Diagnosis and Management of Pituitary Disorders - part 2 docx

Chapter 3 / Metabolic Mechanisms of Muscle Insulin Resistance 37 Furthermore, leptin administration to humans with severe lipodystrophy partially reverses their severe insulin resistance and hyperlipidemia (18). Expression of the insulin-regulated glucose transporter 4 (GLUT-4) is strongly depressed in adipose tissue but is much less reduced in skeletal muscle in animals and humans with type 2 diabetes (19). Because skeletal muscle accounts for approx 80% of glucose disposal in the postprandial state, the diabetes-associated reduction in adipose GLUT-4 did not at first seem highly relevant to metabolic dysregulation. However, subsequent studies showed that mice with adipose-specific knockout of GLUT-4 have impaired insulin sensitivity in muscle and liver (19). The impairment in insulin action is only apparent in tissues in situ and not in excised tissue samples, implying participation of a blood-borne hormone or metabolite that mediates the effect. A subsequent study has demonstrated that mice deficient in adipose GLUT-4 have elevated levels of RBP-4 in blood, due in part to increased production of the hormone by adipose tissue. Furthermore, increases in circulating RBP-4 levels in normal mice induced by infusion or transgenic expression causes insulin resistance (9). Interestingly, food deprivation (fasting) also causes a form of insulin resistance and is associated with a decrease in adipose GLUT-4 expression (20). This raises the possibility that the original purpose of adipocyte-derived insulin-desensitizing molecules, such as RBP-4, TNF and resistin, may have been to prevent hypoglycemia in the fasted state, which with the advent of overnutrition and senescence in modern life has been subverted to create pathophysiology (21). Alterations in metabolic function in liver can also lead to changes in insulin sensitivity in muscle, consti- tuting a second inter-organ signaling network. For example, in rats fed a high-fat diet, hepatic expression of malonyl-CoA decarboxylase (MCD) causes near-complete reversal of severe muscle insulin resistance (22). MCD affects lipid partitioning by degrading malonyl-CoA to acetyl-CoA, thereby relieving inhibition of carnitine palmitoyl transferase-1 (CPT1), the enzyme that regulates entry of long-chain fatty acyl-CoAs (LC-CoAs) into the mitochondria for fatty acid oxidation. In addition, malonyl-CoA is the immediate precursor for de novo lipogenesis. To gain insight into lipid-derived metabolites that might participate in the cross talk between the liver and muscle in the regulation of insulin sensitivity, metabolic profiling of 36 acyl-carnitine species was performed in muscle extracts by tandem mass spectrometry. These studies revealed a unique decrease in the concentration of one lipid-derived metabolite, -OH-butyrylcarnitine, in muscle of MCD-overexpressing animals that likely resulted from a change in intramuscular -oxidation and/or ketone metabolism (22). Our current interpretation of the mechanistic significance of these findings is elaborated further below. Another example of the profound effects of altered lipid partitioning in control of whole-animal metabolic status comes from studies of animals deficient in stearoyl-CoA desaturase-1 (SCD-1) activity in liver. This enzyme catalyzes the conversion of saturated fatty acids (e.g., C16:0, C18:0) to monounsaturated fatty acids (C16:1, C18:1). Knockout of SCD-1 in ob/ob mice reverses obesity and insulin resistance in these animals (23,24). This effect appears to be mediated by enhanced rates of oxidation of saturated versus unsaturated LC-CoAs. There is also evidence to suggest that SCD-1 deficiency results in increased AMPK activity, which further enhances overall rates of fatty acid oxidation (25). Conversely, human studies have shown that high expression and activity of SCD-1 in skeletal muscle of obese subjects contributes to decreased AMPK activity, reduced fat oxidation and increased TAG synthesis (26). Finally, there is growing evidence that adipose tissue and the liver play important roles in the regulation of insulin sensitivity via inflammatory mechanisms (27). At high doses, salicylates (aspirin) reverse insulin resistance and hyperlipidemia in obese rodents while suppressing activation of the NF-B transcription factor (28,29). Subsequently, it has been demonstrated that high-fat diets or obesity result in activation of NF-B and its transcriptional targets in the liver. Overexpression of a constitutively active version of the NF-B activating kinase, IkB kinase catalytic subunit  (IKK-) in liver