© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Actions of insulin on the mammalian heart: metabolism, pathology and biochemical mechanisms
aDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Copp Building, Medical Block ‘A’, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1Z3
bDepartment of Pathology and Laboratory Medicine, The University of British Columbia, Vancouver, B.C, Canada
* Corresponding author. Tel.: +1 (604) 822-3810; Fax: +1 (604) 822-5227; e-mail: rogerb@unixg.ubc.ca
Received 17 December 1996; accepted 24 January 1997
KEYWORDS ACC = acetyl-CoA carboxylase; AMP-PK = protein serine/threonine kinase activated by 5'-AMP; CPT-I = carnitine palmitoyltransferase-I; CREB protein = cyclic AMP-response element binding protein; ERKs = extracellular signal-regulated kinases: the MAP kinases originally identified are now known to represent a sub-set of a wider family; the original members of the family are now referred to as ‘ERKs’ (extracellular signal-regulated kinase); other sub-types of MAP kinases are JNKs (c-Jun, N-terminal kinases) and p38/RK; p125-FAK = 125-kDa focal adhesion (protein tyrosine) kinase; Grb = growth-factor-receptor bound protein; GSK-3 = glycogen synthase kinase-3; HDL = high-density lipoprotein; IRS-1(-2) = insulin receptor substrate-1(-2); HSP = heat shock protein; MAP kinase = initially named for an insulin-stimulated protein Ser/Thr kinase able to phosphorylate microtubule-associated protein-2 (MAP-2); also commonly referred to as ‘mitogen-activated protein kinase’; MAPKAP-kinase = MAP-kinase-activated protein kinase: MAPKAP-K1 is identical to p90rsk, MAPKAP-K2 is distinct and activated downstream from p38/RK; MEKs = dual-specificity MAP kinase- or ERK-kinases (able to phosphorylate the TEY motif of the MAP kinases/ERKs; MEKKs = protein kinases able to phosphorylate and activate MEKs; other mammalian protein kinases which can fulfil this function include the c-Raf family and c-Mos; PDC (PDC-P) = pyruvate dehydrogenase complex (and the inactive phosphorylated form); PDE-III = cyclic nucleotide phosphodiesterase, ‘low Km, particulate’ isoform which is inhibitable by cyclic GMP; PKA = cyclic-AMP-dependent protein kinase; PKB = protein kinase B, also identified as the proto-oncogene akt and as RAC (protein kinase Related to A or C kinase); PKC = protein kinase C; PHAS = 22-kDa, phosphorylated, heat- and acid-stable protein; able to bind to the eukaryotic initiation factor 4, ‘cap-binding’ complex, therefore also designated as eIF-4E:Bp (eukaryotic initiation factor-4E binding protein); PI-3-kinase = phosphatidylinositol-3-OH-kinase (comprised of ‘p85’ and ‘p110’ subunits which are respectively the 85-kDa regulatory, SH2-containing and 110-kDa catalytic subunits); PTB domain = phosphotyrosine binding domain, equivalent in function to SH2 domains but with distinct structure and target-binding characteristics; p21-Ras = 21-kDa GTPase encoded by cellular homologue of rat sarcoma oncogene; Ras-GAP = GTPase-activating protein for p21-Ras; p90rsk = 90-kDa isoform of ribosomal protein S6 kinase; distinct from p70 S6K, the 70-kDa/85-kDa family of ribosomal S6 protein kinases; since p90-rsk is phosphorylated and activated by MAP kinases, it is also recognized as MAPKAP kinase-1; SH2 and SH3 domains = src-homology domains 2 and 3 (sequences related to non-catalytic domains of the protein tyrosine kinase pp60src and which function by binding, respectively, to phosphotyrosine motifs and proline-rich domains of target proteins).; Shc = adaptor protein with homologies to Src and to collagen; Sos = guanine nucleotide exchange factor (GDP-release factor) for p21-Ras, designated ‘son-of-sevenless’ and named from a Drosophila mutant manifest (in development of cell designated ‘R-seven’) due to a defect in signalling downstream from the ‘sev’ receptor tyrosine kinase; STAT = signal transducer and activator of transcription – transcription factors which have SH2 and tyrosyl-phosphorylation domains for dimerization to facilitate translocation from cytosol to nucleus upon cytokine or hormone activation; STZ = streptozotocin; Syp = SH2-containing protein tyrosine phosphatase (SH-PTP2); p60TCF = 60-kDa ‘ternary complex factor’, also designated Elk-1; TOR (mTOR) = target of rapamycin, first defined in yeast (and mammalian homologue); VAMP = vesicle-associated membrane protein; VLDL = very low density lipoprotein
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1 Introduction |
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 Top 1 Introduction 2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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Skeletal muscle, adipose tissue and liver are the quantitatively major targets for insulin action in vivo and regulation of critical steps in intermediary metabolism within these tissues account for many of the impacts of insulin on metabolic homeostasis. Many other tissues including the heart express insulin receptors and their functions may be importantly regulated by insulin. In this review we summarize the evidence that the heart is an important target of insulin action and that abrogation of these actions is important in disease states.
Current understanding of the molecular basis of insulin actions on its target cells is drawn from a large literature emanating from studies of the major target tissues and also from a wide range of other cell types including studies of appropriately transfected and immortalized cell lines [1–4]. We introduce the basic concepts of the molecular basis of insulin actions, not to reiterate the extensive reviews recently published but rather to highlight aspects which are distinctive or which are poorly-defined in the heart. Our understanding of the biochemical mechanisms by which insulin exerts its effects in the heart are still substantially dependent upon extrapolation from studies of other cell types. It is important to recognize where such extrapolations may require qualification; we therefore focus particularly on the specific features of the myocardium which may lead to a distinct perspective of insulin action in this tissue.
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2 The context of insulin actions in the heart |
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Top1 Introduction2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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The actions of any individual external signal must be viewed in the context of a complex set of signals to which cells are exposed in vivo. Three issues are introduced (undoubtedly many more might deserve comment) to illustrate the importance of considering the basis against which we must attempt to judge the actions of insulin in the heart: (i) the constant pumping function of the heart, (ii) the role of intracellular calcium ions in regulating heart function, and (iii) the complex interplay of major systems for cell–cell communication in which the heart plays a central role.
2.1 Insulin actions and the mechanical demands of the heart
The fact that the function of the heart must proceed uninterrupted dictates a perpetual ‘high basal’ level of contractile activity and therefore of energy metabolism. In a human heart, basal cardiac output is generally in the range 5–6 litres/min and may rise by 4–5 times this level in exercise. The function demanded of the heart is reflected by remarkably dense vascularization of the myocardium and a very high reliance on oxidative metabolism indicated by abundance of mitochondria, which occupy close to 30% of myocardial cell volume [5]. The perpetual contractile function of the myocardium poses an intriguing set of challenges to understanding of the regulation of metabolism and other cellular functions. Unlike studies of the function of skeletal muscle at the onset of vigorous contraction, comparable studies of the myocardium must examine the effects of modulators within an especially dynamic context—implying that we must be cautious in the interpretation of results of studies with isolated myocardial cells which perform little or no external work.
