European Journal of Clinical Investigation (2002) 32 (Suppl.

3), 14 23

Free fatty acids in obesity and type 2 diabetes: dening their role in the development of insulin resistance and -cell dysfunction
Blackwell Science, Ltd

G. Boden and G. I. Shulman*

Division of Endocrinology/Diabetes/Metabolism and the General Clinical Research Center, Temple University Hospital, Philadelphia PA, USA, *Howard Hughes Medical Institute, Departments of Internal Medicine and Cellular & Molecular Physiology and the General Clinical Research Center, Yale University School of Medicine, New Haven, CT, USA

Abstract

Plasma free fatty acids (FFA) play important physiological roles in skeletal muscle, heart, liver and pancreas. However, chronically elevated plasma FFA appear to have pathophysiological consequences. Elevated FFA concentrations are linked with the onset of peripheral and hepatic insulin resistance and, while the precise action in the liver remains unclear, a model to explain the role of raised FFA in the development of skeletal muscle insulin resistance has recently been put forward. Over 30 years ago, Randle proposed that FFA compete with glucose as the major energy substrate in cardiac muscle, leading to decreased glucose oxidation when FFA are elevated. Recent data indicate that high plasma FFA also have a signicant role in contributing to insulin resistance. Elevated FFA and intracellular lipid appear to inhibit insulin signalling, leading to a reduction in insulinstimulated muscle glucose transport that may be mediated by a decrease in GLUT-4 translocation. The resulting suppression of muscle glucose transport leads to reduced muscle glycogen synthesis and glycolysis. In the liver, elevated FFA may contribute to hyperglycaemia by antagonizing the effects of insulin on endogenous glucose production. FFA also affect insulin secretion, although the nature of this relationship remains a subject for debate. Finally, evidence is discussed that FFA represent a crucial link between insulin resistance and -cell dysfunction and, as such, a reduction in elevated plasma FFA should be an important therapeutic target in obesity and type 2 diabetes. Keywords insulin secretion, magnetic resonance spectroscopy, nonesteried fatty acids, noninsulin-dependent diabetes mellitus Eur J Clin Invest 2002; 32 (Suppl. 3): 1423

Introduction
Plasma free fatty acid (FFA) concentrations are commonly elevated in obese individuals [1], most likely due to increased FFA release associated with an expansion in fat mass [2,3]. Elevated plasma FFA are common in type 2 diabetes [4] and early changes may be predictive for the transition of patients from impaired glucose tolerance (IGT) to type 2 diabetes [5,6]. Furthermore, studies indicate that
Correspondence to: Dr Guenther Boden, Division of Endocrinology/Diabetes/Metabolism and the General Clinical Research Center, Temple University Hospital, Philadelphia PA 19140, USA. Tel: 215 707 8984; Fax: 2157071560; E-mail: bodengh@tuhs.temple.edu and Dr Gerald I. Shulman, Boyer Center for Molecular Medicine 254, 295 Congress Avenue, New Haven CT 06510, USA. Tel: +1 2037855447, Fax: +1 203737 4059; E-mail: gerald.shulman@yale.edu 2002 Blackwell Science Ltd

elevated circulating FFA may directly contribute to the underlying pathophysiology of type 2 diabetes, in particular, the development of insulin resistance both in the periphery and the liver [7,8]. High plasma FFA concentrations are associated with a number of cardiovascular risk factors linked to insulin resistance including hypertension, dyslipidaemia, hyperuricaemia and abnormal brinolysis [9,10]. Moreover, evidence from in vitro studies supports the suggestion that chronically elevated plasma FFA may contribute to -cell dysfunction in type 2 diabetes [1113]. Despite the link between elevated plasma FFA concentrations and the development of several pathological conditions, the signicant physiological role of FFA tends to be overlooked. FFA constitute an important energy source in most body tissues, representing the primary oxidative fuel for liver, resting skeletal muscle, renal cortex and myocardium [14]. When demand for fuel rises, adipose tissue lipolysis is stimulated, thus increasing systemic FFA availability