of normal rodents to a level designed to mimic the effects of high-fat feeding results in liver and muscle insulin resistance and diabetes (8). In addition, both high-fat feeding and IKK- overexpression increase expression of proinflammatory cytokines such as IL-6, IL-1, and TNF in the liver, and lead to increased levels of these molecules in blood. Antibody-mediated neutralization of IL-6 in these models partially restores insulin sensitivity (8). Interestingly, mice with IKK- knockout in the liver are protected from diet-induced impairment of hepatic insulin action but still develop muscle and adipose insulin resistance (30). In contrast, mice with IKK- knockout in myeloid cells are protected against diet-induced insulin resistance in all tissues (30). These findings suggest the primary mediator of the inflammatory response to elevated lipids may be macrophages that reside within the liver and adipose depots. 38 Muoio et al. How is metabolic fuel overload linked to activation of stress pathways and cytokine production in liver and adipose tissue (or within liver- and adipose-associated immune cells), that leads in turn to development of muscle insulin resistance? One intriguing possibility is that excess lipids may trigger stress responses in the endoplasmic reticulum (ER) (31). Thus, markers of ER stress are elevated in the liver and adipose tissue of genetic or diet-induced forms of obesity, and this in turn is linked to activation of the c-jun amino-terminal kinases (JNK), which are known to interfere with insulin signaling via serine phosphorylation of insulin receptor substrate-1. Moreover, genetic manipulations that relieve ER stress also confer resistance against diet-induced metabolic dysfunction. The question of whether obesity-induced disturbances in ER function stem from chronic lipid overload, the anabolic pressures of hyperinsulinemia, cytokine-induced signaling, mitochondrial dysfunction, and/or other pathophysiological assaults now awaits further investigation. In this regard, it is interesting to note that several of the enzymes responsible for processing excess lipid (e.g., enzymes of lipid esterification) are integral membrane proteins that reside in the ER. METABOLIC ADAPTATIONS LEADING TO INSULIN RESISTANCE IN MUSCLE—A PROBLEM OF IMPAIRED OR INCREASED FATTY ACID OXIDATION? The foregoing sections highlight the important role played by liver and adipose tissue in regulation of muscle insulin sensitivity via two major mechanisms: 1) alteration of fuel delivery to muscle; 2) production of hormones and inflammatory mediators. The remainder of this chapter will focus on key metabolic changes that occur in muscle in response to chronic exposure to elevated concentrations of metabolic fuels, particularly circulating lipids, and how these may contribute to development of muscle insulin resistance. This will include a discussion of the roles of key transcription factors and metabolic regulatory genes in mediating these adaptive changes. We will begin by describing obesity-related changes in intermediary metabolism in skeletal muscle. Fatty acids and glucose constitute the primary oxidative fuels that support skeletal muscle contractile activity, and their relative utilization can be adjusted to match energy supply and demand. Metabolic fuel “switching” is mediated in part by the ability of lipid and carbohydrate catabolic pathways to regulate each other. The idea that elevated fatty acid oxidation inhibits glycolysis and glucose oxidation was first presented in 1963 as the “glucose-fatty acid cycle” (32). Principal elements of this model hold that (a) provision of lipid fuels (fatty acids or ketones) promotes fatty acid oxidation and inhibits glucose metabolism; (b) the inhibitory effects of lipid fuels on glucose oxidation are mediated via inhibition of hexokinase, phosphofructokinase, and pyruvate dehydrogenase. It has further been suggested that these lipid-induced changes in metabolic regulation lead to diminished insulin-stimulated glucose transport (33). Conversely, high glucose concentrations suppress fatty acid oxidation via malonyl-CoA-mediated inhibition of the key enzyme of fatty acid oxidation, CPT1 (34). This pathway represents a near-exact complement to the glucose-fatty acid cycle and is sometimes referred to as the “reverse glucose-fatty acid cycle.” In more recent years the CPT1-malonyl-CoA “partnership” has been featured as a key constituent of the lipotoxicity paradigm (35), in which elevated levels of malonyl-CoA and impaired fatty acid catabolism are thought to encourage cytosolic accumulation of “toxic” lipid species that