2.2 Calcium ions and insulin action in the heart
In the human heart, calcium release and sequestration must occur at a rate of 40–80 cycles/min under resting conditions and as rapidly as 180–220 cycles/min in non-pathological exercise stresses. The potential actions of insulin on heart metabolism or function must therefore be exerted against a ‘calcium background’ which is distinct from that of other insulin target tissues. Many actions of insulin in liver and adipose tissue are achieved with minimal shifts in concentrations of free cytoplasmic Ca2+ and indeed may be antagonised by hormones which patently act through increases in cytoplasmic calcium levels [6–8]. One significant challenge is therefore to understand the features of insulin action in the heart which allow interfacing with potentially antagonistic or interacting signals—for example, is calcium sufficiently localized to prevent inappropriate actions or are the regulatory mechanisms substantially different in the heart?
2.3 Insulin and additional inter-cellular control systems
The actions of insulin in the heart must be exerted within a complex network of local, neural and endocrine signalling systems. Local and neural signals in the heart include those initiated at the sinoatrial node and also by release of sympathetic adrenergic as well as muscarinic neurotransmitters [9]. In turn, myocardial cells may respond with the production of adenosine and other intrinsic feedback modulators [10, 11]. The heart is also a key endocrine element in the regulation of blood pressure and volume in which it interacts with the kidneys and vascular smooth muscle by communications which feature the actions of the cardiac atrial natriuretic peptides, the renin–angiotensin system and aldosterone among others [12, 13]. There is also emerging evidence that insulin may influence tissue blood flow, perhaps by affecting production of nitric oxide in the vascular endothelium [14, 15]. Finally, like other tissues, the heart is sensitive to the nutritional status of the individual and the associated regulatory mechanisms for whole-body fuel homeostasis and for local control of energy metabolism.
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3 Indirect actions of insulin on the heart |
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Top1 Introduction2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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Two issues are emphasized in this section: (i) the effects of insulin on the supply of substrates for cardiac energy metabolism and (ii) the impact of insulin on myocardial perfusion.
3.1 The role of insulin in the supply of metabolic substrates to the heart
Insulin plays a major role in regulating the balance of metabolic fuels received by the myocardium; principally through its actions on adipose tissue, skeletal muscle and liver (Fig. 1). The effects of insulin on protein metabolism, in skeletal muscle and liver may be especially significant in regulating substrate availability to the heart. The ability of insulin to suppress protein degradation (and to promote protein synthesis) profoundly affects overall carbohydrate and nitrogen economy [16, 17]. In insulin deficiency, therefore, the increase in supply of amino acids from net protein catabolism provides precursors for hepatic gluconeogenesis and ketogenesis and contributes to the balance of substrates delivered to the myocardium.
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Also critical is the extent to which insulin inhibits adipose tissue triglyceride hydrolysis (regulating the actions of lipolytic hormones) because this action dictates levels of long-chain fatty acids released to enter a complex with circulating serum albumin. The loss of regulation of adipose tissue lipolysis is most exaggerated in poorly-controlled insulin-dependent diabetes or cachexia, but is also evident in post-absorptive metabolism in the normal 24-hour cycle [16, 18]. The increased availability of fatty acids leads to enhanced β-oxidation at the expense of pyruvate oxidation in cardiac myocytes and other cells with sufficient oxidative capacity. The ‘glucose–fatty acid cycle’ [19, 20] therefore provides a critical mechanism to conserve glucose for cells with less metabolic flexibility (neurones and blood cells, for example).
Increases in circulating free fatty acids in insulin deficiency have important effects on the liver which also contribute to changes in substrate delivery to the heart. Particularly important is the stimulation of synthesis and export of complex lipoproteins by elevated delivery of free fatty acids to the liver [21, 22]. During starvation, the circulating lipoprotein-triglycerides are predominantly accessed by skeletal and cardiac muscles because lipoprotein lipase activity is maintained in these tissues but declines in adipose tissue [23, 24]. In diabetes, however, functional lipoprotein lipase declines in skeletal muscle and heart as well as in adipose tissue—thus contributing significantly to diabetic hyperlipoproteinemia [25]. In extended starvation and most markedly in uncontrolled insulin-dependent diabetes or infection, exaggerated oxidation of fatty acids leads to increases in circulating ketones [16, 26, 27]. Ketones are extensively released by the liver (as a result of the need to conserve levels of free coenzyme A by condensation of acetyl units) and contribute to the exclusion of carbohydrate oxidation by the myocardium. Remarkably, mortality from ketoacidosis in developed countries is still in the range of 2–5% (and as high as 6–24% in developing countries), more than half of the cases of ketoacidosis being accounted for by infection and newly-diagnosed diabetes [28].
3.2 The role of insulin in the regulation of myocardial perfusion
In view of the reliance of the myocardium on oxidative metabolism, a significant restriction to myocardial blood flow has serious consequences for the function and indeed the survival of cardiac myocytes. Acute and chronic effects of insulin may be significant in contributing to the adequacy of perfusion of the coronary circulation. There is increasing evidence that, in the short-term, insulin may stimulate blood flow through skeletal muscle [14] by enhancing the formation of nitric oxide in vascular endothelium [29] and therefore inducing relaxation of the associated vascular smooth muscle. It will be important to establish if similar effects are induced directly in myocardial vascular smooth muscle, but the response systems are certainly present and there appear to be complex functions for nitric oxide within the myocardium [15]. More generally, insulin may contribute to tonic control of vascular smooth muscle and peripheral blood pressure (and therefore the load against which the heart must pump). Certainly, insulin resistance is frequently associated with hypertension [30, 31] and agents directed at correcting insulin resistance may correct defects in blood pressure [32, 33].
In addition to direct effects on vascular tone, defects in insulin secretion or action may contribute significantly to the pathogenesis of macrovascular (atherosclerosis) and microvascular disease [30, 31]. The decline in vascular function progresses over many years, ranging from modest changes in responsiveness and permeability to loss of elasticity and outright occlusion. The consequences of this process include compromised perfusion and function of the heart and finally necrosis of tissue significantly deprived of blood flow. A detailed discussion is beyond the scope of this review, but suffice to say that there is compelling evidence to suggest that loss of insulin sensitivity may play a key role in the development of abnormalities which are major risk factors for atherosclerosis—notably hyperinsulinemia, hypertriglyceridemia, elevated VLDL, reduced HDL (and associated cholesterol levels), impaired glucose tolerance or frank hyperglycemia and hypertension [30, 31, 34, 35]
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4 Direct actions of insulin on the heart |
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Top1 Introduction2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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This section will focus on the role of insulin in the regulation of myocardial energy metabolism and especially on the rapid effects of insulin on the specific activities of key enzymes and transporters (Fig. 2 and Table 1). Effects of insulin on protein expression are also noted, especially where important changes are revealed during starvation or diabetes (Table 2). Of course, effects of starvation or diabetes are confounded by changes in concentrations of hormones in addition to insulin as well as by the effects of hyperglycemia.
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The energy metabolism of the heart is dominated by oxidation of fatty acids, which accounts for more than 80% of ATP production under most physiological conditions; because of its aerobic capacity the heart is also able to utilize lactate [36–38]. Importantly, though glucose is not a dominant fuel in the heart, nevertheless its consumption is still substantial when compared to that of other cell types and may amount to 25–50 g glucose consumed in a 24-hour period in humans.
Our understanding of the mechanisms for the pre-eminence of fatty acids as energy fuel for the myocardium was initiated with the seminal observation that glucose oxidation by the perfused heart was markedly impaired by addition of fatty acids or ketones to the perfusion medium [39–41]. These and subsequent studies led to the concept of the ‘glucose–fatty acid cycle’ [19, 20], which has been validated in numerous studies including those in vivo [42–44].
We now examine selected steps in cardiac metabolism which are insulin-sensitive, notably glucose transport and key enzymes involved in glycogen metabolism, glycolysis, pyruvate oxidation and β-oxidation of fatty acids (Fig. 2 and Table 1).