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and preserving glucose for cerebral requirements. The demand for FFA is particularly high during starvation or exercise; for example, lipid oxidation can account for more than 70% of total body energy expenditure following an overnight fast [15]. Another situation where FFA have an important physiological role is during pregnancy. In the early stages, additional maternal fat is laid down while during late pregnancy, lipolytic hormones stimulate fat breakdown, leading to elevations in plasma FFA concentrations. This induces peripheral insulin resistance and causes a switch in fuel metabolism from carbohydrate to fat oxidation, thus maximizing the availability of glucose for the developing foetus [16]. In addition, FFA have other important physiological functions, for instance, FFA enhance both basal and glucose-stimulated insulin secretion and are essential for stimulussecretion coupling in the pancreatic -cell [17,18]. Different types of FFA inuence glucosestimulated insulin secretion to varying extents, for example longer chain FFA have a greater stimulatory effect on secretion compared with short-chain FFA [19]. Here, we review the inuence of FFA on insulin sensitivity and -cell function in both physiological and pathophysiological circumstances and discuss whether the effect of FFA on these two variables may be linked.

Figure 1 The glucosefatty acid cycle: relationship between glucose and free fatty acid metabolism (Reprinted with permission from Elsevier Science; The Lancet, 1963;1:785 89 [33]).

Role of FFA in the development of insulin resistance

Over 80% of people with type 2 diabetes are obese, while virtually all are insulin resistant [7]. The association between obesity and insulin resistance is likened to a cause and effect relationship since both human and animal studies indicate that weight loss/gain correlates closely with increasing/ decreasing insulin sensitivity, respectively [20 23]. Several factors are proposed to link obesity and insulin resistance and promising candidates include FFA, tumour necrosis factor- (TNF-) and leptin. This review will focus on the potential role of FFA while TNF- and leptin are reviewed elsewhere [2426]. Adipose tissue has been proposed to be a site of insulin resistance [27]. As insulin resistance develops, there is a decrease in insulin-mediated suppression of lipolysis leading to increased circulating FFA and ultimately insulin resistance in skeletal muscle and liver [7,8]. A strong correlation is observed between increased plasma FFA, intramyocellular lipid accumulation and insulin resistance [2831]. A number of hypotheses have been put forward to explain the role of FFA and intracellular lipid in the pathogenesis of type 2 diabetes, but, until recently, a more detailed biochemical explanation for FFA-induced insulin resistance has remained elusive.

Insulin resistance in skeletal muscle Skeletal muscle is a major contributor to insulin resistance in type 2 diabetes. In a study investigating glucose

metabolism in individuals with type 2 diabetes, insulinstimulated total body glucose uptake decreased by 30 40% compared with nondiabetic controls under hyperinsulinaemic conditions [32]. Measurement of peripheral and splanchnic glucose uptake and hepatic glucose output in the same individuals indicated that nearly 90% of this decrease was accounted for by the peripheral tissues (predominantly skeletal muscle) [32]. In 1963, Randle et al. proposed a connection between muscle insulin resistance and elevated FFA concentrations [33]. They demonstrated that FFA compete with glucose as an energy substrate in muscle and adipose tissue, describing the relationship between glucose and FFA metabolism in terms of a glucosefatty acid cycle (Fig. 1). Studying rat cardiac muscle in vitro, they observed that the rate of fat oxidation increased relative to carbohydrate oxidation in response to elevated FFA concentrations [33]. This was associated with a decrease in insulin-stimulated muscle glucose uptake and utilization. Almost 30 years later, Boden et al. observed similar effects in healthy human skeletal muscle [34]. Under euglycaemichyperinsulinaemic clamp conditions, they demonstrated a decrease in carbohydrate oxidation associated with increased fat oxidation following 1 h of lipid infusion [34]. In the same healthy individuals, they observed a signicant inhibition of insulin-stimulated glucose uptake following 34 h of lipid infusion [34]. This observation was conrmed in subsequent studies in healthy volunteers [35,36] and in individuals with type 2 diabetes [37]. In both of these groups, there was a consistent and signicant reduction in insulin-stimulated glucose uptake of 40 55% following lipid infusion compared with controls. Studies in diabetic rats demonstrate that anti-lipolytic agents (nicotinic acid and phenylisopropyladenosine) exert potent blood glucose-lowering effects by decreasing plasma FFA concentrations and thus improving insulin sensitivity [38]. To investigate the effect of anti-lipolytic agents in