disrupt insulin signaling and glucose disposal in muscle. Consistent with this notion, muscle malonyl-CoA concentrations are elevated in several (but not all) models of rodent obesity, and this has been linked with intramyocellular accumulation of LC-CoAs (36,37). Furthermore, knockout mice lacking acetyl CoA carboxylase-2 (ACC2) have decreased muscle malonyl-CoA levels, increased -oxidation, and are protected against diet-induced obesity and insulin resistance (38). It is well documented that with ingestion of high-fat diets and onset of obesity, TAG begin to be stored at sites other than adipose tissue, including skeletal muscle, heart, kidney, liver, and pancreatic islets. Because TAG are a relatively inert intracellular metabolite, attention has turned to other lipid-derived species as potential mediators of lipid-induced tissue dysfunction that often accompanies obesity, eventually leading to metabolic syndrome and type 2 diabetes. For example, insulin resistance in human muscle has been reported to be negatively associated with levels of long chain acyl CoAs (39), and infusion of lipids or ingestion of high fat diets in rodents leads to accumulation of these metabolites in various tissues in concert with development of insulin resistance (40).It has further been suggested that increased cellular fatty acyl CoA and diacylglycerol levels activate PKC-theta, leading in turn to phosphorylation of insulin receptor substrate-1 (IRS-1) on Ser 307 (40). Phosphorylation at Chapter 3 / Metabolic Mechanisms of Muscle Insulin Resistance 39 Ser 307 impairs insulin receptor-mediated tyrosine phosphorylation of IRS-1, and as a consequence, interferes with insulin stimulation of IRS-1-associated PI3-kinase, leading to impaired phosphorylation and regulation of distal components of the pathway such as AKT-1 (41–44). Interestingly, dramatic weight loss induced in morbidly obese subjects by bariatric surgery results in a striking improvement in insulin sensitivity, which is correlated with decreases in the levels of some, but not all long-chain acyl CoA species in skeletal muscle (45). Metabolites that decreased included palmitoyl CoA (C16:0), stearoyl CoA (C18:0), and linoleoyl CoA (C18:2), whereas no significant decreases were observed for palmitoleoyl CoA (C16:1) or oleoyl CoA (C18:1). Sphingolipids have also been implicated in a number of disease states and pathologies. Ceramide is viewed as the “hub” of sphingolipid metabolism, as it serves as the precursor for all complex sphingolipids, and as a product of their degradation (46). Ingestion of high fat diets has been shown to result in accumulation of ceramides in various mammalian tissues, and these metabolites have been implicated in insulin resistance (47,48). Thus, ceramide has been shown to accumulate in insulin-resistant muscles in both rodents and humans, and lipid infusion results in elevated ceramide levels in concert with decreasing insulin sensitivity. Moreover, exercise training, which increases insulin sensitivity, causes clear decreases in muscle ceramide levels (49). When added to cultured adipocytes or myocytes, ceramide causes acute impairment of insulin-stimulated glucose uptake and GLUT4 translocation (50,51). These effects appear to be mediated by effects of ceramide to inhibit tyrosine phosphorylation of IRS-1 and/or activation of Akt/protein kinase B (47,48). All of the foregoing observations would be consistent with a model in which glucose-induced increases in malonyl CoA levels in muscle would lead to reduced rates of fatty acid oxidation, and consequent accumulation of TAG, LC-CoA, diacylglycerol, and ceramides in muscle, possibly contributing to development of insulin resistance. However, in humans, the relationship between malonyl-CoA and insulin resistance is less clear. Although several laboratories have shown that muscle malonyl-CoA content increases in association with decreased fat oxidation during a hyperinsulinemic-euglycemic clamp (52,53), basal levels of malonyl CoA were found to be similar in lean, obese, and type 2 diabetic subjects (54). Moreover, fat oxidation rates during hyperinsulinemic conditions were actually increased in diabetic subjects compared to controls, despite similarly high levels of malonyl-CoA (40,55). Thus, whereas the malonyl-CoA/CPT1 axis plays a key role in regulating muscle lipid oxidation, it is unclear whether disturbances in this system are an essential component of insulin resistance. The broadly accepted idea that obesity-associated increases in malonyl-CoA antagonize fat oxidation, thereby causing insulin-desensitizing lipids to accumulate, seems at