4.1 Glucose transport
Early studies established that insulin stimulated the uptake of glucose by perfused rat hearts [45, 46]. In many of the earlier studies, ‘non-working’ hearts were perfused by the Langendorff method, with glucose as the sole substrate. The sharp rise in intracellular concentrations of glucose following insulin stimulation (from basal levels of <1 mM to as high as 12 mM) suggested that glucose metabolism was limited by glucose transport in the absence of insulin, and that hexokinase (or further ‘downstream’ metabolic enzymes) contributed importantly to flux control following insulin stimulation. This concept has been confirmed in studies of individual myocardial cells [46] although results of further studies indicate that the distribution of control strength is more complex [47].
The currently accepted mechanism of regulation of glucose transport by insulin was proposed on the basis of studies with white adipose cells [48, 49]. The stimulation of glucose transport by insulin is accounted for largely by the movement or translocation of glucose transporter (GLUT) molecules from intracellular membranes to the plasma membrane and in adipocytes the effect of insulin is largely explained by movement of the specific GLUT-4 isoform [50–52]. Studies of specifically labelled GLUTs have been used to discriminate exocytosis from endocytosis and suggest that insulin markedly stimulates the former branch of the cycle while modestly inhibiting the latter and that GLUT molecules likely pass through a complex system of intracellular compartments [53, 54].
Translocation of GLUTs has been demonstrated in perfused hearts [55], in isolated cardiac myocytes [56] and in the heart in vivo [57, 58]. The distinct architecture of muscle cells may require special consideration in interpreting the basic model developed from studies of adipocytes. In muscle cells, the intracellular reserve of GLUTs appears in tubulo-vesicular structures associated with the trans- Golgi network [56, 59]. Further, the T-tubule system of muscle cells may contribute importantly as a destination for insulin-stimulated relocation of GLUTs [60, 61]. Studies of other systems for intracellular trafficking of proteins imply a major role for Rab-family GTPases and other proteins in membrane docking, fusion and budding [62, 63]. In fact, proteins related to the VAMPs of synaptic vesicles [64] and also Rab-family GTPases [57] have been demonstrated in adipocyte membrane fractions which contain GLUT-4, while the p21-Ras GTPase has been implicated in stimulation of GLUT-4 by insulin in cardiac myocytes [65].
Compared to other insulin-responsive tissues, heart muscle expresses among the highest levels so far detected, of the ‘insulin-sensitive’ GLUT-4 isoform [66, 67]. The ratio of GLUT-1/GLUT-4 in heart muscle (approximately 1:3), means that the expression of the GLUT-1 isoform is also substantial in the heart. Activation of myocardial glucose transport by insulin involves translocation of both GLUT-1 and GLUT-4 isoforms [57, 58]. High levels of GLUT-1 in the heart probably contribute to the high rates of glucose transport in the absence of insulin, and the relatively small response to insulin (3–4-fold) compared to skeletal muscle or adipose tissue (10–40-fold). An important caveat, in view of the ability of fatty acids to suppress glucose utilization by heart and skeletal muscle [38–40], is that basal rates of myocardial glucose utilization may be artificially inflated by the absence of lipid substrates in vitro.
An important characteristic of muscle cells is exercise-induced stimulation of GLUT-4 translocation which is insulin-independent. Exercise-induced activation of glucose transport has been best demonstrated in skeletal muscle [68, 69]. Similarly, increases in cardiac work are associated with increased glucose consumption [36, 37] and contraction-induced regulation of GLUT-4 has also been shown in cardiac myocytes and appears to preclude further activation by insulin [70]. The significance of insulin stimulation of myocardial glucose transport must therefore be considered in the context of the effects dictated by mechanical demands of the heart—especially as catecholamines and calcium ions may also activate GLUT-4 translocation independently of insulin [71].
The role of insulin in expression of GLUT proteins has also been studied by examining direct effects of the hormone in vitro and changes seen in starvation and diabetes, when rates of glucose transport into the heart are markedly reduced [45, 72]. The expression of GLUT-1 (but not that of GLUT-4) declined in the heart with 48 hours starvation [67]. In these studies, basal glucose transport was diminished, but maximal stimulation by insulin was not markedly affected. The marginal effects of starvation on cardiac GLUT-4 expression are therefore similar to the effects on skeletal muscle and distinct from changes in adipose tissue, in which GLUT-4 levels decline appreciably in starvation [73]. Induction of diabetes with STZ is also reflected by divergent regulation of GLUT expression in heart and fat. STZ diabetes results in markedly decreased expression of GLUT-4 protein and mRNA in adipose tissue [74, 75] while more variable results have been reported in skeletal muscle [75–78] and essentially no changes detected in the heart, at least over a period of several days after STZ treatment [78, 79]. Distinct regulation of glucose transport within heart compared with skeletal muscle has been reported in studies of human Type-I (insulin-dependent) diabetics analysed by distribution of [18F]deoxyglucose, using positron emission tomography. In these studies, glucose uptake into skeletal muscle was insulin-resistant during euglycemic, hyperinsulinemic clamps but that into heart muscle appeared normal [80].
4.2 Glycogen metabolism
It has long been recognized that the concentration of glycogen in the heart is increased in insulin-dependent diabetes, while the corresponding reserves in liver and skeletal muscle become markedly depleted [81]. Rates of glycogen accumulation in the heart can be increased even without increases in glucose uptake, provided an alternative fuel is available to allow re-direction of glucose from glycolysis to glycogen synthase [82]. This may be an important mechanism to allow glycogen conservation and repletion in the heart during and following exercise, when lactate release from skeletal muscles is likely to be increased [83].
Despite the maintenance of steady-state levels of glycogen in the diabetic heart, the myocardial glycogen pool turns over rapidly even when β-oxidation appears to satisfy a major proportion of the energy demand of the whole heart [84–86]. Additionally, myocardial glycogen is preferentially oxidized compared with exogenous glucose and therefore produces proportionately more ATP per mole of glucose metabolized [84, 86]. The role of insulin in regulating glycogen turnover and in the preferential oxidation of glycogen–glucose remains to be investigated. Cardiac glycogen metabolism is further complicated by a marked transmural gradient of glycogen concentration and glucose metabolism—glycogen concentrations and rates of glucose uptake being highest in the innermost layers of muscle [87, 88]. The differential distribution of glycogen may suggest a critical function in domains of muscle which may be forced to rely less on β-oxidation and indicates that the actions of insulin and other hormones may be expressed differentially through the myocardium.
The major mechanisms for regulation of the key enzymes of glycogen metabolism appear to be similar in heart and skeletal muscle [2, 89]. Cardiac glycogen phosphorylase isozymes differ in some properties from the skeletal muscle form but are still activated both by phosphorylation (by phosphorylase kinase) and by allosteric ligands—particularly by 5'-AMP [90]. Cardiac phosphorylase kinase contains a distinct isoform of alpha-subunit (alpha'), but, like its skeletal muscle counterpart, is phosphorylated and activated by cyclic AMP-dependent protein kinase and by binding of calcium to the intrinsic delta subunit/calmodulin [91]. Cardiac glycogen synthase is activated by glucose-6-phosphate and also subject to multiple-site phosphorylation [92, 93]. Studies with purified heart glycogen synthase confirm phosphorylation and inactivation by PKA, GSK-3, calcium/calmodulin kinase II, PKC and phosphorylase kinase with generation of phosphopeptides identical to those of synthase from skeletal muscle [94]. Insulin induces rapid dephosphorylation and activation of glycogen synthase in perfused hearts, promoting the formation of ‘GS-I’, the form of the enzyme which exhibits full activity independently of glucose-6-phosphate [95, 96]. The results of the studies with isolated perfused hearts are supported by NMR studies in vivo [97]. It is important to note that insulin treatments do not appear to induce changes in cyclic AMP concentrations, the activation state of PKA or the proportion of glycogen phosphorylase in the active form in perfused hearts [98–100], even when hearts are perfused in the presence of fatty acids [98].
