Nucleotide-microarray technology, which allows the simultaneous measurement of the expression of tens of thousands of genes, has become an important tool in the study of disease. In disorders such as malignancy, gene expression often undergoes broad changes of sizable magnitude, whereas in many common multifactorial diseases, such as diabetes, obesity, and atherosclerosis, the changes in gene expression are modest. In the latter circumstance, it is therefore challenging to distinguish the truly changing from non-changing genes, especially because statistical significance must be considered in the context of multiple hypothesis testing. Here, we present a balanced probability analysis (BPA), which provides the biologist with an approach to interpret results in the context of the total number of genes truly differentially expressed and false discovery and false negative rates for the list of genes reaching any significance threshold. In situations where the changes are of modest magnitude, sole consideration of the false discovery rate can result in poor power to detect genes truly differentially expressed. Concomitant analysis of the rate of truly differentially expressed genes not identified, i.e., the false negative rate, allows balancing of the two error rates and a more thorough insight into the data. To this end, we have developed a unique, model-based procedure for the estimation of false negative rates, which allows application of BPA to real data in which changes are modest.

The insulin resistance syndrome refers to a constellation of findings, including glucose intolerance, obesity, dyslipidemia, and hypertension, that promote the development of type 2 diabetes, cardiovascular disease, cancer, and other disorders. Defining the pathophysiological links between insulin resistance, the insulin resistance syndrome, and its sequelae is critical to understanding and treating these disorders. Over the past decade, two approaches have provided important insights into how changes in insulin signaling produce the spectrum of phenotypes associated with insulin resistance. First, studies using tissue-specific knockouts or tissue-specific reconstitution of the insulin receptor in vivo in mice have enabled us to deconstruct the insulin resistance syndromes by dissecting the contributions of different tissues to the insulin-resistant state. Second, in vivo and in vitro studies of the complex network of insulin signaling have provided insight into how insulin resistance can develop in some pathways whereas insulin sensitivity is maintained in others. These data, taken together, give us a framework for understanding the relationship between insulin resistance and the insulin resistance syndromes.

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.

OBJECTIVE: This study characterized the role of insulin receptors and resistance on vascular endothelial growth factor (VEGF) expression and myocardial vascularization in physiological conditions and after ischemia. METHODS AND RESULTS: Cardiac microvascular density was reduced by 30% in insulin-resistant Zucker fatty rats versus lean controls. This was associated with a parallel 40% inhibition of insulin-stimulated activation of both Akt and VEGF expression in the myocardium and cardiomyocytes. In contrast, the activation of Erk1/2 by insulin remained unchanged. In cultured cardiomyocytes, insulin or insulin-like growth factor (IGF)-1 increased VEGF mRNA and protein expression by 2-fold. Inhibition of PI3K/Akt, especially Akt2-mediated cascades but not the Ras/MEK/Erk pathway, using chemical inhibitors, dominant negative adenoviral constructs, or siRNA approaches suppressed VEGF mRNA expression by insulin. Ventricular tissues from muscle insulin receptor knockout (MIRKO) mice, which lack insulin receptors in the myocardium, have significant reductions in insulin but not IGF-1 signaling, VEGF expression, and vascular density before and after ischemia versus controls. CONCLUSIONS: Insulin regulates VEGF gene expression and vascularization in the myocardium specifically via insulin receptors and the activation of PI3K/Akt pathway. Selective inhibition of this pathway may lead to the decreases in VEGF expression and capillary density in the myocardium of patients with insulin resistance.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.

The phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin on liver. Here, we show that mice with a liver-specific deletion of the p85alpha regulatory subunit of PI3K (L-Pik3r1KO) exhibit a paradoxical improvement of hepatic and peripheral insulin sensitivity. Although PI3K enzymatic activity is diminished in L-Pik3r1KO livers because of a reduced level of regulatory and catalytic subunits of PI3K, insulin-stimulated Akt activity is actually increased. This increased Akt activity correlates with increased phosphatidylinositol (3,4,5)-trisphosphate levels which are due, at least in part, to diminished activity of the (3,4,5)-trisphosphate phosphatase PTEN. Thus, the regulatory subunit p85alpha is a critical modulator of insulin sensitivity in vivo not only because of its effects on PI3K activation, but also as a regulator of PTEN activity.

Stearoyl-CoA desaturase (SCD)1 catalyzes the rate-limiting reaction of monounsaturated fatty acid (MUFA) synthesis and plays an important role in the development of obesity. SCD1 is suppressed by leptin but induced by insulin. We have used animal models to dissect the effects of these hormones on SCD1. In the first model, leptin-deficient ob/ob mice were treated with either leptin alone or with both leptin and insulin to prevent the leptin-mediated fall in insulin. In the second model, mice with a liver-specific knockout of the insulin receptor (LIRKO) and their littermate controls (LOXs) were treated with leptin. As expected, leptin decreased SCD1 transcript, protein, and activity by >60% in ob/ob and LOX mice. However, the effects of leptin were not diminished by the continued presence of hyperinsulinemia in ob/ob mice treated with both leptin and insulin or the absence of insulin signaling in LIRKO mice. Furthermore, genetic knockout of sterol regulatory element-binding protein (SREBP)-1c, the lipogenic transcription factor that mediates the effects of insulin on SCD1, also had no effect on the ability of leptin to decrease either SCD1 transcript or activity. Thus, the effect of leptin on SCD1 in liver is independent of insulin and SREBP-1c, and leptin, rather than insulin, is the major regulator of hepatic MUFA synthesis in obesity-linked diabetes.

Although the class I(A) phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin, its mechanism of action is not well understood. To identify the role of the PI3K pathway in insulin regulation of hepatic function, we ablated the expression of both major regulatory subunits of PI3K by crossing mice lacking Pik3r1 in liver with Pik3r2 null mice, creating liver-specific double knockout mice (L-p85DKO). L-p85DKO mice failed to activate PI3K or generate PIP(3) upon insulin stimulation or activate its two major effectors, Akt and PKClambda/xi. Decreased Akt activation resulted in increased gluconeogenic gene expression, impaired glucose tolerance, and hyperinsulinemia, while the defective activation of PKClambda/xi by insulin was associated with hypolipidemia and decreased transcription of SREBP-1c. These data indicate that the PI3K pathway is critical for insulin's actions in the liver in vivo, and that differential regulation by Akt and PKClambda/xi differentially defines specific actions of insulin and PI3K on hepatic glucose and lipid metabolism.

An appropriate beta cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of beta cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in beta cells (betaDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, betaDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old betaDKO mice manifest reduced beta cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised beta cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating beta cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I-dependent pathways are not critical for development of beta cells but that a loss of action of these hormones in beta cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in beta cells might protect against type 2 diabetes.

Obesity, especially central obesity, is a hereditable trait associated with a high risk for development of diabetes and metabolic disorders. Combined gene expression analysis of adipocyte- and preadipocyte-containing fractions from intraabdominal and subcutaneous adipose tissue of mice revealed coordinated depot-specific differences in expression of multiple genes involved in embryonic development and pattern specification. These differences were intrinsic and persisted during in vitro culture and differentiation. Similar depot-specific differences in expression of developmental genes were observed in human subcutaneous versus visceral adipose tissue. Furthermore, in humans, several genes exhibited changes in expression that correlated closely with body mass index and/or waist/hip ratio. Together, these data suggest that genetically programmed developmental differences in adipocytes and their precursors in different regions of the body play an important role in obesity, body fat distribution, and potential functional differences between internal and subcutaneous adipose tissue.