Luciano Rossetti

Endogenous glucose production is inhibited by the adipose-derived protein Acrp30

Intraperitoneal injection of purified recombinant Acrp30 lowers glucose levels in mice. To gain insight into the mechanism(s) of this hypoglycemic effect, purified recombinant Acrp30 was infused in conscious mice during a pancreatic euglycemic clamp. In the presence of physiological hyperinsulinemia, this treatment increased circulating Acrp30 levels by approximately twofold and stimulated glucose metabolism. The effect of Acrp30 on in vivo insulin action was completely accounted for by a 65% reduction in the rate of glucose production. Similarly, glucose flux through glucose-6-phosphatase (G6Pase) decreased with Acrp30, whereas the activity of the direct pathway of glucose-6-phosphate biosynthesis, an index of hepatic glucose phosphorylation, increased significantly. Acrp30 did not affect the rates of glucose uptake, glycolysis, or glycogen synthesis. These results indicate that an acute increase in circulating Acrp30 levels lowers hepatic glucose production without affecting peripheral glucose uptake. Hepatic expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and G6Pase mRNAs was reduced by more than 50% following Acrp30 infusion compared with vehicle infusion. Thus, a moderate rise in circulating levels of the adipose-derived protein Acrp30 inhibits both the expression of hepatic gluconeogenic enzymes and the rate of endogenous glucose production.

A nutrient-sensing pathway regulates leptin gene expression in muscle and fat

Leptin, the protein encoded by the obese (ob) gene, is synthesized and released in response to increased energy storage in adipose tissue. However, it is still not known how incoming energy is sensed and transduced into increased expression of the ob gene. The hexosamine biosynthetic pathway is a cellular ‘sensor’ of energy availability and mediates the effects of glucose on the expression of several gene products. Here we provide evidence for rapid activation of ob gene expression in skeletal muscle by glucosamine. Increased tissue concentrations of the end product of the hexosamine biosynthetic pathway, UDP-N-acetylglucosamine (UDP-GlcNAc), result in rapid and marked increases in leptin messenger RNA and protein levels (although these levels were much lower than those in fat). Plasma leptin levels and leptin mRNA and protein levels in adipose tissue also increase. Most important, stimulation of leptin synthesis is reproduced by either hyperglycaemia or hyperlipidaemia, which also increase tissue levels of UDP-N-acetylglucosamine in conscious rodents. Finally, incubation of 3T3-L1 pre-adipocytes and L6 myocytes with glucosamine rapidly induces ob gene expression. Our findings are the first evidence of inducible leptin expression in skeletal muscle and unveil an important biochemical link between increased availability of nutrients and leptin expression.

Hypothalamic insulin signaling is required for inhibition of glucose production.

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.

Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells

In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemia-induced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or INO-1001. Elevated glucose increased poly(ADP-ribosyl)ation of GAPDH in WT aortae, but not in the aortae from PARP-1-deficient mice. Thus, inhibition of PARP blocks hyperglycemia-induced activation of multiple pathways of vascular damage.

Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats

We investigated the role of hypothalamic insulin signaling in the regulation of energy balance and insulin action in rats through selective decreases in insulin receptor expression in discrete hypothalamic nuclei. We generated an antisense oligodeoxynucleotide directed against the insulin receptor precursor protein and administered this directly into the third cerebral ventricle. Immunostaining of rat brains after 7-day administration of the oligodeoxynucleotide showed a selective decrease of insulin receptor protein within cells in the medial portion of the arcuate nucleus (decreased by ∼80% as compared to rats treated with a control oligodeoxynucleotide). Insulin receptors in other hypothalamic and extra-hypothalamic areas were not affected. This selective decrease in hypothalamic insulin receptor protein was accompanied by rapid onset of hyperphagia and increased fat mass. During insulin-clamp studies, physiological hyperinsulinemia decreased glucose production by 55% in rats treated with control oligodeoxynucleotides but by only 25% in rats treated with insulin receptor antisense oligodeoxynucleotides. Thus, insulin receptors in discrete areas of the hypothalamus have a physiological role in the control of food intake, fat mass and hepatic action of insulin.

