Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing links. The Claude Bernard Lecture 2009

Given the above observations and the negative results of the ACCORD [7], ADVANCE [8] and VADT [9] studies, in which intensified glycaemic control failed to reduce macrovascular complications, it is reasonable to ask what role hyperglycaemia plays in the development of cardiovascular disease (CVD). Epidemiological analysis of the UKPDS [3] demonstrated that rising HbA_1c was associated with increased risk of myocardial infarction and stroke. However, the increased hazard ratio was modest and reduction in HbA_1c following insulin or sulfonylurea therapy did not significantly decrease myocardial infarctions or strokes [10], although long-term follow up did demonstrate a significant reduction in atherosclerotic cardiovascular events [11]. These results suggest a ‘memory’ effect of improved glycaemic control, reminiscent of the Diabetes Control and Complications Trial in type 1 diabetic patients [12].

The negative results of the ACCORD, ADVANCE and VADT [7‐9] studies have also led to questioning of the notion that improved glycaemic control prevents macrovascular complications in type 2 diabetic patients, especially in those with long-standing diabetes. What, for example, could explain the failure to observe a reduction in macrovascular events in these studies (see textbox: Insulin and atherosclerosis)?

First, the underlying hypothesis is not based on sound pathophysiological evidence. Hyperglycaemia is a weak risk factor for CVD compared with other well established risk factors, e.g. dyslipidaemia and hypertension; moreover, most patients in the above studies were being treated with lipid-lowering medications, antihypertensive drugs and anti-inflammatory agents [7‐9]. Second, participants had long-standing diabetes with a prior cardiovascular event or multiple risk factors for CVD. Third, insulin may be the wrong drug for prevention of CVD. Insulin is associated with weight gain [13, 14] and weight gain (obesity) is a major risk factor for CVD [15, 16]. Moreover, even small increments in plasma insulin cause severe insulin resistance [17, 18], which is also a risk factor for CVD. Fourth, the study population, although large in the ACCORD and ADVANCE studies, was inadequate, given the low annual incidence of cardiovascular complications (∼2% per year), to demonstrate a beneficial effect of improved glycaemic control.

Many in vivo and in vitro studies have demonstrated that insulin, especially at high doses, can promote atherogenesis [19‐21] (see textbox: Insulin and atherosclerosis). Insulin promotes de novo lipogenesis and augments hepatic VLDL synthesis [22, 23] via its stimulation of sterol regulatory-element-binding protein-1c and its inhibition of acetyl-CoA carboxylase [24]. In cultured arterial smooth muscle cells, insulin augmented LDL-cholesterol transport [25]. Insulin is a potent growth factor, augments collagen synthesis [26, 27], stimulates arterial smooth muscle cell proliferation [28, 29] and turns on multiple genes involved in inflammation [27].

In vivo studies in dogs [30], rabbits [31] and chickens [32] provide further evidence that insulin promotes atherogenesis. Non-diabetic chickens fed a high-cholesterol diet develop severe atherosclerosis, which regresses when switched to low-cholesterol diet [32]. Insulin administration when the low-cholesterol diet was instituted prevented regression of coronary atherosclerosis. Alloxan-induced diabetic rabbits fed a high-cholesterol diet developed marked hypercholesterolaemia, but their aorta remained free of atherosclerotic plaque [31]. If rabbits are treated with insulin, severe atherosclerosis develops. Dogs receiving a low-dose insulin infusion into a hindlimb artery (to cause local, physiological hyperinsulinaemia) developed severe femoral atherosclerosis, whereas all other arteries remained free of atherosclerotic plaque [30]. Rats chronically (7–10 days) infused with insulin while maintaining euglycaemia became markedly resistant to the stimulation of glucose uptake and suppression of plasma NEFA by insulin [23], and became hypertensive [33].

Two other points about hyperinsulinaemia are noteworthy. In humans with normal glucose tolerance, insulin infusion to raise fasting plasma insulin from 57 to 104 pmol/l for 3 days produced severe insulin resistance [17, 18], a risk factor for CVD (see subsequent discussion). Finally, insulin therapy is invariably associated with weight gain [13, 14]. Most studies designed to reduce HbA_1c <7.0% have required large insulin doses, failed to achieve the targeted HbA_1c goal and resulted in weight gain [13, 14]. The study of Henry et al. [13] is especially noteworthy. HbA_1c was reduced from 7.7% to 5.1%, but this was associated with weight gain of 8.6 kg. This is of major concern since the current diabetes epidemic is being driven by obesity, a major risk factor for CVD [15, 16]. Weight gain promotes atherogenesis via multiple mechanisms including dyslipidaemia and hypertension. Fat deposition in the arterial wall promotes inflammation, which directly accelerates atherogenesis [34‐37]. Interestingly, 27.8% of individuals in the intensive treatment arm of the ACCORD study gained >10 kg [7].