Why are heart glycogen reserves enhanced during diabetes, even though prevailing humoral conditions might be expected to induce glycogen depletion as in skeletal muscle and liver? Glycogen accumulates in the diabetic heart despite the fact that glycogen synthase expression is not elevated in hearts of STZ-diabetic rats [101] or in human Type II diabetics [102]. In addition, the level of GS-I is markedly suppressed [95] and the ability of insulin to induce activation is severely blunted [98, 101, 102]. The defect in activation of glycogen synthase is associated with decreases in the levels and insulin-mediated activation of glycogen synthase phosphatase in experimental and human diabetes [100]; this observation further underlines the importance of the activation of protein serine/threonine phosphatases in insulin action. The accumulation of glycogen when the activation of glycogen synthase is decreased indicates that flux through glycogen phosphorylase must also be diminished, even though glycogen phosphorylase is expressed at near-normal levels in the diabetic heart [98, 100] and is ‘hypersensitive’ to catecholamine-mediated stimulation [99, 103]. Taken together, these observations support an earlier suggestion that the effects of phosphorylation of the glycogen-metabolizing enzymes are likely to be over-ridden by availability of allosteric regulators in the heart, at least in some conditions [45].
4.3 Pyruvate oxidation
The pyruvate dehydrogenase complex (PDC) is a major target for insulin action. The effects of insulin arise through control of delivery of substrates to the myocardium and also by direct effects of insulin on PDC specific activity. The regulation of PDC involves interactions between covalent phosphorylation [104] and non-covalent mechanisms, principally accumulation of reaction products. Most directly, the rise in intramitochondrial acetyl-CoA (and fall in free CoA), occasioned by enhanced rates of oxidation of fatty acids or ketones, causes product inhibition of PDC [105]. In addition, the associated PDC kinase becomes activated and, by phosphorylating three sites on the 
-subunit of the pyruvate dehydrogenase (E1) components of the complex, converts the complex into a phosphorylated form with very low specific activity [106]. The importance of the regulation of PDC activity in overall glucose metabolism is perhaps most clearly revealed by studies with dichloroacetate, which potently inhibits PDC kinase and therefore activates PDC and pyruvate oxidation [107].
In addition to reducing the availability of exogenous fatty acids for oxidation (and thereby reducing the level of intramitochondrial acetyl-CoA), exposure of hearts to insulin leads to rapid and direct activation of PDC [108]. Insulin-mediated activation of PDC persists in isolated mitochondria; however, the mechanism for the effect of insulin is still not fully understood [109]. The activation of the complex is probably explained by increases in PDC-phosphate phosphatase [110], which is dependent on Mg2+ for activity and is further stimulated by Ca2+ [108–111]. Consequently, PDC can be activated by increases in cytoplasmic and hence mitochondrial calcium in the heart [112], but calcium ions alone do not account for the effects of insulin, which acts by a distinct mechanism [113, 114]. Overall, the mechanical demands of the heart dictate the level of activation of the TCA cycle through effects on the intramitochondrial concentrations of NADH/NAD, ATP/ADP and Ca2+ [109, 115]. The primary role of insulin in this context is to induce a switch in selection of substrates used to provide acetyl-CoA.
The regulation of PDC in diabetes involves not only the short-term mechanisms described but also effects mediated by protein synthesis. The expression of PDC itself changes very little but that of PDC kinase is enhanced in starvation and experimental diabetes (Table 2), suggesting that insulin plays a tonic inhibitory role in the expression of this intramitochondrial protein kinase [116, 117]. Taken together, the mechanisms for regulating PDC exert profound effects on overall glucose utilization, including mechanisms to transmit control to additional metabolic enzymes.
4.4 Glycolysis
Regulation of GLUT and PDC activity has an important bearing on flux through glycolysis which is generally coordinated with rates of pyruvate oxidation, increasing in response to insulin and to increased cardiac workload during glucose perfusion [37, 45, 118]. ‘Uncoupling’ of glycolysis and pyruvate oxidation is possible, being most apparent in hypoxia or anoxia (when glycolysis is markedly activated but terminal oxidation of pyruvate and fatty acids is precluded) but also evident even under normoxic conditions. Other conditions, such as reperfusion after ischemia [38] and cardiac hypertrophy (see Section 6), may also disrupt the balance between glycolysis and glucose oxidation.
The regulation of 6-phosphofructo-1-kinase (PFK-1) is critical in the control of glycolysis. Normal tissue levels of key allosteric inhibitors, ATP and citrate, ensure that the enzyme is inactive unless allosteric activators are also available. Fructose-2,6-bisphosphate, the product of the dual function 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, is a critical modulator of cardiac PFK-1 because it is able to overcome the inhibitory effects of ATP [119, 120]. The cardiac form of PFK-2 likely plays a significant role in the activation of glycolysis in response to insulin [121], and although the mechanism is not understood, it appears to be wortmannin-sensitive. Levels of fructose-2,6-bisphosphate are also increased during increases in cardiac workload and contribute to corresponding increases in rates of glycolysis. Importantly, the distinct cardiac PFK-2 isoform is not inhibited upon incubation in vitro in the presence of PKA [122]. This property of the cardiac form of PFK-2 is in sharp contrast to the response of the hepatic isoform, which is markedly inhibited by PKA in concert with the activation of the fructose bisphosphatase active site [120, 123]. Consequently, phosphorylation of hepatic PFK-2 by PKA contributes to inhibition of glycolysis by catecholamines and glucagon and facilitates gluconeogenesis and hepatic glucose output. In the heart, glycolysis is stimulated by catecholamines and by insulin. PFK-2, like PFK-1, is inhibited by citrate, so the accumulation of this one TCA cycle intermediate during fatty acid oxidation [124] serves to directly inhibit PFK-1 and to suppress the formation of the PFK-1 activator, fructose-2,6-bisphosphate.
Insulin also exerts a rapid influence on hexokinase, largely indirectly, through changes in PFK-1 and PDC. Inhibition of PFK-1 and of PDC, when insulin levels are low, lead to accumulation of fructose-6-phosphate and then (via phosphoglucose isomerase) of glucose-6-phosphate which inhibits hexokinase. Stimulation of PFK-1 by insulin therefore relieves inhibition of hexokinase by glucose-6-phosphate. The possibility that insulin might regulate expression of hexokinase was suggested by studies of skeletal muscle [125]. A number of studies have confirmed that expression of cardiac hexokinase (principally the hexokinase-II isoform) is decreased in hearts from alloxan- or STZ-diabetic rats [126, 127]. Although this observation has not always been confirmed [78], a decline in hexokinase activity in the diabetic heart is consistent with the parallel decline in activity of the hexose monophosphate pathway [126].
