The metabolic syndrome

Physiologically important cell-signalling networks are complex, and contain several points of regulation, signal divergence and crosstalk with other signalling cascades. Here, we use the concept of 'critical nodes' to define the important junctions in these pathways and illustrate their unique role using insulin signalling as a model system.

Critical nodes in signalling pathways: Insights into insulin action

The "metabolic syndrome" (MetS) is a clustering of components that reflect overnutrition, sedentary lifestyles, and resultant excess adiposity. The MetS includes the clustering of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure and is associated with other comorbidities including the prothrombotic state, proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders. Because the MetS is a cluster of different conditions, and not a single disease, the development of multiple concurrent definitions has resulted. The prevalence of the MetS is increasing to epidemic proportions not only in the United States and the remainder of the urbanized world but also in developing nations. Most studies show that the MetS is associated with an approximate doubling of cardiovascular disease risk and a 5-fold increased risk for incident type 2 diabetes mellitus. Although it is unclear whether there is a unifying pathophysiological mechanism resulting in the MetS, abdominal adiposity and insulin resistance appear to be central to the MetS and its individual components. Lifestyle modification and weight loss should, therefore, be at the core of treating or preventing the MetS and its components. In addition, there is a general consensus that other cardiac risk factors should be aggressively managed in individuals with the MetS. Finally, in 2008 the MetS is an evolving concept that continues to be data driven and evidence based with revisions forthcoming.

The Metabolic Syndrome

Following the discovery of leptin in 1994, the scientific and clinical communities have held great hope that manipulation of the leptin axis may lead to the successful treatment of obesity. This hope is not yet dashed; however the role of the leptin axis is now being shown to be ever more complex than was first envisaged. It is now well established that leptin interacts with pathways in the central nervous system and through direct peripheral mechanisms. In this review, we consider the tissues in which leptin is synthesized and the mechanisms which mediate leptin synthesis, the structure of leptin and the knowledge gained from cloning leptin genes in aiding our understanding of the role of leptin in the periphery. The discoveries of expression of leptin receptor isotypes in a wide range of tissues in the body have encouraged investigation of leptin interactions in the periphery. Many of these interactions appear to be direct, however many are also centrally mediated. Discovery of the relative importance of the centrally mediated and peripheral interactions of leptin under different physiological states and the variations between species is beginning to show the complexity of the leptin axis. Leptin appears to have a range of roles as a growth factor in a range of cell types: as be a mediator of energy expenditure; as a permissive factor for puberty; as a signal of metabolic status and modulation between the foetus and the maternal metabolism; and perhaps importantly in all of these interactions, to also interact with other hormonal mediators and regulators of energy status and metabolism such as insulin, glucagon, the insulin-like growth factors, growth hormone and glucocorticoids. Surely, more interactions are yet to be discovered. Leptin appears to act as an endocrine and a paracrine factor and perhaps also as an autocrine factor. Although the complexity of the leptin axis indicates that it is unlikely that effective treatments for obesity will be simply derived, our improving knowledge and understanding of these complex interactions may point the way to the underlying physiology which predisposes some individuals to apparently unregulated weight gain.

Leptin: a review of its peripheral actions and interactions.

Insulin is an anabolic hormone with powerful metabolic effects. The events after insulin binds to its receptor are highly regulated and specific. Defining the key steps that lead to the specificity in insulin signaling presents a major challenge to biochemical research, but the outcome should offer new therapeutic approaches for treatment of patients suffering from insulin-resistant states, including type 2 diabetes.

The insulin receptor belongs to the large family of growth factor receptors with intrinsic tyrosine kinase activity. Following insulin binding, the receptor undergoes autophosphorylation on multiple tyrosine residues. This results in activation of the receptor kinase and tyrosine phosphorylation of a family of insulin receptor substrate (IRS) proteins. These substrates are commonly referred to as docking proteins, since several other intracellular proteins bind to the phosphorylated substrates, thereby transmitting the signal downstream. Like other growth factors, insulin uses phosphorylation and the resultant protein–protein interactions as essential tools to transmit and compartmentalize its signal. These intracellular protein–protein interactions are pivotal in transmitting the signal from the receptor to the final cellular effect, such as translocation of vesicles containing GLUT4 glucose transporters from the intracellular pool to the plasma membrane, activation of glycogen or protein synthesis, and initiation of specific gene transcription (Figure ​(Figure1).1). In this article, we review some of our current understanding about early insulin signal transduction through the network of IRS interacting proteins and the mechanisms that may modify insulin signal transduction in insulin-resistant states, especially obesity and type 2 diabetes.



Figure 1

Schematic illustration of major signaling pathways of insulin action. The phosphorylated insulin receptor binds and phosphorylates IRS proteins and Shc, which bind differentially to various downstream signaling proteins. PI3-kinase is critical for metabolic ...