odds with the idea that insulin resistance stems from increased fatty acid oxidation in muscle (the Randle hypothesis) (37,55). Adding further confusion, a survey of the literature reveals reports describing either increased or decreased muscle fat oxidation in association with obesity, thus seeming to support both possibilities. Perhaps neither is entirely correct or incorrect. To reconcile these discrepancies the concept of “metabolic inflexibility” has been proposed, holding that muscles from obese and insulin-resistant mammals lose their capacity to switch between glucose and lipid substrates (56). In support of this idea, skeletal muscle fat oxidation in obese and type 2 diabetic subjects compared with lean subjects is greater in the postprandial state (simulated by hyperinsulinemic, euglycemic clamp) but depressed in the postabsorptive state (57). Thus, whereas control subjects were able to adjust muscle substrate selection in response to a changing nutrient supply, the insulin-resistant subjects were not. In addition, increases in fatty acid oxidation that normally occur in response to fasting, exercise, or -adrenergic stimulation are either absent or less apparent in obese and/or diabetic subjects (58). Many of these metabolic adjustments are mediated at a transcriptional level. Thus, before returning to discuss a unifying theory of muscle insulin resistance that can potentially reconcile the debate about how “toxic” lipid-derived metabolites accumulate in muscle, we will first summarize the role of key transcription factors in metabolic adaptation to overnutrition. TRANSCRIPTION-BASED MECHANISMS OF METABOLIC REPROGRAMMING IN MUSCLE IN RESPONSE TO OVERNUTRITION Understanding of metabolic reprogramming and fuel selection in skeletal muscle under different physiological conditions has deepened as a result of new knowledge about transcription factors that serve as broad metabolic regulators. For example, the family of peroxisome proliferator-activated receptors (PPARs) are powerful global regulators of metabolism according to nutritional status (59–61). The three major PPAR subtypes, PPAR, , 40 Muoio et al. and  have distinct tissue distributions that reflect their discrete but overlapping functions. PPAR is expressed most abundantly in skeletal muscle, the heart, and the liver, where it plays a key role in regulating pathways of -oxidation (61). Although PPAR, the target of the insulin-sensitizing thiazolidinediones, is expressed primarily in adipose tissue (62), recent studies have demonstrated that muscle-specific deletion of PPAR in mice resulted in whole-body insulin resistance, suggesting the low levels of this receptor in muscle are physiologically important (63). PPAR, the most ubiquitous and least characterized of these receptors, has been shown to regulate both fatty acid oxidation and cholesterol efflux, apparently sharing many duties with PPAR (60,64). Recent findings also suggest that PPAR participates in the adaptive metabolic and histologic (fiber-type switching) response of skeletal muscle to endurance exercise (65). Pharmacological activation of either PPAR or PPAR results in the robust induction of genes that influence lipid metabolism, including several associated with lipid trafficking, interorgan lipid transport and cholesterol efflux, fatty acid oxidation, glucose sparing and uncoupling proteins (UCPs) (60,64). Interestingly, a similar set of genes is upregulated by diverse circumstances that raise circulating free fatty acids, including obesity, diabetes, overnight starvation, high-fat feeding, and acute exercise (60,64,66). Studies in PPAR-null mice indicate that this nuclear receptor is essential for regulating both constitutive and inducible expression of genes involved in fatty acid oxidation in the liver and heart (61). However, skeletal muscles from PPAR-null mice are remarkably unperturbed with regard to lipid metabolism, and retain their ability to upregulate several known PPAR-target genes in response to starvation and exercise, perhaps owing to functional redundancy between PPAR and PPAR (60,64). The nutritionally responsive PPAR receptors are themselves regulated by interactions with a variety of co- activators and corepressors. Promiment among these in terms of regulation of skeletal muscle physiology are the PPAR Coactivator-1 (PGC-1) proteins, PGC-1 and PGC-1. PGC1 was originally identified as a PPAR inter- acting protein responsible for regulating mitochondrial replication in brown fat (67). Subsequent studies identified a second isoform (PGC1) and determined that both proteins are widely expressed and function as