Much evidence indicates that insulin resistance per se and associated components of the insulin resistance (metabolic) syndrome (see textbox: Syndrome of insulin resistance) [38‐40] contribute to development of CVD. Studies from our laboratory (Fig. 1) [1, 9, 41‐44] and others [45, 46] have demonstrated that lean type 2 diabetic and obese normal glucose tolerant participants are resistant to insulin and that their insulin resistance primarily affects the glycogen synthetic pathway. Type 2 diabetes [4, 5] and obesity [15, 16] are major cardiovascular risk factors. A common thread linking all components of the insulin resistance (metabolic) syndrome is the basic cellular/molecular cause of insulin resistance [44], which not only promotes inflammation and atherogenesis, but leads to and/or aggravates other components of the syndrome, which themselves are major CVD risk factors.

Hypertension is a central feature of the insulin resistance syndrome. Many studies have demonstrated that hypertensive individuals are resistant to insulin, primarily involving the glycogen synthetic pathway (Fig. 1) [47‐51], and hypertension is a well established risk factor for CVD.

Type 2 diabetic and obese individuals develop dyslipidaemia characterised by hypertriacylglycerolaemia, reduced HDL-cholesterol, and small dense atherogenic LDL particles [39, 40, 52, 53], each of which is a major CVD risk factor. We and others [51, 54‐57] have shown that hypertriacylglycerolaemia, but not hypercholesterolaemia is associated with insulin resistance primarily involving the glycogen synthetic pathway (Fig. 1). The frequency of hypercholesterolaemia is not increased in patients with type 2 diabetes mellitus [53]. However, if elevated, LDL-cholesterol acts synergistically with other risk factors to accelerate atherogenesis [58].

To examine the relationship between insulin resistance and CVD, we compared normal glucose-tolerant individuals with diffuse coronary artery disease with normal glucose-tolerant participants with clean coronary arteries [59]. Individuals with coronary artery disease manifested marked insulin resistance, primarily affecting the glycogen synthetic pathway in skeletal muscle [59] (Fig. 1). Studies by Reaven [40] and Paternostro et al. [60] have demonstrated that non-diabetic individuals with established coronary artery disease are resistant to insulin. Using positron emission tomography, the myocardium of non-diabetic individuals with coronary artery disease [61] and type 2 diabetic patients without coronary artery disease [62] has been shown to be resistant to insulin.

In summary, each component of the ‘metabolic’ syndrome is characterised by insulin resistance involving the glycogen synthetic pathway (Fig. 1). Insulin resistance is present at the stage of impaired glucose tolerance [63, 64] and even before any abnormality in glucose tolerance is observed [65]. Thus, we prefer to refer to the metabolic and cardiovascular cluster of factors that comprise the ‘metabolic’ syndrome as the ‘insulin resistance syndrome’ [20, 21, 66]. This focuses clearly on a specific pathogenic aetiology, rather than on a diffuse cluster of factors that may or may not be related to the same underlying pathophysiology.

Multiple prospective studies have demonstrated that insulin resistance predicts future CVD. In the San Antonio Heart Study [67], insulin resistance was quantitated by HOMA of insulin resistance (HOMA-IR) in 2,564 non-diabetic participants who were followed for 8 years. Individuals in the highest quintile of insulin resistance had an approximately 2.5-fold increased incidence of CVD (Fig. 2a). After adjustment for multiple cardiovascular risk factors, individuals in the highest quintile of insulin resistance still had a twofold increased incidence of CVD. In 3,606 non-diabetic participants followed for 6.9 years in the Botnia Study [68] (Fig. 2b), the metabolic syndrome was associated with a threefold increased risk of CVD. Each component of the metabolic syndrome, as well as insulin resistance (HOMA-IR) itself, was associated with a 1.5- to 2-fold increased incidence of CVD (Fig. 2b). Similar observations have been made in the Bruneck [69], Verona Diabetes [70] and Insulin Resistance Atherosclerosis studies [71]. A strong relationship between HOMA-IR and carotid intimal media thickness has also been demonstrated [72], as has an association between insulin resistance and greater cardiovascular risk factor load [73].