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humans, a long-acting nicotinic acid analogue, Acipimox, was used to lower chronically elevated FFA in obese individuals, with or without type 2 diabetes [39]. After overnight administration of Acipimox, fasting plasma FFA decreased by 6070% in all groups. This was accompanied by a twofold increase in insulin-stimulated glucose uptake in the obese, nondiabetic subgroup, sufcient to normalize glucose uptake to the levels seen in lean, nondiabetic individuals. Insulin-stimulated glucose uptake also improved in obese, diabetic subjects following Acipimox administration, although the compensation was not complete, indicating that FFA was only partially responsible for inducing the insulin resistance observed in type 2 diabetes. Importantly, the improvements in insulin sensitivity detected after overnight Acipimox administration were accompanied by signicant improvements in glucose tolerance [39], consistent with earlier ndings in the diabetic rat model. Hence, there is a gathering body of evidence linking elevated FFA and the development of muscle insulin resistance. Further clarication of the cellular mechanism for skeletal muscle insulin resistance is needed to determine the precise role of FFA. Key ndings to date are outlined below. Reduced glycogen synthesis plays a dominant role in inducing muscle insulin resistance In 1990, Shulman et al. used 13C nuclear magnetic resonance (NMR) spectroscopy to measure the rate of muscle glycogen synthesis in situ [40]. Under hyperglycaemic hyperinsulinaemic clamp conditions, they observed a 60% reduction in subjects with type 2 diabetes relative to nondiabetic, healthy individuals [40]. Muscle glycogen synthesis was estimated to account for 70% of whole-body glucose metabolism and 90% of nonoxidative glucose metabolism [40]. These results indicated that decreased muscle glycogen synthesis plays a central role in the insulin resistance seen in type 2 diabetes, although the precise rate-controlling step was not established. Decreased glucose transport leads to reduced muscle glycogen synthesis Candidate steps in the muscle glycogen synthesis pathway that may be responsible for this decrease include glycogen synthase, hexokinase and glucose transporter enzymes (Fig. 2). If glycogen synthase activity is suppressed in type 2 diabetes, then the rate of glycogen synthesis would be expected to decrease and glucose-6-phosphate would accumulate in the muscle of diabetic individuals in response to insulin. In fact, reductions in both muscle glycogen synthesis and glucose-6-phosphate concentrations have been observed in individuals with type 2 diabetes [40,41], consistent with either hexokinase activity or glucose transport being the rate limiting step in this pathway. In order to differentiate between decreased hexokinase activity and glucose transport as potential rate-limiting steps, the intracellular concentrations of muscle glycogen, glucose-6-phosphate and glucose were measured in patients

Figure 2 Potential rate-controlling steps responsible for reduced insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus; adapted with permission from Shulman G.I. J Clin Invest 2000;106:1716 [8].

with type 2 diabetes and in healthy, nondiabetic subjects using 13C and 31P NMR spectroscopy [42]. Under hyperglycaemic, hyperinsulinaemic conditions, the muscle glycogen synthesis rate and glucose-6-phosphate concentrations were found to be lower in diabetic individuals relative to control subjects, consistent with previous results. Since intracellular glucose represents an intermediate step between glucose transport and hexokinase activity, a substantial accumulation of intracellular glucose would be expected in subjects with type 2 diabetes if hexokinase activity was the rate-controlling step. In fact, the small change in intracellular glucose concentration detected in the diabetic patients was proportional to the observed change in glucose-6-phosphate, indicating that the reduction in muscle glycogen synthesis observed in type 2 diabetes primarily results from decreased insulin-stimulated glucose transport activity [42]. Identifying the role of FFA revisiting Randles hypothesis How are FFA involved in the development of muscle insulin resistance? Several modications have been made since Randle et al. rst proposed that elevated FFA induce insulin resistance in muscle solely by increasing the oxidation of fat relative to carbohydrate [33]. Boden et al. investigated potential mechanisms by which elevated FFA inhibit insulinstimulated glucose uptake both in healthy individuals [35] and in patients with type 2 diabetes (Fig. 3) [37]. In agreement with the previous ndings, they observed an inhibition of carbohydrate oxidation in healthy volunteers after an hour of lipid infusion under euglycaemic, hyperinsulinaemic conditions [35]. This effect alone is unlikely to be responsible for the signicant reduction in insulinstimulated glucose uptake that is observed some 2 3 h later [35]. In a subsequent study of type 2 diabetes patients, they reported that the decrease in insulin-stimulated glucose uptake occurring after 3 4 h lipid infusion is accompanied by a proportional inhibition of both glycogen synthesis and glycolysis [37]. Hence the major inhibitory effect of elevated FFA may occur at an early stage in glucose utilization, i.e. at the level of glucose transport/phosphorylation, since a primary effect on either glycogen synthesis or glycolysis would lead to disproportionate actions on these two