promiscuous coactivators of a number of nuclear hormone receptors, as well as other kinds of transcription factors (68).In addition to its interactions with PPARs to regulate lipid metabolism, PGC1 stimulates mitochondrial biogenesis via coactivation of the nuclear respiratory factor (69) and regulates genes involved in oxidative phosphorylation through interactions with estrogen-related receptor  (70) in muscle. PGC1 also coactivates myocyte enhancer factor-2 (69), a muscle-specific transcription factor involved in fiber-type programming. PGC1 is more abundant in red/oxidative muscle and is induced by exercise, whereas its expression is decreased both by inactivity and chronic high-fat feeding (71,72). In contrast, PGC1 mRNA levels are unaltered by these manipulations. UPREGULATION OF FATTY ACID OXIDATION AS A MECHANISM FOR GENERATING LIPID SPECIES THAT IMPAIR INSULIN ACTION—A UNIFYING HYPOTHESIS? We now return to the issue of how the seemingly discrepant hypotheses of obesity-related muscle insulin resistance (a condition of up-regulated or down-regulated fatty acid oxidation?) can be reconciled. One emergent idea is that lipid-induced upregulation of the enzymatic machinery for -oxidation of fatty acids is not coordi- nated with downstream metabolic pathways such as the tricarboxylic acid (TCA) cycle and electron transport chain (71,73). This idea came to light via the observation that isolated mitochondria from rats fed on a high-fat diet had the same rate of [ 14 C] palmitate oxidation to CO 2 as mitochondria isolated from muscles of standard chow-fed control rats, but with a larger accumulation of radiolabeled intermediates in an acid-soluble pool (71) (Fig. 1A, B). This suggests that insulin resistant muscles from fat-fed rats have a higher rate of “incomplete” fatty acid oxidation. Consistent with this idea is the previously discussed study in which hepatic expression of malonyl-CoA decarboxylase (MCD) caused near-complete reversal of severe muscle insulin resistance in rats fed a high-fat diet (22). In this study, metabolic profiling of 36 acyl-carnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, -OH-butyrylcarnitine (C4-OH), in muscle of MCD-overexpressing animals (22) (Fig. 2A). Moreover, muscle concentrations of this metabolite correlated positively with serum levels of nonesterified fatty acids (Fig. 2B) but not circulating ketones, suggesting that its production occurs locally within the muscle as a consequence of increased lipid delivery. Further studies revealed that exposure of L6 myotubes to elevated concentrations of fatty acids not only induces enzymes of Chapter 3 / Metabolic Mechanisms of Muscle Insulin Resistance 41 Fig. 1. Fatty acid oxidation in rat muscle mitochondria. Mitochondria were isolated from whole gastrocnemius muscles harvested in the ad lib fed or 24 h starved state from rats fed on a either a standard chow (SC) or high fat (HF) diet for 12 wk. Mitochondria were incubated in the presence of 150 M [1- 14 C]palmitate and radiolabel incorporation in CO 2 (A) was determined as a measure of complete oxidation, whereas label incorporation into acid soluble metabolites (ASM) (B) was measured to assess incomplete fatty acid oxidation. Complete and incomplete oxidation rates were normalized to total mitochondrial protein. Data are from Koves et al. (71). fatty acid oxidation, such as CPT-1, but also increases the expression of the ketogenic enzyme, mitochondrial HMG CoA synthase (Fig. 2C), while having no effect on expression of key enzymes of the TCA cycle or the electron transport chain (22). Thus, this work suggests that de novo ketogenesis (typically thought of as a hepatic program) is induced in skeletal muscle to provide an outlet for accumulating acetyl CoA, made necessary by increased -oxidative flux occurring without a coordinated adjustment in TCA cycle activity. The profile of other acylcarnitine species obtained by tandem MS also support the notion of incomplete -oxidation in animal models of insulin resistance. Such profiles demonstrate that multiple fatty acylcarnitine metabolites, including long-chain acylcarnitines such as palmityl- and oleyl-carnitine, were abnormally high in obese compared to lean rats (22,71). Moreover, rats fed a standard chow diet exhibited decreased levels of acylcarnitines in muscle during the transition from the fasted to the fed states, whereas in comparison, rats on the high-fat diet exhibited little or no change (Fig. 3A). Finally, a 3-wk exercise intervention in mice fed on a chronic high-fat diet lowered muscle acylcarnitine levels (Fig. 3B), in association with increased TCA cycle