The analysis by D’Agostino et al. of six prospective studies further supports an independent role for insulin resistance in CVD [74]. It found that, using the Framingham Cardiovascular Risk Engine [75], only 69% of the observed risk for CVD could be explained, leaving 31% unaccounted for (Fig. 3a). Similarly, in the ARIC study [76] (Fig. 3b), only ∼70% of the increase in carotid intimal media thickness could be accounted for by dyslipidaemia, hypertension, glucose intolerance and obesity. We hypothesise that this unexplained risk can be attributed to insulin resistance.

Our studies in man demonstrated that impaired IRS-1 tyrosine phosphorylation/PI-3 kinase activation in lean type 2 diabetic and obese non-diabetic participants causes a profound defect in glucose transport/phosphorylation and glycogen synthesis (Fig. 4b) [104, 105]. Because nitric oxide synthase is activated by the same PI-3 kinase pathway, nitric oxide production is impaired [106, 107], resulting in endothelial dysfunction [108‐110] and accelerated atherosclerosis [110‐113]. This pathogenic sequence establishes the molecular basis linking insulin resistance, inflammation and accelerated atherosclerosis in people with type 2 diabetes mellitus and may help account for the missing 30% CVD risk that cannot be explained by circulating cardiovascular risk factors [74, 76] (Fig. 4b). This pathogenic sequence also explains why insulin resistance is a strong predictor of CVD [67‐73].

Once the insulin signalling defect becomes established, it initiates a reverberating negative feedback cycle. Impaired glucose utilisation causes hyperglycaemia, which stimulates insulin secretion (Fig. 4b). Because the IRS-1/PI-3 kinase pathway is defective, the MAP kinase pathway is excessively stimulated since it is normally sensitive to insulin. Increased IRS-1 serine phosphorylation, induced by metabolic/inflammatory abnormalities present in the diabetic state (reviewed by Draznin [114]), further impairs insulin signalling through the PI-3 kinase pathway [115, 116]. In diabetic and obese patients, continued MAP kinase pathway stimulation [94] causes vascular smooth muscle proliferation, increased collagen formation and excessive production of growth factors/inflammatory cytokines, contributing to accelerated atherosclerosis. The same insulin signalling defects that we have demonstrated in skeletal muscle of type 2 diabetic patients have been demonstrated in arterial vascular smooth muscle cells (VSMC) in animal models of diabetes and in humans [92, 117, 118].

Draznin and colleagues [93, 114] have provided additional evidence for this ‘dual insulin signalling hypothesis’. In cultured VSMCs and endothelial cells treated with PI-3 kinase inhibitors, insulin cannot activate nitric oxide synthase to generate nitric oxide and these cells are no longer protected from the detrimental effects of vascular endothelial growth factor, platelet-derived growth factor (PDGF) and other inflammatory peptides. Insulin continues to stimulate VSMC proliferation/migration even though the PI-3 kinase pathway has been inhibited and increases prenylated Ras (rat sarcoma) and Rho (Ras related homologue), leading to augmented VSMC response to the growth-promoting effects of IGF-1, epidermal growth factor, PDGF and angiotensin II. These effects are enhanced when PI-3 kinase is inhibited [119, 120]. The sensitising effect of VSMCs to angiotensin II is of particular importance, since hyperinsulinaemia doubles the ability of angiotensin II to transactivate NF-kB [120], a powerful nuclear transcription factor that activates multiple inflammatory pathways involved in atherogenesis [121, 122] and causes IRS-1 serine phosphorylation, which inhibits insulin signalling [123]. Angiotensin II also serine phosphorylates IRS-1 in aortic smooth muscle and skeletal muscle cells [124]. This provides a pathophysiological link between insulin resistance, atherogenesis and essential hypertension.

To examine whether the insulin signalling defect is genetic or acquired, we studied lean normal glucose-tolerant offspring of two diabetic parents [65]. These offspring are severely insulin-resistant [65, 103] and at high risk of developing diabetes. Insulin-stimulated glucose disposal was markedly reduced despite increased insulin receptor tyrosine phosphorylation [103]. Basal and insulin-stimulated IRS-1 tyrosine phosphorylation/PI-3 kinase activity were markedly reduced. From these observations five points ensue: (1) early in the natural history of type 2 diabetes insulin receptor activation is normal (Fig. 4b); (2) the rate-limiting step for insulin signalling resides at IRS-1; (3) molecular abnormalities responsible for insulin resistance are present long before onset of overt diabetes or impaired glucose tolerance; (4) insulin normally augments MAP kinase, but not PI-3 kinase, demonstrating dissociation between regulation of PI-3 kinase and MAP kinase pathways; and (5) tissues of offspring with normal glucose tolerance are being ‘incubated’ in a ‘sea’ of molecular insulin resistance and atherogenicity from an early age, explaining in part why clinically evident CVD is present in 15 to 20% of individuals at initial diagnosis [125] and why insulin resistance and CVD are closely linked [19, 20, 44, 67‐72]. Only thiazolidinediones simultaneously augment the PI-3 kinase (metabolic) pathway while inhibiting the MAP kinase (atherogenic) pathway [126, 127]. Thiazolidinediones also enhance nitric oxide synthase activity, increasing nitric oxide production [128‐130]; they also reduce high-sensitivity C-reactive protein levels and improve multiple cardiovascular risk factors in type 2 diabetic participants [131].