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Figure 3 Inhibitory effects of FFAs on glucose utilization. The inhibition of carbohydrate oxidation (1) was the earliest demonstrable effect. It developed during the initial 2 h of lipid infusion but did not inhibit insulin-stimulated glucose uptake or glycolysis. The inhibition of glucose transport /phosphorylation (2) developed after 3 4 h, whereas the inhibition of glycogen synthesis (3) developed after 4 6 h of high plasma levels of free fatty acids; adapted with permission from Boden G. Endocrine Practice 2001;7:44 51 [93].

glucose transport /phosphorylation rather than at glycogen synthesis. It should be noted, however, that studies from another laboratory indicate that elevated FFA signicantly increase glucose-6-phosphate concentrations in healthy subjects [35]. One possible reason for this inconsistency is that the observed increase is an artefact caused by glycogen hydrolysis in the muscle biopsy sample that can be avoided by performing 31pNMR measurements in situ [36]. To differentiate between an action of FFA on phosphorylation or glucose transport activity, intramuscular glucose concentrations were measured using a novel 13C NMR technique as described in Cline et al. [42]. As before, if hexokinase activity is the rate-limiting step, a signicant accumulation of intracellular glucose is expected in response to high FFA concentrations. In fact, intracellular glucose concentrations decreased signicantly in healthy volunteers following a 5-h infusion of lipid, supporting the hypothesis that elevated FFA induce insulin resistance principally at the level of glucose transport [43]. Elevated FFA inhibit glucose transport by inhibiting insulin signalling How do elevated plasma FFA inhibit glucose transport? One possibility is a direct effect on the GLUT-4 glucose transporter, for example, FFA-induced changes in GLUT4 synthesis or vesicle trafcking, budding, or fusion [44]. Alternatively, elevated FFA may indirectly affect GLUT-4 activity by modifying upstream insulin signalling events. To examine the latter possibility, Dresner et al. [43] investigated the impact of raising FFA concentrations on phosphatidylinositol 3-kinase (PI 3-kinase), an important component of the insulin signalling cascade involved in regulating GLUT-4 translocation [45,46]. They found that the increase in PI 3-kinase activity observed in response to insulin stimulation in control subjects was virtually abolished in individuals given lipid infusions [43]. It is not clear from this study whether elevated plasma FFA inhibit insulin-stimulated PI 3-kinase activity directly or by modifying other components of the insulin signalling pathway. For a comprehensive review of the insulin signalling cascade, see White [47]. Subsequent experiments demonstrated that high FFA concentrations may affect several upstream proteins in the pathway, including insulin receptor substrate 1 (IRS-1) and protein kinase C theta (PKC; Fig. 4) [48]. In rats infused with lipid under euglycaemic hyperinsulinaemic conditions, a 50% decrease in PI 3kinase activity was observed compared with control rats. In these animals, insulin-stimulated IRS-1 tyrosine phosphorylation decreased, and membrane-bound PKC concentrations increased, relative to controls [48]. These results suggested that increased circulating FFA may induce insulin resistance in skeletal muscle by activating PKC, conrming previous observations where alterations in PKC and PKC were linked to the development of muscle insulin resistance in high-fat-fed rats [49]. It is proposed that the resulting decrease in IRS-1 tyrosine phosphorylation suppresses PI 3-kinase activity and decreases GLUT-4 translocation, culminating in a reduction of glucose transport [8].

