Malonyl CoA: Altered fatty acid partitioning between the mitochondria and cytoplasm as a cause of insulin resistance was first suggested by studies in denervated rat muscle, in which enhanced diacylglycerol (DAG) synthesis and PKC activation were observed,¹⁰⁰ and later by similar findings in muscle of obese, insulin-resistant KKA^y mice.¹⁰¹ In these and other instances, insulin resistance in rodent muscle correlated with an increase in the concentration of malonyl CoA,⁷⁶ an allosteric inhibitor of carnitine palmitoyltransferase. As shown in Figure 18-4, an increase in malonyl CoA by decreasing the oxidation of cytosolic FACoA would increase its availability for the formation of DAG, triglycerides, ceramide, and possibly other factors linked to insulin resistance. In keeping with such a mechanism, it has been demonstrated by McGarry^99,102 that treatment with etomoxir, a pharmacologic CPT1 inhibitor, concurrently increases triglyceride accumulation and causes insulin resistance in rat skeletal muscle. Also supporting this notion are a number of other findings, including the following: mice lacking functional acetyl CoA carboxylase 2 (ACC₂, the isoform that generates the malonyl CoA that regulates CPT₁) are more insulin sensitive than control rats^103,104; the administration of an ACC inhibitor diminishes obesity and insulin resistance in fat-fed rats¹⁰⁵; and antisense oligonucleotides directed at hepatic ACC1 and ACC2 diminish both hepatic steatosis and insulin resistance in mice fed a high-fat diet.¹⁰⁶ Finally, low rates of fatty acid oxidation have been reported in pre-obese humans,^107,108 Zucker diabetic rats,¹⁰⁹ and interleukin-6 (IL-6) knockout mice¹¹⁰ prior to the onset of diabetes and obesity. Malonyl CoA was not assayed in any of these studies; however, in the two rodent models, a decrease in the activity of AMP-activated protein kinase (AMPK) was found, suggesting that its concentration was elevated^111,112 (see section on AMPK).

Mitochondrial Dysfunction: Altered fatty acid partitioning in muscle and other tissues could also occur if fatty acid oxidation is depressed as a consequence of mitochondrial dysfunction. Decreases in mitochondrial function, and in some instances number and size, have been found in muscle of individuals with type 2 diabetes associated with obesity,¹¹³ in lean insulin-resistant elderly individuals,¹¹⁴ and in lean insulin-resistant offspring of diabetic parents.⁷ In the latter case, the reduction in mitochondrial function could be attributed to a similar reduction in mitochondrial content.¹¹⁵ Likewise, decreases in the mRNA for PGC1α, a transcriptional coactivator that enhances genes for mitochondrial biogenesis and function, have been observed in some¹¹⁶ but not all¹¹⁵ studies of nondiabetic first-degree relatives of patients with type 2 diabetes, as well as in individuals with type 2 diabetes and impaired glucose tolerance.¹¹⁷ Whether these mitochondrial changes are hereditary or secondary to metabolic events (e.g., lipotoxic changes due to abnormalities in intracellular lipid metabolism) or abnormalities in AMPK regulation (see next section) remains to be determined. However, given the key role for increased intracellular fatty acid metabolites in mediating insulin resistance, decreased mitochondrial function leading to decreased fatty acid oxidation will certainly exacerbate the problem. Also to be determined is whether the changes observed in the offspring of diabetic parents reflect a difference in muscle fiber type, since the ratio of mitochondrial-rich type 1 fibers to glycolytic type 2 fibers may be decreased in these individuals.^7,118

AMP-Activated Protein Kinase: AMPK (Box 18-1) is a fuel-sensing enzyme that appears to play a key role in regulating both cellular metabolism and mitochondrial function. In addition, an increasing body of evidence has suggested that its dysregulation could be a cause of the metabolic syndrome (animal studies) as well as a target for its prevention and therapy (human and animals studies).^3,24,75

