Insulin Resistance

Ingestion of high fat diets and development of obesity is associated with increased triglyceride storage at sites other than adipose tissue, including skeletal muscle, the heart, kidney, and liver. These changes are often associated with chronic elevations in circulating free fatty acids and TAG. This has led to the widely accepted notion that obesity-associated tissue dysfunction, including insulin resistance and cell death, is a direct consequence of chronic exposure of tissues to elevated lipids and resultant accumulation of toxic by-products of lipid metabolism. Whereas much evidence continues to support this concept, as will be highlighted in later sections of this chapter, recent work suggests the presence of other, more indirect pathways for development of muscle insulin resistance driven by events in distant tissues such as liver and adipose.

Once considered a passive energy reservoir, adipose tissue is now recognized as an important endocrine organ that informs the brain and peripheral tissues of changes in whole-body energy status. The endocrine function of the adipocyte came to light with the hallmark discovery of leptin as the mutated gene in homozygous ob/ob mice, which exhibit an obesity syndrome characterized by severe adiposity, hyperphagia, hyperlipidemia, hyperinsulinemia, and insulin resistance in multiple tissues, including skeletal muscle (1; 2). Leptin replacement in ob/ob mice restores energy balance by acting on central and peripheral receptors that mediate changes in feeding behavior and systemic fuel metabolism (1; 2). The ensuing decade of research has revealed that adipose cells also produce a variety of other hormones and cytokines (referred to collectively as "adipokines"). These include peptide hormones such as adiponectin (also Acrp30) and resistin, and proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor a (TNFa). Many of these adipocyte-secreted peptides regulate both lipid and glucose metabolism (3), and as result, may play a direct role in development of insulin resistance and diabetes (4-6). For example, the two best-characterized antidiabetic adipokines, leptin and adiponectin, have been shown to decrease triglyceride synthesis, promote fatty acid catabolism, and enhance insulin action in both skeletal muscle and the liver. Although information on the signaling mechanisms that mediate these actions is still unfolding, growing evidence indicates that both leptin and adiponectin cause activation of 5' AMP kinase, an enzyme that normally mediates responses to conditions of energy deficit (sensed as a fall in ATP and a rise in AMP levels) by activating fatty acid oxidation. Interestingly, leptin levels are increased, and adiponectin levels are decreased in insulin resistant, obese human subjects and animal models, suggesting that obesity leads to a state of leptin resistance, and one of adiponectin deficiency. In addition to perturbations relating to leptin and adiponectin, insulin resistance is associated with increased production of resistin, IL-6, TNFa, and retinol-binding protein 4 (RBP-4) by adipose tissue, all of which have been shown to induce insulin resistance in both muscle and the liver (4,7-9).

Also consistent with a critical role of adipose tissue in control of metabolic function in muscle and other peripheral tissues are the effects of lipoatrophy. For example, several strains of transgenic mice have been created with ablation or loss of function of white adipose tissue (10-12). Animals lacking white adipose tissue have severe hepatic and muscle insulin resistance, occurring in concert with large increases in triglyceride stores in both tissues (13). Moreover, transplantation of normal fat tissue into such mice restores insulin sensitivity (14), which appears to be mediated by redistribution of fat from the liver and muscle to the adipose depot and via endocrine factors produced by the transplanted fat tissue. Most evidence points to leptin as the key endocrine mediator in these studies. Thus, leptin infusion ameliorates insulin resistance in lipoatrophic mice (15,16), whereas transplantation of fat from leptin-deficient mice into such animals fails to improve insulin sensitivity (17).

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, TNFa 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, constituting 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, P-OH-butyrylcarnitine, in muscle of MCD-overexpressing animals that likely resulted from a change in intramuscular P-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-kB transcription factor (28,29). Subsequently, it has been demonstrated that high-fat diets or obesity result in activation of NF-kB and its transcriptional targets in the liver. Overexpression of a constitutively active version of the NF-kB activating kinase, IkB kinase catalytic subunit P (IKK-P) 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-P overexpression increase expression of proinflammatory cytokines such as IL-6, IL-1P, and TNFa 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-P 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-P 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.

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 CPTl-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

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.

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