4.5 Fatty acid metabolism
We have discussed the roles of insulin in regulating the supply of exogenous fatty acids to the myocardium—notably at the level of adipose tissue and liver to control circulating levels of free fatty acids and lipoprotein triglycerides. Insulin also regulates fatty acid metabolism within the heart itself. The regulation of glucose transport and of PDC provide important means to modulate fatty acid oxidation, the latter being notably revealed by the actions of pyruvate [128, 129] and dichloroacetate [107]. There are additional steps which also contribute to the regulation of fuel selection in the myocardium. Here we discuss the effects of insulin on triglyceride lipases and on carnitine palmitoyl transferase-I (CPT-I), critical enzymes of lipid metabolism in the heart.
Fatty acids may be generated in the myocardium by release from triglycerides in the plasma lipoproteins as well as from endogenous tissue triglyceride stores [130]. The hydrolysis of plasma triglycerides is effected at the luminal surface of vascular endothelial cells, but the lipoprotein lipase (LPL) which is responsible is synthesized in cardiac myocytes and this process is regulated by insulin. The expression of cardiac LPL is sustained during moderate starvation (while the adipose tissue enzyme is repressed) and this contributes to the channelling of lipid fuels from adipose tissue to muscle [23, 24]. In contrast, the expression of LPL is inhibited in experimental diabetes in heart and skeletal muscle as well as in fat tissue [25, 131]. The effects of insulin on lipoprotein lipase are complex and incompletely understood—indeed, the steps involved in the maturation and processing of LPL from synthesis to localization in vascular endothelial cells are still not fully defined [132]. Heparin-releasable LPL at the vascular endothelium is reduced in perfused hearts from diabetic rats [23, 24, 133]. The decline in LPL levels, coupled with altered properties of the circulating lipoproteins, combines to markedly reduce the utilization of circulating triglycerides by the diabetic heart [133, 134]. The LPL activity recovered from cardiomyocytes is also reduced by STZ-diabetes [135], the reduction being explained partly by a decline in general protein synthesis and partly by a defect in processing of the enzyme to the fully active form. Interestingly, no changes in LPL mRNA abundance or turnover were detected during 4–5 days of STZ-diabetes [136].
The role of endogenous triglycerides as an energy fuel was suggested by the fact that isolated perfused rat hearts continue beating for 1–2 h in the absence of exogenous substrates [41, 81, 137]. The utilization of endogenous cardiac triglyceride was directly demonstrated from mass measurements [138] and by prelabelling with [14C]palmitate [128]. The size and turnover of the cardiac triglyceride reserve are both increased in hearts from diabetic rats [130, 138–140], reflecting enhanced hydrolysis and re-esterification. Myocardial triglyceride lipolysis is limited by the activity of hormone-sensitive lipase, which is similar to the adipose tissue enzyme in that it is activated by catecholamines and inhibited by insulin and nicotinic acid [141–143]. Studies of immunoreactivity and of phosphorylation sites confirm that cardiac and adipose hormone-sensitive lipases are indeed very similar [144] and activated by PKA-mediated phosphorylation of a single phosphorylation site [145, 146]. It is likely that the actions of insulin on cardiac lipolysis are mediated principally by activation of protein phosphatases and of PDE-III. Feedback inhibition of hormone-sensitive lipase by fatty acids might account for the conservation of myocardial triglycerides when exogenous fatty acids are abundant [45, 130, 147]. However, increases in esterification rates, normally limited by availability of fatty acyl-CoA esters, would also offer an attractive explanation of the maintenance of triglyceride reserves with high turnover [138, 148].
Fatty acid oxidation is also regulated in the heart—especially by control of carnitine palmitoyl transferase-I (CPT-I). A critical mechanism for regulating CPT-I, through allosteric inhibition by malonyl CoA, was elucidated as a result of studies of hepatic fatty acid oxidation and ketogenesis [149]. The distinct ‘M’ (‘muscle’) isoform of CPT-I represents 98% of the total activity in adult heart [150]; it exhibits a higher Km for carnitine and lower IC50 for malonyl-CoA than the liver (‘L’) isoform [151]. The sensitivity of CPT-I to malonyl-CoA is diminished in the liver in starvation [152] and in STZ-diabetes [153], but opinions are divided about the importance of this mechanism in the heart [151–154].
It is most likely that the rapid effects of insulin on CPT-I activity are mediated by changes in the concentration of malonyl-CoA in cells. The only source of malonyl-CoA in mammalian cells is the reaction catalysed by acetyl-CoA carboxylase, an enzyme which is markedly activated by insulin in adipose tissue and liver [155]. The heart expresses a distinct 280-kDa isoform of acetyl-CoA carboxylase [156, 157] which appears to play a significant role in regulating fatty acid oxidation in the heart, judging by the inverse relationship between rates of fatty acid oxidation and tissue concentrations of malonyl-CoA [38, 158]. The larger 280-kDa isoform of acetyl-CoA carboxylase displays a higher Km for acetyl-CoA than the 265-kDa isoform found in adipose tissue and liver [156] and substantial differences in primary structure have been detected [159]. The mechanism by which acetyl-CoA carboxylase might be regulated by insulin in the heart is not resolved—no effects of insulin on the activity of the cardiac enzyme have yet been demonstrated to persist following enzyme extraction or purification. It has been suggested that the supply of acetyl-CoA might limit the formation of malonyl-CoA in the heart [38, 160], but cytoplasmic acetyl-CoA levels are highest in the heart when fatty acid oxidation is predominant (and when the ACC activator citrate is most abundant). These observations suggest that some additional mechanism must restrict cardiac acetyl-CoA carboxylase in starvation and diabetes. The multiple-site phosphorylation of the 265-kDa form of acetyl-CoA carboxylase [155, 161, 162] suggests that this mechanism might also be significant in regulating the 280-kDa isoform of the enzyme in the heart. Our own recent studies have demonstrated the phosphorylation of acetyl-CoA carboxylase within rat cardiac myocytes and shown that the 280-kDa isoform is indeed phosphorylated at multiple sites [Boone, Rodrigues and Brownsey, unpublished observations]. It will be important to establish which cellular protein kinases are most important in the phosphorylation of the cardiac ACC isoform and what effects, if any, are induced by insulin. In adipose tissue and liver, acetyl-CoA carboxylase is phosphorylated and inhibited in response to catecholamines and glucagon, most likely through the actions of the AMP-activated protein kinase [161–164]. This protein kinase may also play a significant role in regulating acetyl-CoA carboxylase in the heart. For example, in hearts subjected to ischemia and reperfusion, formation of malonyl-CoA is decreased in parallel with enhanced rates of fatty acid oxidation and activation of AMP-activated protein kinase [165].
Additional mechanisms for regulating fatty acid oxidation through the availability of malonyl-CoA are also possible. For example, it has been suggested that only a small proportion of total tissue malonyl-CoA may be accessible to CPT-I, raising the possibility that compartmentation or binding of this inhibitor could be a regulated process [166]. Finally, since the heart appears to express no fatty acid synthase [167], a mechanism for malonyl-CoA removal must be available and may be regulated. Malonyl-CoA decarboxylase has been described in mitochondria from various rat tissues [168] and activity in the heart appears to be increased during reperfusion following ischemia [169]. A detailed examination of cardiac malonyl-CoA decarboxylase and of its regulatory properties is clearly merited.
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5 Intracellular signalling pathways in insulin action |
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Top1 Introduction2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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From the foregoing discussions, it is clear that the effects of insulin on key metabolic enzymes and transporters and on myocardial metabolic pathways are well defined. It is therefore important to establish the intracellular signalling mechanisms which may lead to the regulation of glucose transport proteins and enzymes important in the control of cardiac energy metabolism. Recent reviews have provided detailed accounts of our current understanding of the molecular basis for insulin action [1–4]. Our goal is to outline the major features of insulin action established as a ‘consensus’ from diverse cell types and examine the evidence which indicates if the same or distinct signalling elements operate in the heart. We first discuss the activity of the insulin receptor and the proteins with which it directly communicates (Fig. 3); we then evaluate distinct pathways for intracellular signalling which are likely to be required to explain the pleiotropic effects of insulin (Table 3).