As shown in Fig. 18-4, when AMPK is activated (e.g., during exercise or in some tissues by caloric deprivation), it phosphorylates and inhibits acetyl CoA carboxylase (ACC), the enzyme that catalyzes the synthesis of malonyl CoA. By a still undetermined mechanism, it activates malonyl CoA decarboxylase, an enzyme that catalyzes malonyl CoA degradation.¹¹⁹ In addition, AMPK concurrently inhibits the use of cytosolic FACoA for diacylglycerol, triglyceride, and ceramide synthesis, and at least in endothelial cells¹²⁰ and adipocytes (see Adipose Tissue), it diminishes the generation of lipid peroxides and the activation of NF-κB caused by elevated concentrations of fatty acids.^{75,120,121(and unpublished data)} Thus AMPK appears to protect cells against lipotoxicity and to do so by multiple actions. In addition to modulating these events acutely, AMPK chronically regulates the effects of transcriptional regulators (e.g., SREBP1C) that govern the synthesis of acetyl CoA carboxylase and other key enzymes. Of specific relevance to this chapter, decreases in AMPK activity and increases in malonyl CoA are both associated with insulin resistance in muscle (Table 18-1) and liver in many situations, and failure to activate AMPK in the fat cell during lipolysis is associated with increases in oxidative stress and inflammation.^{121(and unpublished observations)} In addition, hormones and other factors that decrease AMPK in peripheral tissues, such as glucocorticoids,¹²² TNF-α,¹²³ and resistin,¹²⁴ cause insulin resistance. Conversely, numerous endogenous hormones (e.g., adiponectin and leptin) and pharmacologic agents (e.g., metformin, thiazolidinediones, α-lipoic acid) that diminish insulin resistance have been demonstrated to activate AMPK and diminish the concentration of malonyl CoA in experimental animals, as do exercise and treatment with the AMPK activator AICAR (5-aminoimidazole-4-carboxamide riboside)⁷⁵ (Table 18-2).

Closely linked to mitochondrial theories of insulin resistance is PGC1α (PPARγ-coactivator 1α), a transcriptional coactivator whose expression is increased when AMPK is activated (e.g., by exercise or AICAR).125–128 In skeletal muscle, and likely in other tissues, PGC1α regulates mitochondrial biogenesis and the expression of multiple genes governing oxidative phosphorylation, including those for citric acid cycle enzymes and electron transport proteins.^116,117,129 PGC1α polymorphisms have been reported in populations with type 2 diabetes in Denmark¹³⁰ and Japan.¹³¹ Also, modest but coordinated decreases in the expression of many PGC1α-mediated genes have been reported in muscle of overweight/obese middle-aged individuals with type 2 diabetes or impaired glucose tolerance.^116,117 A decreased capacity for exercise (VO_{2 max}) was also observed in these subjects, in keeping with the findings of earlier studies in similar patient groups.^{118,132–134}

In keeping with its effect on PGC1α, it has been shown that AMPK is necessary for mitochondrial biogenesis.¹²⁸ Whether the various hormones (e.g., adiponectin, leptin, catecholamines) and pharmacologic agents (thiazolidinediones, metformin) reported to activate AMPK in peripheral tissues also increase PGC1α expression and stimulate mitochondrial biogenesis and enzyme activity in skeletal muscle and other tissues, or whether agents that decrease AMPK (e.g., endocannabinoids and glucocorticoids) have the opposite effect, are active areas for study. Also requiring study is the relative role of Ca²⁺ calmodulin–dependent protein kinase, which like AMPK is activated during exercise and increases PGC1α expression.¹³⁵

Overview: A number of lines of investigation have linked abnormalities in adipose tissue to the pathogenesis of the metabolic syndrome. First, as already noted, elevated plasma FFA levels, attributable to an increase in their release from adipocytes in obese or centrally obese individuals, correlate with the presence of insulin resistance in most patients.85,136 Second, when the function of the adipocyte as a store for lipid is impaired,^137,138 as it is in many insulin-resistant obese individuals and people with various lipodystrophies (see later discussion), fatty acids are deposited as triglyceride in ectopic sites such as muscle, liver, and visceral fat, and this is associated with insulin resistance, inflammation, and other manifestations of cellular dysfunction (lipotoxicity).¹³⁹ Recent studies suggest that markedly obese individuals with diminished levels of the lipid droplet proteins CIDE A and perilipin in adipose tissue have an impaired ability to deposit lipid in their adipocytes, a higher rate of lipolysis, and they are insulin resistant.⁹⁷ In contrast, in comparably obese individuals with normal levels of these lipid droplet proteins, increased lipolysis and insulin resistance were not observed. Thus differences in the capacity of the adipocyte to store lipid may be a key determinant of whether obesity leads to the metabolic syndrome. Finally, the adipocyte can release hormones such as leptin and adiponectin that in multiple tissues activate AMPK, diminish ectopic lipid, and enhance insulin action. In addition, it can release proinflammatory cytokines such as TNF-α,¹⁴⁰ resistin,¹²⁴ and plasminogen activator inhibitor (PAI)¹² that could cause insulin resistance.¹⁴¹ As already noted, diminished AMPK activity, which results in large increases in oxidative stress and possibly inflammatory changes in the adipocyte when lipolysis is stimulated, might contribute to the latter.^{121(and unpublished data)}