pathways. There may also be a secondary abnormality in glycogen synthesis caused by elevated FFA concentrations, since a reduction in muscle glycogen synthase activity was observed following 4 6 h lipid infusion that was not apparent after 3 4 h [35]. From these data, as well as from measurements of intramuscular glucose-6-phosphate concentrations, these workers suggested that two different defects contribute to the impairment of glycogen synthesis depending on the FFA concentration. At FFA concentrations of 075 m, they found increased intramuscular glucose-6-phosphate concentrations suggesting a FFA-induced inhibition of glycogen synthase activity, whereas at FFA concentrations of 05 m, they were not able to nd any difference in intramuscular glucose-6-phosphate. From these data, the authors concluded that, at this lower FFA concentration, decreased muscle glucose transport/phosphorylation activity accounted for the reduced rates of muscle glucose uptake. Roden et al. conrmed that an increase in plasma FFA (2 m) caused a profound inhibition of insulin-mediated glucose uptake in skeletal muscle following 4 6 h of lipid infusion [36]. Since glucose-6-phosphate lies between glycogen synthase and hexokinase in the glycogen synthetic pathway (Fig. 2), accumulation of glucose-6-phosphate in response to lipid infusion would indicate an effect at the level of glycogen synthesis. In fact, under euglycaemic hyperinsulinaemic clamp conditions, they reported a signicant decrease in intramuscular glucose-6-phosphate concentrations in healthy individuals as measured by 31P NMR spectroscopy in response to lipid infusion associated with a 50% reduction in insulin-stimulated muscle glycogen synthesis as measured by 13C NMR spectroscopy [36]. These results denitively demonstrate that elevated plasma FFA inhibit insulin-stimulated glucose uptake at the level of

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contrast, liver-LPL-overexpressing mice had a twofold increase in liver triglyceride content and were insulin resistant due to the impaired ability of insulin to suppress endogenous glucose production, which was associated with defects in insulin activation of IRS-2-associated PI 3-kinase activity. These defects in insulin action and signalling were associated with increases in intracellular fat metabolites (diacylglycerol, fatty acyl coenzyme A). These ndings suggest a direct and causative relationship between intracellular fat metabolites and insulin resistance mediated via alterations in the insulin signalling pathway.

Insulin resistance in liver

Figure 4 Proposed alternative mechanism for fatty acid-induced insulin resistance in human skeletal muscle; adapted with permission from Shulman G.I. J Clin Invest 2000;106:1716 [8].

If this proposal is true, then it is conceivable that increased intracellular lipid accumulation may inhibit insulin signalling. This is supported by evidence from A-ZIP/ F-1 transgenic mice. A-ZIP/F-1 is a protein that inhibits the function of transcription factors critical for adipose tissue development. Overexpression of this protein in mice causes abnormal fat deposition or lipodystrophy [50,51]. These lipodystrophic, or fatless, mice are severely insulin resistant and have a reduced response to insulin-stimulated IRS-1 and PI 3-kinase activity [51]. In addition, there is a twofold increase in intracellular lipid content in both muscle and liver. When normal adipose tissue is transplanted into these mice, insulin signalling and intracellular lipid content are simultaneously restored to normal levels, supporting the proposal that intracellular lipid accumulation may lead to abnormal insulin signalling and insulin resistance [51]. The introduction of normal adipose tissue into lipodystrophic mice may be analogous to the mechanism by which the thiazolidinediones (troglitazone, rosiglitazone and pioglitazone) improve insulin sensitivity [8]. For example, rosiglitazone activates peroxisome proliferator-activated receptor gamma (PPAR) in adipose tissue, leading to increased adipocyte differentiation, redistribution of fat from muscle and liver to subcutaneous adipose tissue and improved insulin sensitivity [52,53]. Specically, these changes may be mediated by alterations in muscle PKC and PKC activity, as evidenced by data from rosiglitazonetreated high-fat-fed rats [54]. Research is currently underway to investigate further how fat redistribution mediated by the thiazolidinediones affects the insulin signalling pathway and thus improves insulin sensitivity. Further evidence for the role of intracellular lipid in mediating insulin resistance has been obtained from transgenic mice with muscle-specic and liver-specic overexpression of lipoprotein lipase (LPL) [55]. Muscle-LPL-overexpressing mice had a threefold increase in muscle triglyceride content and were insulin resistant due to decreases in insulinstimulated glucose transport in skeletal muscle and insulin activation of IRS-1 associated PI 3-kinase activity. In