activity and restoration of glucose tolerance (71). These studies also highlighted important roles for PGC1 and PPAR transcription factors in mediating lipid- induced metabolic adaptations (71). Similar to muscle mitochondria from high-fat fed rats, L6 myocytes exposed to increasing fatty acid concentrations exhibited disproportionate increases in the rates of incomplete (assessed by measuring incorporation of the label from [ 14 C] oleate into acid-soluble -oxidative intermediates) relative to complete (label incorporation into CO 2 ) -oxidation of fatty acids. Overexpression of PGC1 in lipid-cultured L6 cells caused production of 14 CO 2 to increase and maintain pace with production of [ 14 C]-labeled acid-soluble - oxidative intermediates (Fig. 4A). In other words, the ratio of complete to incomplete -oxidation was dramatically increased by PCG1 expression (Fig. 4B). Consistent with these functional assessments, cDNA microarray analyses showed that fatty acid exposure in the context of low PGC1 activity resulted in the induction of classic PPAR-targeted genes involved in lipid trafficking, glucose sparing and -oxidation, but with little or no change in other downstream pathways that regulate respiratory capacity. In contrast, high PGC1 expression enabled the coordinated induction of -oxidative enzymes with equally important downstream targets (e.g., TCA cycle, ETC, and NADH shuttle systems). These findings imply that PGC1 enables tighter coupling between -oxidation and the TCA cycle. Taken together, these metabolic studies underscore several important points. First, the accumulation of fatty acylcarnitines in muscle of obese/insulin resistant rats implies increased rather than decreased rates of 42 Muoio et al. Fig. 2. Reversal of insulin resistance corresponds with reduced -OH-butyryl-carnitine levels in muscle. A) Tandem mass spectrometry-based analysis of short (SC), medium (MC) and long (LC) chain acyl carnitine species in gastrocnemius muscles. Wistar rats were fed on a high-fat diet for 11 wk before virus treatment and muscles were harvested 5 d after injections of adenoviruses encoding active malonyl-CoA decarboxylase (AdCMV-MCD 5) or an inactive mutated form of the enzyme (AdCMV-MCD mut ). B) Linear regression analysis of -OH-butyrate (C4-OH) levels in muscle versus serum free fatty acids (FFA). C) Semiquantitative RT-PCR analysis of HMG-CoA synthase 2 (HS2) mRNA, normalized to glucose-6-phosphate dehydrogenase, G6PDH mRNA, in fully differentiated rat L6 myotubes incubated without (L6-control) or with 500 μM oleate (L6-FA) for 24 h. RNA from liver of fasted rats was analyzed as a positive control. Data are from An et al. (22).  Fig. 4. PGC1 enhances complete oxidation of fatty acids. Fatty acid oxidation was evaluated in rat L6 myocytes treated with recombinant adenoviruses encoding -galactosidase (-gal) or PGC1, compared against a no virus control (NVC) group. Forty eight h after addition of virus, cells were incubated 3 h with 100-500 μM [ 14 C]oleate. A) Complete fatty acid oxidation was determined by measuring 14 C-label incorporation into CO 2 . B) The relationship between incomplete and complete fatty acid oxidation was expressed as a ratio of label incorporated into acid soluble metabolites (ASM) divided by labeling of CO 2 . Differences among groups were analyzed by ANOVA and Student’s t-test, * indicates P < 005 comparing PGC1 to NVC and -gal treatments, ‡ indicates P < 005 comparing low and high FA conditions. Data are from Koves et al. (71). Chapter 3 / Metabolic Mechanisms of Muscle Insulin Resistance 43 Fig. 3. Muscle acylcarnitine profiling in diet-induced insulin resistance and exercise training. A) Gastrocnemius muscles were harvested from rats fed ad libitum (fed) or starved 24 h after 12 wk on either a standard chow (SC) or high fat (HF) diet. B) Gastrocnemius muscles were harvested from mice fed on standard chow (SC) or high fat (HF) diets for 14 wk. During the final 2 wk of the diet half of the mice in each group were kept sedentary (Sed) or exercise trained (Ex) by running wheel. Muscle acylcarnitine profiles were evaluated by tandem mass spectometry and are expressed as a percent of SC-fed controls. Data are from Koves et al. (71). 