The term ‘lipotoxicity’ was coined by Unger to describe the deleterious effect of tissue fat accumulation on glucose metabolism [132]. However, lipotoxicity has assumed added significance (see textbox: Lipotoxicity). Experimental NEFA elevation to reproduce levels in type 2 diabetes causes severe muscle/liver insulin resistance [133‐135] and inhibits insulin secretion [136], reproducing the three basic core defects of type 2 diabetes. Elevated plasma NEFA impair glucose oxidation/glycogen synthesis [133] and decrease glucose transport/phosphorylation [104, 135]. Most importantly, lipid infusion to increase plasma NEFA levels in participants with normal glucose tolerance caused a dose–response inhibition of insulin receptor/IRS-1 tyrosine phosphorylation and PI-kinase activity, which correlate closely with reduced insulin-stimulated glucose disposal [137].

Magnetic resonance spectroscopic studies have demonstrated that intramyocellular and intrahepatic fat accumulation are closely associated with organ-specific insulin resistance [138‐141]. Intracellular toxic metabolites of triacylglycerol and NEFA metabolism (fatty acyl CoA, diacylglycerol, ceramides) cause severe insulin resistance by impairing insulin signalling and multiple intracellular steps of glucose metabolism [133, 137, 142]. To examine the causality of this association, we treated type 2 diabetic patients with acipimox, an inhibitor of lipolysis. The decline in plasma NEFA (563 to 230 mmol/l) was associated with decreased muscle long-chain fatty acyl CoA [143], which correlated closely with enhanced insulin-stimulated muscle glucose uptake (Fig. 5). The deleterious effect of increased intracellular fat on insulin sensitivity is supported by the work of Kim et al. [144], who overexpressed lipoprotein lipase in muscle or liver in mice. In muscle this caused a marked increase in fat content, severe insulin resistance and impaired PI-3 kinase activity, without affecting hepatic insulin sensitivity. Lipoprotein lipase overexpression in liver increased hepatic fat content, induced severe hepatic insulin resistance and impaired hepatic insulin signalling, but had no effect on muscle [144]. Long-chain fatty acyl CoAs cause IRS-1 serine phosphorylation, thereby inhibiting the PI 3 kinase pathway [145, 146].

In animals [147] and humans [148], plasma NEFA elevation by lipid infusion also increased intramyocellular diacylglycerol, a potent activator of protein kinase C (PKC) isoforms, which inhibit insulin signalling through serine phosphorylation of IRS-1 [149]. Muscle [150] and plasma [151] ceramide levels are also increased in obese and type 2 diabetic individuals, correlating closely with severity of insulin resistance.

In summary, much evidence supports the notion that increased plasma NEFA/intramyocellular levels of toxic lipid metabolites (long-chain fatty acyl CoAs, diacylglycerol, ceramides) play a role in the pathogenesis of muscle/liver insulin resistance.

Type 2 diabetes and obesity are characterised by low-grade, chronic inflammation [152‐154], which could contribute to accelerated atherosclerosis. Increased activity of IkB/NFkB provides a molecular mechanism responsible for inflammation and insulin resistance in type 2 diabetes mellitus [91, 148, 152, 155]. Under basal conditions, NFkB is associated with IkB in the cytoplasm (Fig. 6). Upon activation by inflammatory factors, including fatty acyl CoAs, IkB kinase phosphorylates IkB, causing IkB polyubiquitination and degradation. The free NFkB translocates to the nucleus, where it binds to target genes, stimulating inflammatory mediators (TNFα, IL-1B, IL-6, PKC) involved in atherogenesis [152, 156, 157]. TNFα, IL-6 and PKC, as well as IK kinase, cause serine phosphorylation of IRS-1, inhibiting insulin signalling and causing insulin resistance [123, 158].

