157 We found that the insulin-inducedsuppression of NEFA was inversely related to intraabdominalfat volume but not to subcutaneous fat,80 which supports theimportant role of visceral fat as a source of NEFA flux to theliver.158,159 Indeed, the contribution of visceral adipose tissuelipolysis to NEFA increases as a function of visceral fat.159

However, even in viscerally obese people, 50% to 60% ofNEFA entering the liver is from the systemic circulation.159

De Novo LipogenesisIn fasting healthy human subjects, DNL in the liver contrib-utes less than 5% to VLDL-triglyceride content.150 By con-

Figure 4. Sources of fatty acids for liver and VLDL-triglycerides.Fatty acids (FA) may enter the liver through 4 different path-ways: NEFA derived from adipose tissue (1), hepatic DNL (2),spillover of FA from lipolysis of chylomicron (CM) triglycerides(TG) into the NEFA pool (3), and uptake of CM remnants (4).NEFA are taken up by the liver (5) and esterified to TG (6). Glu-cose serves as carbon donor for DNL and, together with insulin,upregulates DNL (7). Liver TG (6) may be temporarily stored (8)before used as an energy source through �-oxidation in themitochondria (9). The majority of lipids are secreted as VLDL (10).




trast, DNL has been shown to account for approximately 25%of liver and VLDL-triglyceride content in hyperinsulinemicsubjects with NAFLD.151 In healthy subjects, DNL is ele-vated after meals,160 which can be accounted for by eleva-tions in the circulating levels of lipogenesis precursors.However, in NAFLD, DNL is already elevated in the fastedstate, and further postprandial elevation is not observed.151

Indeed, constant elevation of DNL was also observed incontrol subjects fed a diet high in simple carbohydrates for 25days.161 A recent study showed that insulin-resistant leansubjects produce 60% less glycogen than insulin-sensitivelean subjects after receiving 2 high-carbohydrate meals.162

The energy is diverted to increased hepatic DNL and resultsin increased plasma triglyceride and reduced HDL cholesterollevels.162 These observations reflect the sustained elevation offactors involved in hepatic DNL,150,151 such as SREBP1-c,163,164 the carbohydrate response element-binding protein(ChREBP),165 and PPAR�.166–168

Chylomicron Synthesis and Other SourcesAfter entering the blood stream through chylomicron synthe-sis in the intestine, dietary fatty acids can be taken up by liveras chylomicron remnants.150 Alternatively, LPL catalyzes therelease of fatty acids from the chylomicrons at a rate thatexceeds tissue uptake, resulting in spill over of these fattyacids into the plasma NEFA pool.150

The contribution of dietary fatty acids to liver triglyceridesdepends on the fat content of the diet. Patients with NAFLDoften consume significantly more saturated fats comparedwith control subjects matched for age, sex, and body massindex (BMI).169 However, studies of patients with NAFLDwho had consumed a standardized 30% fat diet for thepreceding 4 days showed that only 15% of the liver triglyc-erides were derived from dietary fatty acids.151

During the same 4-day study, the source of 10% to 20% ofVLDL-triglycerides was not accounted for by 1 of the 4recognized pathways.151 The source of these fatty acids hasbeen proposed to be either hepatic storage or visceral fat thatis transported to the liver through the portal vein.151 Indeed,liver biopsies in these subjects showed that only 38% of livertriglycerides could be accounted for by the recognized path-ways during the 4-day study period.151 The estimated turnoverrate of liver fat in these subjects was 38 days,151 compared withestimates of 1 to 2 days in normal individuals.170 Interestingly,Vedala et al have presented kinetic evidence for a significantcontribution from a slow-turnover hepatic cytosolic triglyc-eride storage pool to fasting VLDG-triglycerides.171 Theturnover time for this pool was longer in hypertriglyceridemic(HPTG) subjects with diabetes compared with nondiabeticsubjects with and without HPTG, and it is likely that thecontribution of the slow-turnover hepatic cytosolic triglycer-ide storage pool can explain the lack of accountability of livertriglycerides in NAFLD subjects.151

Lipid Management of DyslipidemiaCompared with nondiabetic individuals, patients with type 2diabetes are at a much greater risk for CVD. Consequently,the treatment of CVD risk factors is a healthcare priority inthis patient population. A number of clinical trials with