44 Muoio et al. mitochondrial fatty acid uptake and -oxidation. Second, experiments in isolated mitochondria from high-fat rats suggest that PPAR-mediated increases in -oxidative activity exceeded the capacity of the TCA cycle to fully oxidize the incoming acetyl-CoA. This supports the idea that assessment of complete fat oxidation via measurement of CO 2 production provides only a partial view of lipid catabolism. Lastly, the acylcarnitine profiles from fed and fasted rats suggested that mitochondria from obese animals were unable to appropriately adjust mitochondrial fatty acid influx in response to nutritional status, thus supporting the observation of metabolic inflexibility in humans (57). The foregoing findings now provide a potential reconciliation of current prominent hypotheses of metabolic perturbations leading to muscle insulin resistance (summarized schematically in Fig. 5). The new model holds that fuel oversupply to muscle results in enhanced fatty acid -oxidation due both to transcriptional regulation and increased substrate supply. However, in the absence of work (i.e., exercise), the TCA cycle not only remains Fig. 5. Proposed model of lipid-induced insulin resistance in skeletal muscle. During conditions of overnutrition, starvation and/or inactivity, fatty acid influx and peroxisome proliferator-activated receptor (PPAR)-mediated activation of target genes ( in yellow) promotes -oxidation without an accompanying increase in tricarboxylic acid (TCA) cycle enzymes. TCA cycle flux and complete fat oxidation is further hampered by a high energy redox state (rising NADH/NAD and acetyl-CoA/free CoA ratios). As a result, metabolic by-products of incomplete fatty acid oxidation (acylcarnitines, ketones and reactive oxygen species (ROS)) accumulate, which in turn gives rise to the accumulation of LC-CoA species and subsequent production of other lipid-derived metabolites, such DAG, ceramide and IMTAG. Together, these mitochondrial and lipid-derived stresses impinge upon insulin signal transduction, thus inhibiting glucose uptake and metabolism (in blue). Exercise combats lipid stress by activating PPAR  coactivator 1  (PGC1), which coordinates increased -oxidation with the activation of downstream metabolic pathways (in orange), thereby promoting enhanced mitochondrial function and complete fuel oxidation. Tighter coupling of -oxidation and TCA cycle activity alleviates mitochondrial stress, lowers intramuscular lipids and restores insulin sensitivity. Abbreviations: ACS; acyl-CoA synthase, -Oxd; -oxidative enzymes, CD36/FAT; fatty acid transporter, CPT1; carnitine palmitoyltransferase 1, DAG; diacylglycerol, ETC; electron transport chain; Glut4; glucose transporter 4, HS2; mitochondrial HMG-CoA synthase, IMTG; intramuscular triacylglycerol, IR; insulin receptor, LC-CoAs; long-chain fatty acyl-CoAs; PDH; pyruvate dehydrogenase; PDK; pyruvate dehydrogenase kinase, ROS, reactive oxygen species, TF; transcription factor. Chapter 3 / Metabolic Mechanisms of Muscle Insulin Resistance 45 inactivated at a transcriptional level, but moreover, flux through the pathway is inhibited by the high energy redox state that prevails under circumstances of overnutrition. As a result, acetyl CoA accumulates and forces accumulation of other acyl CoA species (as reflected by acylcarnitine profiling). This leads in turn to increased production of other lipid-derived molecules, including TAG, diacylglycerol, ketones, ceramides and reactive oxygen species, as well as other yet unidentified metabolites that could contribute to or reflect mitochondrial stress. An important question remaining is whether the high rates of fatty acid catabolism in the obese state are insufficient to compensate for increased lipid delivery, thereby allowing excess lipid-derived metabolites to impair insulin signaling, or alternatively, whether persistently high rates of mitochondrial -oxidation directly contribute to the development of insulin resistance. These possibilities are not necessarily mutually exclusive. Assuming that insulin resistance originally evolved as a survival mechanism, it is likely that nature has devised several distinct metabolic and molecular roadways leading to the same (dys)functional endpoint. Future studies are certain to reveal new clues as to how these pathways intersect, and perhaps more importantly, how they can be circumvented by behavioral and/or pharmacological therapies. ACKNOWLEDGEMENTS Studies cited from the authors’ laboratories were supported by NIH grants PO1 DK58398 (to C.B.N.), K01 DK56112 (D.M.M.), and the American Diabetes Association (D.M.M.). REFERENCES 1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432. 2. Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269:540–543. 3. Moller DE. Potential role of TNF-alpha in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol Metab 2000;11(6):212–217. 4. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes. Nature 2001;409:307–312. 5. 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