Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, ␤ cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. ␤ cell-and hepatocytespecific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/ enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a ␤ cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in ␤ cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the ␤ cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both ␤ cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Liraglutide, with diet and exercise, maintained weight loss achieved by caloric restriction and induced further weight loss over 56 weeks. Improvements in some cardiovascular disease-risk factors were also observed. Liraglutide, prescribed as 3.0 mg per day, holds promise for improving the maintenance of lost weight.

Weight gain induced by an energy-dense diet is hypothesized to arise in part from defects in the neuronal response to circulating adiposity negative feedback signals, such as insulin. Peripheral tissue insulin resistance involves cellular inflammatory responses thought to be invoked by excess lipid. Therefore, we sought to determine whether similar signaling pathways are activated in the brain of rats fed a high-fat (HF) diet. The ability of intracerebroventricular (icv) insulin to reduce food intake and activate hypothalamic signal transduction is attenuated in HF-fed compared with low-fat (LF)-fed rats. This effect was accompanied by both hypothalamic accumulation of palmitoyl- and stearoyl-CoA and activation of a marker of inflammatory signaling, inhibitor of kappaB kinase-beta (IKKbeta). Hypothalamic insulin resistance and inflammation were observed with icv palmitate infusion or HF feeding independent of excess caloric intake. Last, we observed that central IKKbeta inhibition reduced food intake and was associated with increased hypothalamic insulin sensitivity in rats fed a HF but not a LF diet. These data collectively support a model of diet-induced obesity whereby dietary fat, not excess calories, induces hypothalamic insulin resistance by increasing the content of saturated acyl-CoA species and activating local inflammatory signals, which result in a failure to appropriately regulate food intake.

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In peripheral tissues, insulin signaling involves activation of the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K) enzyme system. In the hypothalamus, insulin functions with leptin as an afferent adiposity signal important for the regulation of body fat stores and hepatic glucose metabolism. To test the hypothesis that hypothalamic insulin action involves intracellular PI3K signaling, we used histochemical and biochemical methods to determine the effect of insulin on hypothalamic IRS-PI3K activity. Here, we report that insulin induces tyrosine phosphorylation of the insulin receptor and IRS-1 and -2, increases binding of activated IRS-1 and -2 to the regulatory subunit of PI3K, and activates protein kinase B/Akt, a downstream target of PI3K. Using an immunohistochemical technique to detect PI 3,4,5-triphosphate, the main product of PI3K activity, we further demonstrate that in the arcuate nucleus, insulin-induced PI3K activity occurs preferentially within cells that contain IRS-2. Finally, we show that the food intake-lowering effects of insulin are reversed by intracerebroventricular infusion of either of two PI3K inhibitors at doses that have no independent feeding effects. These findings support the hypothesis that the IRS-PI3K pathway is a mediator of insulin action in the arcuate nucleus and, combined with recent evidence that leptin activates PI3K signaling in the hypothalamus, provide a plausible mechanism for neuronal cross-talk between insulin and leptin signaling. Diabetes 52:227-231, 2003 I n peripheral tissues (e.g., liver, muscle, and fat), the binding of insulin to its receptor stimulates autophosphorylation and activation of the insulin receptor intrinsic tyrosine kinase (1), leading to the recruitment and phosphorylation of members of the insulin receptor substrate (IRS) protein family (IRS1-4) (1). Tyrosine-phosphorylated IRS proteins, in turn, bind src homology 2 domain-containing signaling proteins, such as the p85 regulatory subunit of the type IA phosphatidylinositol 3-kinase (PI3K) (1), which activates the p110 catalytic subunit of PI3K. Activated PI3K rapidly mediates the phosphorylation of PI 4,5-biphosphate to PI 3,4,5-trisphoshpate (PIP 3 ) (2), a key signaling intermediate that recruits and activates downstream molecules, including serine-threonine kinases, tyrosine kinases, GTPases, and others (1). One key downstream target of PI3K is protein kinase B (PKB)/Akt, which is activated via serine and threonine phosphorylation (1).Interestingly, homologues of the insulin receptor, IRS, PI3K, and PKB/Akt proteins are present in the nervous system of evolutionarily distant organisms such as Caenorhabditis elegans (3) and Drosophila melanogaster (4), in which they play an essential role in regulation of energy balance, reproductive fitness, and longevity. That an analogous role is played by this signaling pathway in mammalian brain (5) is suggested by the expression of these proteins in hypothalamic areas, such as the arcuate nucleus, that participate in the control of e...

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To investigate whether phosphatidylinositol-3 kinase (PI3K) signaling mediates the metabolic effects of hypothalamic leptin action, adenoviral gene therapy was used to direct expression of leptin receptors to the area of the hypothalamic arcuate nucleus (ARC). This intervention markedly improved insulin sensitivity in genetically obese, leptin-receptor-deficient Koletsky (fa(k)/fa(k)) rats via a mechanism that was not dependent on reduced food intake but was attenuated by approximately 44% by third-ventricular infusion of the PI3K inhibitor LY294002. Conversely, ARC-directed expression of a constitutively active mutant of protein kinase B (PKB/Akt, an enzyme activated by PI3K) mimicked the insulin-sensitizing effect of restored hypothalamic leptin signaling in these animals, despite having no effect on food intake or body weight. These findings suggest that hypothalamic leptin signaling is an important determinant of glucose metabolism and that the underlying neuronal mechanism involves PI3K.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-␥1 (PLC-␥1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (؊͞؊) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-␥1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.Phosphoinositide-specific phospholipase C (PLC) activity catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to the second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerol. In mammalian cells, this hydrolytic activity is provoked by a substantial number of hormones and growth factors that regulate distinct PLC isozymes (1). To date, 10 PLC isozymes have been identified and classified into ␤, ␥, and ␦ subfamilies based on overall sequence similarities and the conservation of sequence motifs X and Y that together form a catalytic domain. Hormones that signal through heterotrimeric G proteins modulate the activity of PLC-␤ isozymes. The PLC-␥ isozymes uniquely contain two SH2 domains, which mediate association with receptor autophosphorylation sites, plus an SH3 domain of unclear function. Tyrosine kinasedependent signaling pathways, such as growth factors and certain oncoproteins, phosphorylate and activate PLC-␥1, which is expressed ubiquitously. T and B cell receptors use soluble tyrosine kinases to activate PLC-␥1 as well as PLC-␥2, whose expression is most abundant in the lymphoid system. The mechanism by which PLC-␦ isozymes are regulated is unclear.