3-hydroxy-3-methylglutaryl (HMG) CoA (HMG-CoA) re-ductase inhibitors (statins) have shown significant CVD riskreduction through LDL cholesterol lowering in patients withdiabetes,172–177 mainly through increased LDL-receptor activ-ity.178 Increased LDL-receptor activity may also correctchylomicron metabolism.179 Indeed, the recently publishedCollaborative Atorvastatin Diabetes Study (CARDS), aplacebo-controlled trial of patients with type 2 diabetes, wasterminated 2 years earlier than its anticipated length owing tothe significant reduction in number of CVD events observedin patients randomized to receive low-dose atorvastatin ver-sus placebo.172 The statin therapy in this trial resulted insignificant reduction of CVD events in patients with type 2diabetes without previous CVD or high levels of LDL choles-terol and indicated that patients with type 2 diabetes may becandidates for statin therapy regardless of LDL cholesterol levelor absence of a previous CV event. Thus, statins are safe andefficacious in reducing CVD events in patients with type 2diabetes, but recent clinical trials have demonstrated that statinsfail to completely reverse the increased risk of CVD in subjectswith type 2 diabetes.172,173

Fibrates are associated with positive lipid-modifying prop-erties (discussed earlier), and it was anticipated that fibrateswould significantly reduce CVD risk in high-risk people withfeatures of the metabolic syndrome. Indeed, results ofposthoc analyses from the Veterans Affairs High-DensityLipoprotein Intervention Trial (VA-HIT) and the BezafibrateInfarction Prevention (BIP) study indicated that treatmentwith fibrates is beneficial in individuals with diabetes and themetabolic syndrome.180 However, results from the FenofibrateIntervention and Event Lowering in Diabetes (FIELD) studywere not as favorable as expected.181,182 The current recom-mendations for fibrates in the management of dyslipidemia inthe metabolic syndrome are the topic of a separate review inthis series.180

Niacin (nicotinic acid) lowers VLDL, LDL, and sdLDLand raises HDL cholesterol levels, and thus has an overallbeneficial effect on the lipoprotein profile.183 This effectresults from inhibition of lipolysis in adipose tissue via Gprotein–coupled receptors, leading to a reduction of plasmaNEFA183,184 and thus limiting the substrate for VLDL secre-tion. Clinical trials suggest that niacin is a powerful strategyfor raising HDL cholesterol.185,186 However, higher doses ofniacin (�1.5 g/d) may worsen glycemic control in individualswith type 2 diabetes, and recommendations are to use lowerdoses and combine with statins.187

Diabetic dyslipoproteinemia is exacerbated by hepaticoverproduction of large triglyceride-rich VLDL1, but neitherstatins nor fibrates reduce the flux of NEFA to the liver orreduce liver fat. Interestingly, the PPAR� agonist pioglita-zone has been reported to reduce plasma triglyceride levelsby increasing VLDL-triglyceride clearance rate with no effecton hepatic secretion of VLDL apoB.188 The effect on clear-ance rate can be explained by an increase of LPL mass and areduced apoCIII production in response to pioglitazone.188

Recently, PPAR� agonists have been reported to markedlyimprove dyslipidemia and also reduce liver fat in obesemen.189 Potential future targets include several transcriptionfactors (eg, Foxa2 and Pgc-1b),87,88 key regulatory enzymes




in hepatic lipid metabolism (eg, PKC-�, DGAT2 and SCD1),lipid intermediates that interfere with insulin signaling,114 andthe hypothalamic glucose-sensing mechanisms that regulateliver VLDL-triglyceride production.97,98 Novel targets alsoinclude the cannabinoid type 1 (CB1) receptor in the liver: theCB1 receptor antagonist rimonabant has been reported tosuppress lipogenesis, lower plasma triglycerides, and raiseHDL cholesterol.190

ConclusionSeveral kinetic studies support the view that production ofVLDL1 and VLDL2 can be independently regulated,48,77,80,101

and these lipoproteins have different effects on metabo-lism.191 Overproduction of VLDL1 alters the composition ofHDL, which ultimately leads to an increased catabolism of theseparticles,62 and is closely associated with formation ofsdLDL.7 Thus, it is important to realize that VLDL is not ahomogenous pool of lipoprotein particles. We are beginningto understand the molecular mechanism(s) involved in theinability of insulin to suppress VLDL1 production in themetabolic syndrome. Hopefully, clarification of these molec-ular mechanisms will be translated into targeted treatment fordyslipidemia, which is of key importance given the high riskfor CVD in patients with the metabolic syndrome.

AcknowledgmentsThe authors thank Dr R.S. Perkins for expert editing of the manuscript.

Sources of FundingThis work was supported by the Swedish Research Council, theSwedish Heart-Lung Foundation, the Goran Gustafsson Foundation,the Swedish Foundation for Strategic Research, the Torsten andRagnar Soderberg Foundation, European Atherosclerosis Society,Sigrid Juselius Foundation, Clinical Research Institute HUCH Ltd,and HEPADIP (EU-project code: ESHM-CT-2005-018734).

DisclosuresM.R.T. has received honoraria from Sanofi-Aventis, Lilly, andMerck Sharp & Dohme, has advisory board appointments forSanofi-Aventis, Merck Sharp & Dohme, Novartis, and AstraZeneca,and has speakers’ bureau appointments for Merck Sharp & Dohme.J.B. has advisory board appointments for Sanofi-Aventis.

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