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5.1 The insulin receptor—subunit structure and hormone binding
The structure and function of the insulin receptor has been intensively studied and new insights continue to emerge [4, 170]. The functional tetrameric receptor is comprised of two dimers, each made up of
-(135-kDa) and β-(95-kDa) subunits. The - and β-subunits are derived from a single, contiguous precursor [171, 172] by proteolytic cleavage and processing, which includes complex glycosylation. A disulfide bond (likely involving Cys-647) couples the C-terminal domain of each -subunit to the small exofacial domain of the corresponding partner β-subunit [1, 170]; further disulfide bonds also form between the cysteine-rich domains of the two -subunits of each intact receptor, so that the disulfide bridging pattern of each tetramer is β–––β [1, 4, 170]. Once the receptor is assembled in the plasma membrane, the -subunits are entirely extracellular and bind insulin by a mechanism which displays apparent negative cooperativity [173]. Cross-linking studies with photo-activated insulins suggest important determinants for insulin binding are found in both the N-terminal and the C-terminal domains of the receptor -subunits [174, 175].
Among two possible receptor isoforms expressed in mammalian cells, the higher-affinity ‘A’-isoform is by far the predominant form expressed in the heart, contributing more than 95% of the total [176]. The A-isoform of the insulin receptor differs from the alternate B isoform because of differential splicing of a common mRNA which may lead to the deletion (in isoform A) or inclusion (in isoform B) of exon 11 and the corresponding 12-amino-acid residues beginning at position 717 of the -subunit C-terminus. Alterations in expression of the receptor isoforms has been reported in skeletal muscle of patients with Type II diabetes, in which the B-isoform is expressed at unusually high levels [177]. The possibility that receptor isoform ‘switching’ may occur in the heart has not been explored, but could be important given the distinct properties of the isoforms; the A-isoform (-exon 11) displays approximately 2-fold higher sensitivity to insulin, but the B isoform expresses substantially higher intrinsic and insulin-stimulated protein tyrosine kinase activity [176, 178].
Determination of the abundance of functional insulin receptors in myocytes within the intact heart has been complicated by the substantial numbers of endothelial receptors and the requirement for trans-endothelial transport of the hormone to reach the surface of myocardial cells [179]. However, many in vivo and in vitro studies attest to the sensitivity of the heart to insulin over a physiological concentration range of 10–200 µU/ml, or 0.1–2 nM [97, 101, 180]. Since tissue sensitivity to insulin is dictated by receptor abundance [181] the density of functional insulin receptors in the heart is probably in the range found in other insulin-sensitive cell types (10 000–100 000 per cell).
5.2 Activation of the insulin receptor protein tyrosine kinase
The insulin receptor is a transmembrane allosteric enzyme in which insulin binding induces autophosphorylation and activation of the protein tyrosine kinase domain on the cytoplasmic aspect of the transmembrane β-subunits. The fact that limited trypsin treatment can cause receptor activation and that mutant receptors with truncated -subunits (as well as isolated kinase domains) are constitutively active suggest that the -subunits exert a tonic inhibitory effect on the protein tyrosine kinase of the β-subunit [1, 4, 170].
On the basis of experimental studies, it is evident that most aspects of insulin signalling appear to depend on a functional receptor kinase [1, 4, 170]. In addition, a number of clinical cases of severe insulin resistance have been described which appear to be accounted for by natural mutations in the insulin receptor gene which are associated with loss of receptor kinase activity [182]. A triplet of closely-spaced tyrosines (residues Y-1158, Y-1162 and Y-1163) in the receptor β-subunits become phosphorylated in the full activation of the kinase domain [183, 184]. Recent crystallographic studies of the isolated kinase subdomain reveal that the activation of the receptor kinase requires the removal of an autoinhibitory loop from the active site, associated with the autophosphorylation by the kinase domain of the partner (‘trans’) β-subunit [185]. Additional sites for tyrosyl and seryl/threonyl phosphorylation also occur in the juxtamembrane and C-terminal domains of the β-subunits, notably tyrosines 953, 960, 1328 and 1334 plus Ser/Thr residues 1272, 1305/1306 and 1327 [170]. The functional significance of these additional receptor phosphorylation sites is discussed below; it is clearly vital to establish if the sites play equivalent roles in the heart.
5.3 Intracellular proteins which interact with the insulin receptor
Among the immediate targets of the insulin receptor, the most extensively studied has been IRS-1—the ‘insulin-receptor substrate 1’ [1, 186]. Originally observed as a prominent 185-kDa phosphoprotein [187], this soluble protein becomes phosphorylated in response to insulin on at least 8 out of 20 potential tyrosine phosphorylation sites. These sites occur within Y–X–X–M motifs which are recognized by several defined proteins which contain SH2 (src-homology 2) domains. IRS-1 therefore appears to act as a nucleation site for the assembly of subsequent downstream signalling proteins or complexes (Fig. 3). Proteins with SH2 domains which associate with IRS-1 include the adaptor proteins Grb2 and Nck, the protein tyrosine phosphatase SH-PTP2/Syp and ‘p85’, the 85-kDa regulatory subunit of PI-3-kinase [188–190]. Studies of transgenic mice in which IRS-1 is ablated suggested that a related ‘IRS-2’ protein must also play a critical role in insulin actions in vivo [191]. The structure of IRS-2 has been reported but details of its functions have not yet been established [192, 193].
In addition to IRS proteins, other recognized targets of the insulin receptor exist (Fig. 3). One of the additional receptor targets for which a functional role has been established is Shc, which binds directly to activated insulin receptors and becomes tyrosine-phosphorylated in response to insulin [194]. Among several isoforms of Shc the insulin receptor may favour the 52-kDa isoform [195]. The identification of Shc isoforms, together with IRS proteins and the newly-emerging ShcB, ShcC and Grb10, shows that the range of insulin receptor targets is likely to be appreciable [196, 197]. It will be an important challenge to determine which of these receptor targets play critical roles in mediating the effects of insulin in the heart.
Signal transducing ‘adaptor’ proteins such as IRS and Shc can form cross-links between two or more proteins because they contain multiple interaction domains. In IRS and Shc, for example, the PTB domains contact the insulin receptor juxtamembrane domain while SH2 binding sites can be generated within these adaptors when they become tyrosine-phosphorylated. Shc may additionally attract SH3-containing proteins because of its proline-rich motifs. The 85-kDa subunit of PI-3-kinase [198] or the tyrosine phosphatase Syp [199] may also serve to cross-link IRS-1 with the insulin receptor because they each have two SH2 domains. An important consequence of the multiple protein–protein contacts described is the possibility of the formation of multimolecular signalling complexes at the plasma membrane. These complexes might pre-exist in a non-active conformation in the absence of hormone stimulation or they might assemble only upon binding of hormone to the receptor.
5.4 IRS proteins and multiple metabolic signalling pathways
Reluctantly, we must concede that our understanding of the signal transmission pathways which mediate the metabolic effects of insulin are poorly understood even in muscle, fat and liver. In particular, it is becoming evident that several routes for metabolic signal transmission can be discriminated, so that reagents which interfere with certain insulin actions do not necessarily affect others. The complexity of the array of proteins which can interact at the plasma membrane with the insulin receptor (directly, or in larger complexes) may indeed be giving us a foresight of the complexity of post-receptor pathways.