Protein–protein interactions
Some of the best-characterized protein interaction domains involved in insulin signaling are the PH (pleckstrin homology), PTB (phosphotyrosine binding), SH2, and SH3 domains (1) (Table ​(Table1).1). Other, less-characterized domains (e.g., LIM, PDZ, NOTCH, and WW) may also prove to be relevant (2). These interaction domains exist in the natural tertiary structure of proteins. In other cases, the domains for interaction are created by posttranslational covalent modification of the protein. The most common examples of the latter are the effects of phosphorylation of proteins on tyrosine or serine/threonine residues and the lipid modification by prenylation or fatty acid acylation



Table 1

Classes of domains involved in protein–protein interaction in insulin signaling



Figure ​Figure22 illustrates how signal transduction is transmitted from the receptor downstream using different types of domains. PH domains, which are found in most of the proteins that interact with the insulin receptor, bind to charged headgroups of specific phosphatidylinositides and are thereby targeted preferentially to membrane structures. PH domains in the IRS proteins target the proteins to the membrane adjacent to the insulin receptor (3). PTB domains, also found in IRS proteins, recognize the phosphotyrosine in the amino acid sequence asparagine-proline-any amino acid-phosphotyrosine (NPXpY), often present in tyrosine kinase receptors, including insulin receptor (4, 5).



Figure 2

Protein–protein interaction domains involved in insulin signal transduction. A typical IRS protein contains a PH domain, which targets the protein in plasma membrane, and a PTB domain, which binds to NPXpY motif in the β subunit of the ...



Most of the intracellular partners of the insulin receptor substrates contain Src homology 2 (SH2) domains. These domains are also phosphotyrosine binding cassettes but tend to have higher binding affinity than PTB domains and recognize specific amino acid patterns, making a more rigid protein–protein interaction possible. SH2 domains consist of roughly 100 amino acids, including a highly conserved phosphotyrosine binding pocket (FLAVRES sequence) (6). The specificity of the binding is regulated by the few amino acids COOH-terminal to the phosphotyrosine. The SH2 domains of PI3-kinase recognize at least four pYMXM motifs in tyrosine-phosphorylated IRS-1. The SH2 domain of the adaptor protein Grb-2 and the SH2 domain of the phosphotyrosine phosphatase SHP-2 bind other sequences, including pYVNI, pYIDL, and pYASI sequences (1). In addition to these adaptor proteins, other SH2 proteins, such as Crk (adaptor), Nck (adaptor), Fyn (tyrosine kinase), and Csk (tyrosine kinase), bind to tyrosine residues on IRS proteins through their specific SH2 domains. SH3 domains are often present in these SH2 adaptor proteins. SH3 domains bind to proline-rich sequences with the consensus sequence PXXP with a specific helix structure and provide a link between the adaptor protein and its downstream targets or associated catalytic subunits (6).

It should be kept in mind that many of these recognition elements bind signaling proteins to a specific target, be it another protein, a membrane or cytoskeletal structure, or, as with transcription factors, a specific nucleotide sequence, with amazing accuracy. Many signaling proteins contain more than one of these domains, as well as other recognition elements. There are numerous proteins, which bind to other proteins with unknown mechanisms or undefined domains. These include the 14-3-3 proteins, the transforming protein simian virus 40 (SV40) large T antigen, Ca2+ ATPase, and β integrin, discussed in detail below.


Lessons from knockout animal models
Evaluating how insulin sensitivity affects whole body glucohomeostasis has been a major challenge for diabetes research. One approach has been to use targeted disruption of several insulin signaling proteins, such as the insulin receptor, IRS-1, and IRS-2. Targeted disruption of the insulin receptor is lethal after a few days of birth in homozygous animals, whereas heterozygous animals have almost no phenotype (161, 162). Disruption of IRS-1 results in retarded growth (due to IGF-1 resistance), but only mild insulin resistance and impaired glucose tolerance without diabetes (163, 164). Disruption of IRS-2 also leads to insulin resistance, but in this case, there is also a reduction in β-cell mass, leading to diabetes (165). Combination of the heterozygous insulin receptor knockout with the heterozygous IRS-1 knockout leads to more severe insulin resistance and a phenotype that includes a delayed onset of diabetes similar to human type 2 diabetes (166). This is a pure form of insulin resistance with elevated insulin levels. Interestingly, only about 40% of these mice develop diabetes, suggesting the importance of additional background genes.

Tissue-specific disruption of the insulin receptor gives a more detailed view of how glucose homeostasis is regulated in a tissue-specific manner in vivo. Muscle-specific disruption of the insulin receptor (MIRKO) in mice results in severe insulin resistance in isolated skeletal muscles, but at the whole body level, glucose tolerance is near normal (74). Indeed, the major metabolic disturbances found in the MIRKO mouse are increased body fat, elevated triglycerides, and slightly elevated free fatty acid levels. β cell–specific disruption of the insulin gene (βIRKO) has a more severe phenotype than the MIRKO, with severely impaired glucose tolerance and a loss of first-phase insulin secretion in the glucose tolerance test (167). These animal models demonstrate that insulin serves an important role in β-cell function and suggest that insulin resistance at the β-cell level may contribute to the altered insulin secretion in type 2 diabetes. Additional data from these and other models should help us to clarify how glucose homeostasis is regulated and define some defects in signaling proteins responsible for insulin resistance in type 2 diabetes.