It is commonly accepted that endogenous glucose production (mainly contributed by the liver) is increased in type 2 diabetes and a close correlation has been observed between fasting plasma glucose concentrations and the rate of hepatic glucose production [56,57]. While endogenous glucose production is reported to be 25% higher in individuals with poorly controlled type 2 diabetes relative to healthy controls [58,59], there has been much controversy as to whether changes in gluconeogenesis or glycogenolysis are responsible for this increase. In order to address this question, Magnusson et al. used 13C NMR spectroscopy to assess directly the rates of net hepatic glycogenolysis and gluconeogenesis in control and poorly controlled type 2 diabetic subjects. Using this approach, they found that the increased rates of glucose production in the diabetic subjects could be entirely accounted for by a 60% increase in the rate of gluconeogenesis [58]. These data are supported by recent evidence using other approaches to assess gluconeogenesis in severely hyperglycaemic type 2 diabetes patients [60,61]. Although little is known concerning the mechanism of insulin resistance in the liver, a role for elevated plasma FFA has been postulated. While raised FFA concentrations promote gluconeogenesis in rat liver [62], the effect in humans has only been examined recently. Nicotinic acid was administered to healthy individuals from 16 to 20 h of a 24-h fast to lower plasma FFA [63]. Nicotinic acid discontinuation at 20 h resulted in a rebound in plasma FFA concentrations above basal fasting levels. Changes in gluconeogenesis mirrored the changes in plasma FFA, while glycogenolysis was inversely correlated with FFA concentrations. Overall, these changes in plasma FFA concentrations had little effect on endogenous glucose production as a balance was maintained between changes in gluconeogenesis and glycogenolysis due to hepatic autoregulation [63]. A similar approach was used in subjects with type 2 diabetes [64]. As before, reducing plasma FFA inhibited, while increasing FFA stimulated, gluconeogenesis but the impact on glycogenolysis differed to that in healthy subjects. When plasma FFA concentration decreased with nicotinic acid administration, glycogenolysis also decreased, causing a signicant overall reduction in endogenous glucose production. These observations support the idea that autoregulation of endogenous glucose production in

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response to decreasing gluconeogenesis is abnormal in subjects with diabetes. Hence pharmacological agents that effectively lower FFA in people with type 2 diabetes are likely to have a signicant effect in reducing fasting plasma glucose [64]. In addition to stimulating gluconeogenesis, elevated FFA also affect insulin secretion [65] and hepatic insulin clearance [66], both of which affect endogenous glucose production. Further experiments are required to investigate the role of elevated FFA in suppressing insulinstimulated inhibition of endogenous glucose production.

Therapeutic approaches to lowering FFA/insulin resistance As outlined above, elevated plasma FFA contribute signicantly to skeletal muscle insulin resistance. While data concerning the involvement of FFA in hepatic insulin resistance appear contradictory, there is a suggestion that high circulating FFA may also stimulate endogenous glucose production. Therefore, it appears logical that lowering FFA should be a therapeutic target in obesity and type 2 diabetes. Weight loss leads to decreased circulating FFA and improved insulin sensitivity in individuals with type 2 diabetes [2123,67]. However, patient compliance to diet and exercise therapy is often poor and weight loss may not be maintained in the longer term, so additional therapeutic options are required [68]. Several pharmacological agents, including the thiazolidinediones, reduce elevated FFA and/or insulin resistance in people with type 2 diabetes [69]. For example, insulinstimulated glucose disposal improved by 40 50% following troglitazone therapy in subjects with type 2 diabetes [59,70], while a similar increase was demonstrated in individuals treated with pioglitazone [71]. Recent data indicate that the same is true for rosiglitazone [72]. It is proposed that the thiazolidinediones reduce insulin resistance by enhancing insulin-stimulated glucose transport, leading to increased rates of muscle glycogen synthesis and glucose oxidation [73]. Two main effects of these drugs on glucose transport are proposed: rstly, a direct increase in the expression of glucose transporters in skeletal muscle [73] and secondly, a reduction in circulating FFA concentrations leading to enhanced insulin signalling and increased GLUT-4 translocation [8]. In addition to their primary action in adipose tissue leading to important effects in skeletal muscle, the thiazolidinediones also improve insulin sensitivity in the liver. For example, high doses of troglitazone (600 mg) induced a small but signicant decrease in endogenous glucose production in subjects with type 2 diabetes, possibly as a result of decreased systemic FFA [59]. Recently, rosiglitazone treatment was shown to stimulate fat redistribution from the liver to subcutaneous tissue [74,75], indicating a potential mechanism by which the thiazolidinediones may improve hepatic insulin sensitivity [8]. These agents may also be of use in the treatment of nonalcoholic steatohepatitis (NASH) or fatty liver, a condition closely associated with abdominal obesity and insulin resistance. NASH is common in individuals with type 2 diabetes and is characterized