Considerable interest has been generated by studies employing wortmannin and other agents (including LY294002) which profoundly inhibit many metabolic effects of insulin, including glucose transport, glycogen synthesis, lipogenesis and antilipolysis [200–202]. Wortmannin potently inhibits PI-3-kinase, though it is not completely selective. Studies in which cells have been transfected with constitutively active or dominant negative mutants of PI-3-kinase confirm that the stimulation of GLUT-4 is strictly dependent on PI-3-kinase itself; in contrast, other insulin responses including activation of glycogen synthesis and of p70-S6 kinases involve wortmannin-sensitive steps distinct from PI-3-kinase [201, 202].
Events downstream from PI-3-kinase are still poorly defined. PKC-
may be a cellular target of PI-3,4,5-trisphosphate, one of the major products of the PI-3-kinase reaction [203], but the consequences of activation of this PKC isoform by insulin have not been established. Another possibility is the recently-discovered protein serine/threonine kinase PKB, which is rapidly activated by insulin in fat and muscle cells [204] and may be a critical mediator of insulin signals from PI-3-kinase to glycogen synthase kinase-3 [205]. The activation of PKB is dependent upon its phosphorylation [206], perhaps by PI-3-kinase which expresses protein serine as well as lipid kinase activity [207] but more likely through an intermediate ‘PKB-kinase’ [205]. Although we currently understand little of the mechanism of activation of PKB or of its major substrates, nevertheless, the expression of PKB in insulin-sensitive cells including the heart [Zhande and Brownsey, unpublished observations], its sensitivity to wortmannin, ability to influence GSK-3 and ability to stimulate GLUT-4 and lipogenesis suggest it could be critical in metabolic signalling and merits further investigation in the heart [205, 208].
5.5 Shc and the MAP kinase (ERK) signalling pathway
Shc plays a critical role in mediating the mitogenic or growth-promoting effects of insulin, IGF-1 and EGF, at least in part through activating the p21-Ras/MAP kinase (ERK) pathway [209]. Corresponding experiments with heart cells have not yet been reported, although the possibility that insulin might act as a signal for hypertrophy in some situations is discussed below (Section 6). An important SH2-containing target attracted by tyrosine-phosphorylated Shc is Grb-2 which also contains SH3 domains able to contact the proline-rich sequences of Sos [210]. While complexed with Grb2, Sos acts as a guanine nucleotide exchange factor of the p21-Ras GTPase [211]. Binding of Grb/Sos to Shc at the plasma membrane ‘receptor complex’ thus brings about the activation of p21-Ras by enhancing release of GDP and its replacement with GTP.
The activation of p21-Ras promotes the activation of a sequence of protein kinases, known as the ‘MAP kinase cascade’ [2, 212, 213]. Because the MAP kinases were initially discovered as the activity in 3T3-L1 adipocytes responsible for the insulin-stimulated phosphorylation of the microtubule-associated protein (MAP)-2 [214], this system has considerably influenced our thinking about insulin signal transduction. p21-Ras(GTP) is able to bind directly (through its N-terminal domain) to the protein serine/threonine kinase c-Raf-1 [215] as well as to a functional equivalent of Raf-1 named ‘MEKK1’ [216]. The interaction with p21-Ras leads to recruitment of Raf-1 to the plasma membrane [217], but this is not sufficient for Raf-1 activation. PKC isoforms have been shown to directly phosphorylate Raf-1 in vitro and might play a role in its activation; however, it has recently been proposed that Raf-1 activation is explained by oligomerization and association with 14-3-3 proteins [218, 219]. Consequently, insulin signalling from the receptor to Raf-1 must likely occur at the plasma membrane and further steps are required before signals may be emitted to the cytosol or beyond. Following activation, Raf-1 is then able to phosphorylate and activate MAP kinase kinases (MEKs) which are consequently activated [220]. MEK-1 and MEK-2 are dual-specificity protein kinases, able to phosphorylate threonine and tyrosine residues of the TEY motifs of their specific target MAP kinases, ERK-1 and ERK-2 [221].
The components of the response pathways for Ras-dependent and PKC-dependent activation of the MAP kinases are present and functional in the mature heart. MAP kinase function in the heart has been best studied in response to the endothelins, angiotensin and other growth-promoting stimuli [222, 223], but responsiveness to insulin has not been established in the heart.
5.6 ERK-1 and ERK-2—a major role in rapid metabolic actions of insulin?
Initial studies indicated that glycogen metabolism could be regulated through ERK activation. MAPKAP kinase-1, the mammalian 
90-kDa ribosomal protein S6 kinase (p90rsk, or RSK2) was found to be phosphorylated and activated by ERKs [2, 224] and, in turn, could phosphorylate a specific site on the glycogen-binding subunit of protein phosphatase-1 [2]. The effect of this regulatory phosphorylation was to ensure recruitment of the phosphatase to glycogen particles where it dephosphorylates its key targets to activate net glycogen synthesis [2]. Despite this attractive hypothesis and the fact that the activation of the ERKs may be inhibited by wortmannin [225, 226], increasing evidence suggests that the metabolic effects of insulin, including the activation of glycogen synthesis, are not dependent upon the classical ERK pathway [227]. For example, activation of ERKs in adipocytes and 3T3-L1 cells by EGF [228] or in PC-12 cells by NGF [229] is not accompanied by insulin-like activation of glucose transport or synthesis of glycogen or lipids. We have observed analogous uncoupling of fatty acid biosynthesis and ERK activation in mature adipose tissue exposed to differing extracellular osmolarities [230]. Finally, several metabolic responses of fat and other cells to insulin are unaffected by a specific MEK inhibitor [231, 232]. It is therefore clear that post-receptor systems other than the ERK pathway are required for metabolic signalling in response to insulin in many cell types. Since the metabolic effects of insulin are paramount in the heart, the possibility that the MAP kinase pathway may not play a significant role has important implications for future studies of insulin action in this tissue.
5.7 MAP kinase (ERK) signalling, myocardial gene expression and protein synthesis
The MAP kinase (ERK) signalling pathway plays a significant role in mediating responses associated with growth, differentiation or developmental processes [213, 233]. In many cases, these cellular responses involve changes in specific gene expression and in rates of protein synthesis. More than 50 insulin-responsive genes have been identified [234]. A much smaller number of cardiac muscle genes have been identified which are regulated by insulin (some are mentioned above) and the mechanisms involved in activation of this subset of genes have not been defined. On the basis of studies with other cell types, insulin may exert effects on gene expression by several distinct mechanisms. In some cases, genes are under ‘metabolite control’, so that changes in intracellular glucose, glucose-6-phosphate or some other metabolite may represent a critical signal for transcription factor modulation. Other genes are under the control of ‘insulin response elements’, for which consensus sequences have been identified and appropriate binding proteins (activating or repressing factors) are being sought [234]. At least two general classes of ERK-sensitive transcription factors have been identified. Transcription factors which may be directly phosphorylated by ERKs include p60TCF/Elk-1 which cooperates with the serum response factor in stimulating the induction of c-fos and AP-1 activity [235, 233]. In addition, the MAP kinase pathway may affect transcription from cyclic-AMP-response elements (CRE) through activation of p90rsk, which is able to phosphorylate the CREB (CRE-binding) proteins [236]. Finally, the ability of insulin to stimulate the serine phosphorylation of STAT3 may provide a particularly direct mechanism for insulin signalling to the nucleus [237].