Adiponectin: Of the various adipokines, adiponectin, also referred to as ACRP30, has been most closely linked to insulin resistance in humans. It is produced exclusively or at least predominantly in adipose tissue and is present in the circulation in trimeric, hexameric, and high-molecular-weight (HMW) forms. The biological relevance of the three oligomers142,143 and still other forms of adiponectin is incompletely understood. These considerations aside, there is abundant evidence that low immunoassayable adiponectin levels in plasma (accounted for mainly by the HMW form) are present in obese individuals and in individuals at risk for type 2 diabetes^144,145 and coronary heart disease,¹⁴⁶ even in the absence of overt obesity. In addition, polymorphisms of the adiponectin gene have been associated with the metabolic syndrome in some populations and a predisposition to type 2 diabetes in others.¹⁴⁴

In keeping with these findings in humans, in mice fed a high-fat diet, genetically knocking out adiponectin causes glucose intolerance and insulin resistance¹⁴⁷; and conversely, overexpression of full-length¹⁴⁸ or globular¹⁴⁹ adiponectin attenuates the severity of atherosclerosis in apoE knockout mice. In addition, the administration of full-length (HMW) adiponectin and the globular subunit have been shown respectively to diminish hepatic lipid accumulation in ob/ob mice with non-alcoholic fatty liver disease (NAFLD)¹⁵⁰ and insulin resistance in fat-fed rats.¹⁵¹

Adiponectin, like exercise, activates AMPK and stimulates AMPK-mediated events such as glucose transport and fatty acid oxidation in muscle152,153 and inhibition of glucose production by liver.¹⁵⁴ In addition, adiponectin has antiinflammatory actions.¹⁵⁵ Whether the insulin-sensitizing effect of adiponectin is AMPK-mediated has not been proven definitively; however, treatment with thiazolidinediones increases plasma adiponectin, and the insulin-sensitizing effect of these agents and their ability to activate AMPK are both markedly attenuated in adiponectin knockout mice.^156,157

Leptin: Since its discovery by Friedman and his co-workers,¹⁵⁸ interest in leptin has for the most part focused on its role as an appetite suppressant. However, it has also been recognized for some time that leptin increases oxidative metabolism and fatty acid oxidation in peripheral tissues, owing both to a direct action¹⁵⁹ and to an effect on the hypothalamus that appears to be mediated by the sympathetic nervous system.¹⁶⁰ As first reported by Minokoshi and Kahn and their co-workers, both the direct and centrally-mediated effects of leptin on peripheral tissues are associated with AMPK activation,¹⁶¹ whereas its action on hypothalamic nuclei is associated with a decrease in AMPK activity.¹⁶²

When leptin is lacking or its receptor is not functioning, lipid accumulates in many tissues, and cellular damage may result. Unger et al.163,164 have reported that in the Zucker diabetic fatty rat (ZDF), such ectopic lipid accumulation occurs in liver, muscle, and the pancreatic β cell, and that it antedates the presence of diabetes and pancreatic β-cell apoptosis. They coined the term lipotoxicity to describe this phenomenon and have proposed that a major action of leptin is to prevent the accumulation of ectopic lipid and related events (e.g., increased ceramide and oxidant stress) that cause lipotoxicity (apoptosis, mitochondrial dysfunction, inflammation, insulin resistance). Of particular note for this review, decreases in AMPK activity and an increased concentration of malonyl CoA have been found in tissues of the fa/fa and the ZDF rat, rodents with a functionally deficient leptin receptor, and the ob/ob mouse, which does not synthesize leptin.¹¹² Furthermore, treatment with the AMPK activator, AICAR,¹¹² as well as the TZD troglitazone¹⁶⁵ and other means of AMPK activation including exercise,¹⁶⁶ prevent the ectopic lipid accumulation, pancreatic β-cell damage, and the development of diabetes in the ZDF rat.