Mice Lacking Adiponectin Show Decreased Hepatic Insulin Sensitivity and Reduced Responsiveness to Peroxisome Proliferator-activated Receptor γ Agonists

The adipose tissue-derived hormone adiponectin improves insulin sensitivity and its circulating levels are decreased in obesity-induced insulin resistance. Here, we report the generation of a mouse line with a genomic disruption of the adiponectin locus. We aimed to identify whether these mice develop insulin resistance and which are the primary target tissues affected in this model. Using euglycemic/insulin clamp studies, we demonstrate that these mice display severe hepatic but not peripheral insulin resistance. Furthermore, we wanted to test whether the lack of adiponectin magnifies the impairments of glucose homeostasis in the context of a dietary challenge. When exposed to high fat diet, adiponectin null mice rapidly develop glucose intolerance. Specific PPARγ agonists such as thiazolidinediones (TZDs) improve insulin sensitivity by mechanisms largely unknown. Circulating adiponectin levels are significantly up-regulated in vivo upon activation of PPARγ. Both TZDs and adiponectin have been shown to activate AMP-activated protein kinase (AMPK) in the same target tissues. We wanted to address whether the ability of TZDs to improve glucose tolerance is dependent on adiponectin and whether this improvement involved AMPK activation. We demonstrate that the ability of PPARγ agonists to improve glucose tolerance in ob/ob mice lacking adiponectin is diminished. Adiponectin is required for the activation of AMPK upon TZD administration in both liver and muscle. In summary, adiponectin is an important contributor to PPARγ-mediated improvements in glucose tolerance through mechanisms that involve the activation of the AMPK pathway.

Hypothalamic K(ATP) channels control hepatic glucose production.

Obesity is the driving force behind the worldwide increase in the prevalence of type 2 diabetes mellitus. Hyperglycaemia is a hallmark of diabetes and is largely due to increased hepatic gluconeogenesis. The medial hypothalamus is a major integrator of nutritional and hormonal signals, which play pivotal roles not only in the regulation of energy balance but also in the modulation of liver glucose output. Bidirectional changes in hypothalamic insulin signalling therefore result in parallel changes in both energy balance and glucose metabolism. Here we show that activation of ATP-sensitive potassium (KATP) channels in the mediobasal hypothalamus is sufficient to lower blood glucose levels through inhibition of hepatic gluconeogenesis. Finally, the infusion of a KATP blocker within the mediobasal hypothalamus, or the surgical resection of the hepatic branch of the vagus nerve, negates the effects of central insulin and halves the effects of systemic insulin on hepatic glucose production. Consistent with these results, mice lacking the SUR1 subunit of the KATP channel are resistant to the inhibitory action of insulin on gluconeogenesis. These findings suggest that activation of hypothalamic KATP channels normally restrains hepatic gluconeogenesis, and that any alteration within this central nervous system/liver circuit can contribute to diabetic hyperglycaemia.

SirT1 Gain of Function Increases Energy Efficiency and Prevents Diabetes in Mice

In yeast, worms, and flies, an extra copy of the gene encoding the Sirtuin Sir2 increases metabolic efficiency, as does administration of polyphenols like resveratrol, thought to act through Sirtuins. But evidence that Sirtuin gain of function results in increased metabolic efficiency in mammals is limited. We generated transgenic mice with moderate overexpression of SirT1, designed to mimic the Sirtuin gain of function that improves metabolism in C. elegans. These mice exhibit normal insulin sensitivity but decreased food intake and locomotor activity, resulting in decreased energy expenditure. However, in various models of insulin resistance and diabetes, SirT1 transgenics display improved glucose tolerance due to decreased hepatic glucose production and increased adiponectin levels, without changes in body weight or composition. We conclude that SirT1 gain of function primes the organism for metabolic adaptation to insulin resistance, increasing hepatic insulin sensitivity and decreasing whole-body energy requirements. These findings have important implications for Sirtuin-based therapies in humans.

Adipose-derived resistin and gut-derived resistin-like molecule–β selectively impair insulin action on glucose production

The adipose-derived hormone resistin is postulated to link obesity to insulin resistance and diabetes. Here, the infusion of either resistin or the resistin-like molecule-beta (RELMbeta) rapidly induced severe hepatic but not peripheral insulin resistance. In the presence of physiologic hyperinsulinemia, the infusion of purified recombinant resistin, increasing circulating resistin levels by approximately twofold to 15-fold, inhibited glucose metabolism such that lower rates of glucose infusion were required to maintain the plasma glucose concentration at basal levels. The effects of resistin and RELMbeta on in vivo insulin action were completely accounted for by a marked increase in the rate of glucose production. These results support the notion that a novel family of fat- and gut-derived circulating proteins modulates hepatic insulin action.

Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo β-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.