As with regulation of transcription factors, effects of insulin on protein synthesis have been recognized but are incompletely defined [17, 238]. Several proteins important in regulating mRNA translation are phosphorylated in response to insulin, including the S6 protein of the 40S ribosomes, initiation factors eIF-2B and eIF-4F (including the 4A, 4- and 4-
subunits), the eIF-4E binding proteins (eIF-4E:Bp, also called ‘PHAS’) and elongation factor eEF-2. The phosphorylation of the ribosomal S6 protein is most likely accounted for by the p70/p85 S6 kinases [239], but the functional effects of phosphorylation are still not clear. The phosphorylation of the eIF-4F subunits is particularly attractive, because the 4-subunit initiates binding to the 5'-methyl guanosine ‘cap’ of mRNA and can potentially play a role in global and selective mRNA translation; this could be especially important in attempts to understand how insulin can specifically enhance translation of particular mRNA species [240]. So far, the protein kinases involved in the phosphorylation of the initiation and elongation factors have not been identified.
5.8 Summary of the impact of signalling pathways on key metabolic proteins
(i) A number of routes for signal transduction from the insulin receptor have been deduced from studies with ‘selective’ inhibitors of signalling proteins (notably wortmannin, rapamycin and MEK inhibitors) as well as the use of transgenic animals or transfected cells which express mutated signalling proteins.
(ii) The regulation of PDC stands alone, so far, among metabolic responses to insulin, being insensitive to wortmannin. Understanding signal transmission from the insulin receptor to the matrix of mitochondria remains a significant challenge. Fundamental studies of PDC kinase and especially of PDC-P phosphatase will be central to this endeavour.
(iii) The MAP kinase (ERK) pathway and its activation via Shc and/or IRS proteins is rather well defined, though the outputs from this pathway are still being identified. A major role for this pathway in mediating effects of insulin on gene transcription through phosphorylation of transcription factors such as Elk-1 and the CREBs seems likely, though the myocardial genes which might be controlled remain to be defined. ERK-mediated regulation of gene transcription might be complemented by insulin effects on the JNK pathway, but these kinase cascades appear to play a minor role, if any, in mediating rapid regulation of insulin-sensitive metabolism.
(iv) Several discrete signalling routes are inhibited by wortmannin and related inhibitors. One route, to the activation of the p70-S6 kinase, is also inhibited by rapamycin and contributes to the regulation of mRNA translation, through phosphorylation of the ribosomal protein S6 and of the initiation factor 4E-binding proteins. Other targets downstream from p70-S6 kinases may yet become apparent.
(v) The wortmannin-sensitive responses to insulin which are unaffected by rapamycin can also be sub-divided according to the species of protein inhibited by wortmannin. The activation of glucose transport by insulin is mediated specifically by the well-characterized (p85/p110) PI-3-kinase. Other insulin responses, including activation of PKB and glycogen synthase, appear to be mediated by wortmannin-sensitive proteins distinct from the established PI-3-kinase. The steps downstream from the wortmannin-sensitive proteins are still poorly defined and yet account for some of the major metabolic actions of insulin, involving the regulation of PFK-2, glycogen synthase, acetyl-CoA carboxylase, PDE-III, hormone-sensitive lipase and the appropriate modulating protein kinases and phosphatases.
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6 Clinical significance of defective insulin signalling |
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Top1 Introduction2 The context of...3 Indirect actions of...4 Direct actions of...5 Intracellular signalling...6 Clinical significance of...7 Concluding commentsReferences |
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Alterations in the availability of insulin or in the functions of its signalling pathways may be involved in a number of physiologic and pathologic processes in the heart including the changes in metabolic function of the heart in the neonatal period [241, 242], insulin resistance associated with aging [243], obesity [244], non-insulin-dependent diabetes mellitus [245], essential hypertension [245, 246] and cardiac hypertrophy [247] as well as the cardiomyopathy observed in patients with Type I and Type II diabetes mellitus [248, 249]. Several of these topics are discussed elsewhere in this issue, so here we limit our discussion to brief comments to illustrate the actions of insulin in the setting of hypertension and cardiac hypertrophy.
Basal glucose uptake is enhanced into the myocardium of patients with hypertension [250] or experimental animals with cardiac hypertrophy [247, 251]. However, insulin responses are diminished in hypertrophied hearts from spontaneously hypertensive rats [247]. These findings indicate that insulin-independent glucose uptake is accelerated and that insulin-dependent glucose uptake is impaired in hearts exposed to pressure overload. This conclusion is also supported by the decreased expression of GLUT-4 [247] and increased ratio of GLUT-1 to GLUT-4 mRNA [252] in hypertrophied hearts. Similarly, we have found that rates of glycolysis of exogenous glucose are accelerated in isolated working hypertrophied hearts from aortic-banded rats [253–255]. The observed increases in insulin-independent glucose uptake and glycolysis are consistent with the demonstration that the activity of glycolytic enzymes is increased [256, 257] and that the patterns of expression of the isoenzymes of lactate dehydrogenase [256] and enolase [242] in hypertrophied hearts reflect those seen in fetal, anaerobic hearts rather than normal adult hearts.
Accelerated rates of glycolysis of exogenous glucose in hypertrophied hearts are not accompanied by corresponding changes in glucose oxidation, and instead lead to an uncoupling between rates of glycolysis and oxidation of exogenous glucose that is significantly greater in the hypertrophied heart than the normal heart [253–255]. So far, the mechanisms responsible for the exaggerated uncoupling of glycolysis from glucose oxidation in hypertrophied hearts are not yet known. An important therapeutic point that arises from the fact that hearts exposed to a pressure overload are resistant to the metabolic effects of insulin, is that glucose–insulin–potassium infusion is unlikely to alter myocardial glucose utilization in this setting. Any benefits to myocardial metabolism of this therapeutic approach would more likely occur indirectly as a result of inhibition of adipose tissue hormone-sensitive lipase and consequent decreases in plasma concentrations of free fatty acids.
In addition to its metabolic effects, the actions of insulin on cellular gene expression (Section 5) may also contribute to cardiac myocyte growth in hypertrophy and other clinical settings. Insulin resistance in patients with essential hypertension and non-insulin-dependent diabetes mellitus is characteristically accompanied by hyperinsulinemia [245], raising the possibility that elevated levels of insulin play a role in the development of cardiac hypertrophy in these settings. Experimental observations which provide support for this concept include those showing direct effects of insulin on protein synthesis in isolated perfused hearts [258] and in cardiac myocytes [259]. Further, insulin increases the expression of cardiac hexokinase II [127] and contributes to determination of enolase isoenzyme expression [242]. These effects of insulin on glycolytic enzymes are in keeping with the recognized metabolic phenotype of hearts which exhibit hypertrophy as a result of pressure overload. Lastly, it has recently been shown that transgenic mice deficient in the GLUT-4 hexose transporter are hyperinsulinemic and develop significant cardiac hypertrophy [260].
The mechanisms by which insulin mediates its growth-related effects in the heart are not yet established, but an important clue may be provided by the recent observation that rapamycin, which inhibits activation of p70-S6 kinase, also blocks the stimulation of overall protein synthesis induced by exposure of neonatal rat ventricular myocytes to angiotensin II, an important mediator of hypertrophic growth in the heart [261]. Since the activation of p70-S6 kinase by insulin is also rapamycin-sensitive [262], it is tempting to speculate that the increased protein synthesis observed in cardiac myocytes exposed to insulin is, in part,