Atherosclerotic cardiovascular disease is the leading cause of death in insulin-resistant (type 2) diabetes. Vascular endothelial dysfunction paves the way for atherosclerosis through impaired nitric oxide availability, inflammation, and generation of superoxide. Surprisingly, we show that ablation of the three genes encoding isoforms of transcription factor FoxO in endothelial cells prevents atherosclerosis in Low-density lipoprotein receptor knockout mice by reversing these sub-phenotypes. Paradoxically, the atheroprotective effect of FoxO deletion is associated with a marked decrease of insulin-dependent Akt phosphorylation in endothelial cells, owing to reduced FoxO-dependent expression of the insulin receptor adaptor proteins, Irs1 and Irs2. These findings support a model in which FoxO is the shared effector of multiple atherogenic pathways in endothelial cells. FoxO ablation lowers the threshold of Akt activity required for protection from atherosclerosis. The data demonstrate that FoxO inhibition in endothelial cells has the potential to mediate wide-ranging therapeutic benefits for diabetes-associated cardiovascular disease.

Leptin is a circulating molecule for the regulation of food intake and body weight suggested to be mediated in the hypothalamus via Ob-Rb receptor, which activates Janus kinase-signal transducer and activator of transcription (STAT) pathways. Although leptin receptors exist in many regions of the brain, there have been few in vivo functional studies of leptin's target site other than the hypothalamus. We report here that peripherally applied leptin increased STAT3 phosphorylation not only in the hypothalamus but also in the brain stem as assessed by Western blotting. Moreover, administration of leptin induced expression of the suppressor of cytokine signaling 3 mRNA, a negative feedback regulator of leptin signaling, in the brain stem as well as in the hypothalamus. Using immunohistochemistry, we observed phosphorylated STAT3-immunoreactive cells in the arcuate nucleus, ventromedial hypothalamus, lateral hypothalamic area of the hypothalamus, and the nucleus of the tractus solitarius, dorsal motor nucleus of the vagus nerve, lateral parabrachial nucleus, and central gray of the brain stem of leptin-injected mice. These findings represent physiologically functional leptin Ob-Rb receptor in the brain stem as well as in the hypothalamus. It is suggested that circulating leptin may directly act in the brain stem to elicit autonomic and neuroendocrine control of food intake and energy expenditure.

Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity following weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by FoxO1. We show that FoxO1 ablation in pro–opiomelanocortin (Pomc) neurons (Pomc–Foxo1−/−) reduces food intake without affecting energy expenditure. Analyses of hypothalamic neuropeptides in Pomc–Foxo1−/− mice reveal selective increases of α–Msh and COOH–cleaved β–endorphin, the products of Carboxypeptidase E (Cpe)–dependent processing of Pomc. We show that Cpe is decreased in diet–induced obesity, and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc–Foxo1−/− mice represents a model for therapeutic intervention in obesity, and raises the possibility of targeting Cpe to develop weight loss medications.

Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, Galphaq and Galpha11 were transcribed predominantly in the brain. The highest transcriptional level of Galphaq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Galpha11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Galphaq and Galpha11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Galphaq/Galpha11 was extensively colocalized with metabotropic glutamate receptor mGluR1alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Luján et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Galphaq and/or Galpha11, leading to the activation of the intracellular effector, phospholipase Cbeta.

Insulin receptor substrate (IRS)-2؊/؊ mice develop diabetes because of insulin resistance in the liver and failure to undergo ␤-cell hyperplasia. Here we show by DNA chip microarray analysis that expression of the sterol regulatory element-binding protein (SREBP)-1 gene, a downstream target of insulin, was paradoxically increased in 16-week-old IRS-2 ؊/؊ mouse liver, where insulin-mediated intracellular signaling events were substantially attenuated. The expression of SREBP-1 downstream genes, such as the spot 14, ATP citratelyase, and fatty acid synthase genes, was also increased. Increased liver triglyceride content in IRS-2 ؊/؊ mice assures the physiological importance of SREBP-1 gene induction. IRS-2 ؊/؊ mice showed leptin resistance; low dose leptin administration, enough to reduce food intake and body weight in wild-type mice, failed to do so in IRS-2 ؊/؊ mice. Interestingly, high dose leptin administration reduced SREBP-1 expression in IRS-2 ؊/؊ mouse liver. Thus, IRS-2 gene disruption results in leptin resistance, causing an SREBP-1 gene induction, obesity, fatty liver, and diabetes.The pathogenesis of type 2 diabetes involves complex interactions among multiple physiological defects. Transgenic and knockout technology to create animal models of type 2 diabetes have had a major impact on assessment of the function of newly identified molecules implicated in the regulation of glucose homeostasis in vivo (1). The insulin receptor substrate (IRS) 1 proteins play a key role in signal transduction from the insulin receptor (reviewed in Refs. 2-4). These molecules are major intracellular phosphorylation targets of activated insulin receptor tyrosine kinase. The mammalian IRS protein family contains at least four members, ubiquitous IRS-1 (5) and IRS-2 (6), adipose tissue-predominant IRS-3 (7), and IRS-4, which are expressed in thymus, brain, and kidney (8). The physiological roles of each protein have been evaluated by gene targeting strategies. IRS-1 Ϫ/Ϫ mice are growth-retarded and insulin-resistant (9, 10) but do not develop diabetes, because an alternate substrate IRS-2 (10) or pp190 (11) compensates for the lack of IRS-1 in liver (11) and, at least in part, in skeletal muscle (12). In addition, hyperinsulinemia associated with ␤-cell hyperplasia effectively countervailed the insulin-resistant states (13). IRS-2 Ϫ/Ϫ mice, however, developed diabetes because of inadequate ␤-cell proliferation combined with liver-insulin resistance (14 -16). Mice lacking IRS-3 or IRS-4 had milder phenotypes (17, 18).Liver is a major target organ for insulin action, contributing to energy storage in the fed state by regulating catabolic and anabolic pathways. Liver-specific insulin receptor knockout mice exhibit dramatic insulin resistance (19). Insulin decreases gluconeogenic enzyme mRNAs (20) and increases lipogenic enzyme mRNAs. A transcription factor of sterol regulatory element-binding protein 1c (SREBP-1c) (21-23) or adipocyte differentiation and determination factor (24) plays a central role in insulin-mediated lipogen...