by hyperinsulinaemia and an increased supply of FFA to the liver [76]. In contrast to the thiazolidinediones, metformin appears to act primarily in the liver. The major effect of metformin is a reduction in endogenous glucose production [70,77]. Following some debate as to whether decreased endogenous glucose production results from a direct effect of metformin on gluconeogenesis [77] or glycogenolysis [78], recent studies by Hundal et al. using 13C NMR to measure directly the rates of net hepatic glycogenolysis and using 2H2O to measure directly the rates of gluconeogenesis found that metformin lowers the rates of glucose production exclusively by inhibiting gluconeogenesis [61]. In addition, metformin therapy appears to cause a small increase in peripheral glucose disposal [70]. Since chronic hyperglycaemia may contribute to peripheral insulin resistance [79], the effect of metformin on glucose disposal may be a secondary effect in response to reduced glucose toxicity [70].

Impact of FFA on insulin secretion

It is commonly accepted that changes in plasma FFA concentrations inuence insulin secretion. While it is well documented that acute FFA administration leads to stimulation of glucose-stimulated insulin secretion [80 84], the impact of chronic FFA exposure remains controversial. A growing body of evidence supports the hypothesis that chronically elevated FFA have a lipotoxic effect on the pancreas [11,12,85]. For example, prolonged lipid infusion has been shown to have a biphasic inuence on insulin secretion from the perfused pancreas of normal rats [13]. Following an initial stimulation of glucose-induced insulin secretion in response to lipid after 3 h, a marked decrease in secretion was observed following 48 h of lipid infusion. The observed inhibitory effect of lipid only affected glucose-induced secretion, was coupled to decreased glucose oxidation and appeared to be dependent on fatty acid oxidation [13]. The importance of FFA was later conrmed in isolated rat islets, where long-term exposure to FFA substantially inhibited glucose-stimulated insulin secretion by decreasing glucose oxidation [86]. Further support for the lipotoxicity hypothesis comes from experiments investigating the effect of troglitazone in islets from Zucker Diabetic Fatty (ZDF) rats [87]. These rats are hyperglycaemic with signicant abnormalities in insulin secretion that have been attributed to a 50-fold increase in islet triglyceride content relative to nondiabetic controls [88]. The inhibitory effect of triglyceride was conrmed by the improvement in insulin secretion following troglitazone incubation, which simultaneously reduced islet triglyceride content [87]. These data support the hypothesis that increased circulating FFA and islet lipid accumulation are responsible for the suppression of insulin secretion seen in ZDF rats. One possible mechanism for the proposed lipotoxicity is that surplus unoxidized FFA (characterized by elevated islet triglyceride content) increase the formation of nitric oxide, which induces -cell apoptosis [85].

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In contrast, data in humans indicate an increase in glucose-stimulated insulin secretion following prolonged FFA administration [65]. In healthy volunteers, plasma glucose was clamped at a level that would stimulate insulin secretion and lipid was infused continuously for 48 h. Contrary to in vitro observations in rats [13], lipid administration increased the mean insulin secretion rate by almost 50% over the duration of the study compared with low FFA controls [65]. However, when these data were adjusted for insulin resistance [89], the enhanced insulin secretion rate was found to compensate precisely for the FFA-induced insulin resistance. Nevertheless, it may be necessary to study the effect of FFA over a more prolonged period. Since it is not feasible in humans to perform lipid infusions for periods longer than 48 h, other methods are needed that more closely relate to human obesity. One approach involved using nicotinic acid or Acipimox to lower elevated FFA concentrations in obese subjects with and without diabetes [17,39]. In these subjects, signicant decreases in insulin secretion (30 50%) were observed, indicating that basal plasma FFA support between 30 and 50% of basal insulin levels. These data suggest that physiological increases in plasma FFA concentrations in humans potentiate glucosestimulated insulin secretion and are unlikely to be lipotoxic to cells.