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Protein–protein interaction in insulin signaling and the molecular mechanisms of insulin resistance

Decreased transport of leptin across the blood-brain barrier in rats lacking the short form of the leptin receptor

Strohl, Kingman P., Agnes J. Thomas, Pamela St. Jean, Evelyn H. Schlenker, Richard J. Koletsky, and Nicholas J. Schork.Ventilation and metabolism among rat strains. J. Appl. Physiol. 82(1): 317–323...

Ventilation and metabolism among rat strains

Objective To review previous work and present additional evidence characterizing the I1-imidazoline receptor and its role in cellular signaling, central cardiovascular control, and the treatment of metabolic syndromes. Second-generation centrally-acting antihypertensives inhibit sympathetic activity mainly via imidazoline receptors, whereas first-generation agents act viaα2-adrenergic receptors. The I1 subtype of imidazoline receptor resides in the plasma membrane and binds central antihypertensives with high affinity.

Methods and results Radioligand binding assays have characterized I1-imidazoline sites in the brainstem site of action for these agents in the rostral ventrolateral medulla. Binding affinity at I1-imidazoline sites, but not at other classes of imidazoline binding sites, correlates closely with the potency of central antihypertensive agents in animals and in human clinical trials. The antihypertensive action of systemic moxonidine is eliminated by the I1/α2-antagonist efaroxan, but not by selective blockade of α2-adrenergic receptors. Until now, the cell signaling pathway coupled to I1-imidazoline receptors was unknown. Using a model system lacking α2-adrenergic receptors (PC12 pheochromocytoma cells) we have found that moxonidine acts as an agonist at the cell level and I1-imidazoline receptor activation leads to the production of the second messenger diacylglycerol, most likely through direct activation of phosphatidylcholine-selective phospholipase C. The obese spontaneously hypertensive rat (SHR; SHROB strain) shows many of the abnormalities that cluster in human syndrome X, including elevations in blood pressure, serum lipids and insulin. SHROB and their lean SHR littermates were treated with moxonidine at 8 mg/kg per day. SHROB and SHR treated with moxonidine showed not only lowered blood pressure but also improved glucose tolerance and facilitated insulin secretion in response to a glucose load. Because α2-adrenergic agonists impair glucose tolerance, I1-imidazoline receptors may contribute to the multiple beneficial effects of moxonidine treatment.

Conclusion The I1-imidazoline receptor is a specific high-affinity binding site corresponding to a functional cell-surface receptor mediating the antihypertensive actions of moxonidine and other second-generation centrally-acting agents, and may play a role in countering insulin resistance in an animal model of metabolic syndrome X.

/pdf/the-i1-imidazoline-receptor-from-binding-site-to-therapeutic-19t50noslb.pdf

The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease.

Insulin resistance is associated with both obesity and hypertension. However, the cellular mechanisms of insulin resistance in genetic models of obese-hypertension have not been identified. The obj...

Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat

The SHROB rat is a unique strain with genetic obesity, hypertriglyceridemia, hyperinsulinemia, renal disease with proteinuria, and genetically determined hypertension, characteristics paralleling human Syndrome X. The obese phenotype results from a single homozygous recessive trait, designated faK, and is allelic with the Zucker fatty trait (fa), but of distinct origin. The faK mutation is a premature stop codon in the extracellular domain of the leptin receptor, resulting in a natural receptor knockout. The SHROB are glucose intolerant compared to heterozygous or wild-type SHR, but retain fasting euglycemia even on a high sucrose diet, suggesting that diabetes requires polygenic interaction with additional modifier genes. Insulin-stimulated phosphorylation of tyrosine residues on the insulin receptor and on the associated docking protein IRS-1 are reduced in skeletal muscle and liver compared to SHR, due mainly to diminished expression of insulin receptor and IRS-1 proteins. Despite multiple metabolic derangements and severe insulin resistance, hypertension is not exacerbated in SHROB compared to SHR. Thus, insulin resistance and hypertension are independent in this model. Increased activity of the sympathetic nervous system may be a common factor leading by separate pathways to hypertension and to insulin resistance. We studied the chronic effects of sympathetic inhibition with moxonidine on glucose metabolism in SHROB. Moxoni-dine (8 mg/kg/day), a selective I1-imidazoline receptor agonist, not only reduced blood pressure but also ameliorated glucose intolerance. Moxonidine reduced fasting insulin by 47% and plasma free fatty acids by 30%. Moxonidine enhanced expression and insulin-stimulated phosphorylation of IRS-1 in skeletal muscle by 74 and 27%, respectively. Thus, central sympatholytic therapy not only counters hypertension but also insulin resistance, glucose tolerance, and hyperlipidemia in the SHROB model of Syndrome X.

Richard J. Koletsky

Papers

Decreased transport of leptin across the blood-brain barrier in rats lacking the short form of the leptin receptor

Ventilation and metabolism among rat strains

The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease.

Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat

Molecular pathology in the obese spontaneous hypertensive Koletsky rat: a model of syndrome X.