Skeletal muscle IkB content is decreased in type 2 diabetic patients [159], indicative of increased NFkB activity. We have confirmed this by demonstrating increased NF-kB p50–p65 DNA binding activity in type 2 diabetic and obese individuals (P Tantiwong, K Shammugasumdaran, A Monroy, S Ghosh, E Cersosimo, A Sriwijitkamol, RA DeFronzo, S Mohan, N Musi, unpublished results). Reduced IkB and increased NFkB p50–p65 binding activity were strongly correlated with reduced insulin sensitivity.

Multiple stimuli [152], including lipids, activate IkB kinase/NFkB, causing insulin resistance in cultured myocytes [160] and muscle from rodents [155, 161] and humans [162]. In human myocytes, palmitate enhances NFkB activity and increases IL6 mRNA, a downstream gene regulated by NFkB. Transfection of myocytes with an adenovirus-IkB super repressor mutant blocked the augmentation of NFkB activity and of IL6 mRNA expression by palmitate [162].

Circulating NEFA also cause inflammation and insulin resistance by directly activating plasma membrane receptors, such as toll-like receptor 4 [162] (Fig. 7). In muscle of obese and type 2 diabetic individuals, toll-like receptor 4 mRNA/protein content is increased and correlates closely with HOMA-IR [162] and IkB/NFkB activation, providing another pathway via which NEFA incite inflammation and promote atherogenesis.

NEFA also stimulate formation of collagen, an integral component of the atherosclerotic plaque [163, 164]. Physiological NEFA elevation in participants with normal glucose tolerance increases mRNA expression of collagen, fibronectin and lumican, as well as connective tissue growth factor [134], a member of the CCN gene family, which mediates fibrotic responses [163]. Increased connective tissue growth factor levels are a characteristic finding in complicated atherosclerotic plaques and induce mononuclear cell chemotaxis [164].

Plasma NEFA exert profound effects on endothelial function. Insulin-stimulated blood flow is mediated via nitric oxide [86] and is impaired in obese and type 2 diabetic individuals [108, 165]. Experimental plasma NEFA elevation in participants with normal glucose tolerance reduced endothelium-dependent blood flow, without altering endothelium-independent blood flow [166]. Endothelial dysfunction is associated with accelerated atherosclerosis and insulin resistance [110, 111], providing another mechanism via which NEFA promote CVD in type 2 diabetes mellitus.

‘Adiposopathy’ is another form of lipotoxicity [131]. Fat cells produce adipocytokines, which travel to distant sites (muscle, liver, arterial tissue) where they exert deleterious effects on metabolism and vascular function. Adipose tissue of obese and type 2 diabetic individuals is infiltrated by mononuclear cells and is in a state of chronic inflammation [167]. The adipocytes and infiltrated macrophages secrete proinflammatory/prothrombotic cytokines (TNFα, resistin, IL-6, plasminogen activator inhibitor-1, angiotensinogen) that promote atherogenesis and cause insulin resistance (reviewed by Bays et al. [131]). Adipocytes also under-produce adiponectin, a potent insulin-sensitising, anti-atherogenic cytokine [131].

Accumulation of fat within the intraabdominal cavity, i.e. visceral fat (Fig. 8), is associated with insulin resistance and CVD [131, 168, 169]. Although the mechanism(s) for this association remain(s) unclear, visceral obesity is part of the insulin resistance (metabolic) syndrome [38‐40]; however, the individual components of the insulin resistance syndrome, not the increase in visceral fat, may account for the increased incidence of CVD.

Tissue fat overload is explained by: (1) excessive energy intake; (2) increased lipolysis in insulin-resistant adipocytes, leading to increased plasma NEFA and enhanced influx into muscle/liver; and (3) decreased fat oxidation. Weight loss (energy-intake restriction) has proven effective in reducing total body fat content [170]. Glucagon-like peptide-1 analogues are potent appetite suppressants and effectively promote weight loss [171].

Thiazolidinediones exert the following unique actions that ameliorate lipotoxicity (Textbox 3): (1) decrease plasma NEFA by inhibition of lipolysis; (2) reduction of muscle long-chain fatty acyl CoA levels; (3) redistribution of fat within the body; and (4) amelioration of adiposopathy.

Insulin resistance in the PI-3 kinase pathway with normal insulin sensitivity in the MAP kinase pathway plays an important role in accelerated atherogenesis in patients with type 2 diabetes mellitus. Lipotoxicity is a major cause of insulin resistance and impaired beta cell function in type 2 diabetes mellitus and plays a central role in the accelerated CVD. Interventions that mobilise fat out of tissues (restriction of energy intake, thiazolidinediones, glucagon-like peptide-1 analogues) are likely to produce a durable reduction in glycaemia and reduce atherosclerotic cardiovascular events.