reects a genetic predisposition to -cell dysfunction. Despite their elevated plasma FFA concentrations, these 20% are unable to compensate for insulin resistance efciently, resulting in a decit in insulin secretion relative to insulin resistance and, ultimately, the onset of hyperglycaemia. The remaining 80%, while insulin resistant, can compensate by stimulating the pancreas via FFA to secrete more insulin and so do not progress to diabetes. Inevitably, there is a slight overcompensation and this leads to the hyperinsulinaemia that is characteristic in these individuals. If this theory is correct, then the insulin secretory response to FFA is likely to be reduced in diabetic individuals. However, preliminary experiments designed to test this hypothesis indicate a normal insulin secretory response in individuals with type 2 diabetes, either following lipid infusion or during the FFA rebound that occurs after discontinuation of nicotinic acid administration [94]. Interestingly, a twofold increase in ketone bodies was also observed in the diabetic subjects [94]. Since ketone bodies also act as insulin secretagogues [95 97], these data suggest that individuals with type 2 diabetes may increase plasma ketone bodies in order to compensate for the abnormal insulin secretory response to FFA. Validation of the relationship between FFA, ketone bodies and insulin resistance is required in order to explore this hypothesis further.

Do FFA provide the link between insulin resistance and -cell dysfunction?
It is now well established that high plasma FFA concentrations have a major inuence on the underlying pathophysiology of type 2 diabetes, primarily by inducing insulin resistance in the periphery. Recent evidence indicates that insulin resistance is also present in the cell and may contribute to the abnormalities in insulin secretion observed in type 2 diabetes [90,91]. Since high plasma FFA and increased intracellular lipid inhibit insulin signalling in muscle [48,49,51], it is possible that elevations in FFA may also contribute to -cell insulin resistance, representing a secondary pathogenetic mechanism. According to the lipotoxicity hypothesis, chronically elevated FFA may exert a direct toxic action on the cells of the pancreas by increasing the rate of nitric oxide formation [11,12,85]. In addition, evidence from transgenic mice expressing human islet amyloid polypeptide (IAPP) indicates that increased dietary fat consumption may play a role in the development of -cell dysfunction by inducing islet amyloid deposition [92]. However, despite growing support from in vitro studies, there is no substantial in vivo evidence to support the existence of lipotoxicity in humans as yet. The concept that chronically elevated FFA concentrations are solely responsible for both insulin resistance and -cell dysfunction is problematic. If this were true, since obesity is commonly associated with increased plasma FFA concentrations, all obese individuals would ultimately have type 2 diabetes. In fact, only approximately 20% of obese people go on to develop the condition [93] and this probably

Conclusion
Recent ndings support the idea of a causal relationship between elevated FFA and peripheral insulin resistance. Under specic circumstances, a rise in FFA concentration has important physiological consequences, for example, during pregnancy, elevated FFA induce insulin resistance and so valuable glucose is conserved for the developing foetus. The divide between physiology and pathophysiology appears to be narrow and high plasma FFA are intimately associated with obesity and type 2 diabetes. In obese individuals, plasma FFA concentrations are chronically elevated and, besides producing insulin resistance in skeletal muscle, may have additional actions in the liver and pancreas that may contribute to the development of type 2 diabetes. Although the exact relationship between elevated FFA, insulin resistance and -cell dysfunction requires further investigation, there is mounting evidence that elevated FFA should represent an important therapeutic target in obesity and type 2 diabetes. Agents such as the thiazolidinediones that provide long-term reductions in plasma FFA concentrations may be of considerable benet in the management of these and related conditions.

Acknowledgement
Dr G. Bodens work was supported by US Public Health Service Grants RO1AG 07988, RO1OK 588895 and MO1RR00349 (GCRC Branch of NCRR).

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