SirT1 inhibition reduces IGF-I/IRS-2/Ras/ERK1/2 signaling and protects neurons

Sirtuins are known to protect cells and extend life span, but our previous studies indicated that S. cerevisiae Sir2 can also increase stress sensitivity and limit life-span extension. Here we provide evidence for a role of the mammalian Sir2 ortholog SirT1 in the sensitization of neurons to oxidative damage. SirT1 inhibition increased acetylation and decreased phosphorylation of IRS-2; it also reduced activation of the Ras/ERK1/2 pathway, suggesting that SirT1 may enhance IGF-I signaling in part by deacetylating IRS-2. Either the inhibition of SirT1 or of Ras/ERK1/2 was associated with resistance to oxidative damage. Markers of oxidized proteins and lipids were reduced in the brain of old SirT1-deficient mice, but the life span of the homozygote knockout mice was reduced under both normal and calorie-restricted conditions. These results are consistent with findings in S. cerevisiae and other model systems, suggesting that mammalian sirtuins can play both protective and proaging roles.     

Inhibition of PKCepsilon improves glucose-stimulated insulin secretion and reduces insulin clearance

In type 2 diabetes, pancreatic beta cells fail to secrete sufficient insulin to overcome peripheral insulin resistance. Intracellular lipid accumulation contributes to beta cell failure through poorly defined mechanisms. Here we report a role for the lipid-regulated protein kinase C isoform PKCepsilon in beta cell dysfunction. Deletion of PKCepsilon augmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCepsilon-inhibitory peptide improved insulin availability and glucose tolerance in db/db mice with preexisting diabetes. Functional ablation of PKCepsilon selectively enhanced insulin release ex vivo from diabetic or lipid-pretreated islets and optimized the glucose-regulated lipid partitioning that amplifies the secretory response. Independently, PKCepsilon deletion also augmented insulin availability by reducing both whole-body insulin clearance and insulin uptake by hepatocytes. Our findings implicate PKCepsilon in the etiology of beta cell dysfunction and highlight that enhancement of insulin availability, through separate effects on liver and beta cells, provides a rationale for inhibiting PKCepsilon to treat type 2 diabetes.     

Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production

Insulin action in the central nervous system regulates energy homeostasis and glucose metabolism. To define the insulin-responsive neurons that mediate these effects, we generated mice with selective inactivation of the insulin receptor (IR) in either pro-opiomelanocortin (POMC)- or agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus. While neither POMC- nor AgRP-restricted IR knockout mice exhibited altered energy homeostasis, insulin failed to normally suppress hepatic glucose production during euglycemic-hyperinsulinemic clamps in AgRP-IR knockout (IR^ΔAgRP) mice. These mice also exhibited reduced insulin-stimulated hepatic interleukin-6 expression and increased hepatic expression of glucose-6-phosphatase. These results directly demonstrate that insulin action in POMC and AgRP cells is not required for steady-state regulation of food intake and body weight. However, insulin action specifically in AgRP-expressing neurons does play a critical role in controlling hepatic glucose production and may provide a target for the treatment of insulin resistance in type 2 diabetes.     

Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice

Liver X receptors (LXRs) are nuclear receptors that play crucial roles in lipid metabolism in vivo and are activated by oxysterol ligands in vitro. The identity of the ligand that activates LXRs in vivo is uncertain. Here we provide two lines of evidence that oxysterols are LXR ligands in vitro and in vivo. First, overexpression of an oxysterol catabolic enzyme, cholesterol sulfotransferase, inactivates LXR signaling in several cultured mammalian cell lines but does not alter receptor response to the nonsterol agonist T0901317. Adenovirus-mediated expression of the enzyme in mice prevents dietary induction of hepatic LXR target genes by cholesterol but not by T0901317. Second, triple-knockout mice deficient in the biosynthesis of three oxysterol ligands of LXRs, 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, respond to dietary T0901317 by inducing LXR target genes in liver but show impaired responses to dietary cholesterol. We conclude that oxysterols are in vivo ligands for LXR.     

The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man

FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARα in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARα activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.     

Central melanocortin signaling restores skeletal muscle AMP-activated protein kinase phosphorylation in mice fed a high-fat diet

Little is known about the role of the central melanocortin system in the control of fuel metabolism in peripheral tissues. Skeletal muscle AMP-activated protein kinase (AMPK) is activated by leptin and serves as a master regulator of fatty acid beta-oxidation. To elucidate an unidentified role of the central melanocortin system in muscle AMPK regulation, we treated conscious, unrestrained mice intracerebroventricularly with the melanocortin agonist MT-II or the antagonist SHU9119. MT-II augmented phosphorylation of AMPK and its target acetyl-CoA carboxylase (ACC) independent of caloric intake. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the coadministration of SHU9119 or in KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat-diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin but was markedly restored by MT-II. Our data provide evidence for the critical role of the central melanocortin system in the leptin-skeletal muscle AMPK axis and highlight the system as a therapeutic target in leptin resistance.     

Dynamic functional relay between insulin receptor substrate 1 and 2 in hepatic insulin signaling during fasting and feeding

Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.     

Mode of ATM-dependent suppression of chromosome translocation

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.     

Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62

Signaling cascades that control adipogenesis are essential in the regulation of body weight and obesity. The adaptor p62 controls pathways that modulate cell differentiation. We report here that p62(-/-) mice develop mature-onset obesity, leptin resistance, as well as impaired glucose and insulin intolerance. The metabolic rate was significantly reduced in p62(-/-) nonobese mice, which displayed increased mRNA levels of PPAR-gamma and reduced levels of UCP-1 in adipose tissue. Basal activity of ERK was enhanced in fat from nonobese mutant mice. Embryo fibroblasts from p62(-/-) mice differentiated better than the wild-type controls into adipocytes, which was abrogated by pharmacological inhibition of the ERK pathway. p62 is induced during adipocyte differentiation and inhibits ERK activation by direct interaction. We propose that p62 normally antagonizes basal ERK activity and adipocyte differentiation and that its loss leads to the hyperactivation of ERK that favors adipogenesis and obesity.     

FXR signaling in metabolic disease

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to be important in controlling numerous metabolic pathways; these include roles in maintaining bile acid, lipid and glucose homeostasis, in preventing intestinal bacterial infection and gallstone formation and in modulating liver regeneration and tumorigenesis. The accumulating data suggest that FXR may be a pharmaceutical target for the treatment of certain metabolic diseases.     

Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease

Age-related disease, not aging per se, causes most morbidity in older humans. Here we report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in three mouse models. In a longevity study, median survival was increased in UCP mice (animals with skeletal muscle-specific UCP1 expression), and lymphoma was detected less frequently in UCP female mice. In apoE null mice, a vascular disease model, diet-induced atherosclerosis was decreased in UCP animals. In agouti yellow mice, a genetic obesity model, diabetes and hypertension were reversed by induction of UCP1 in skeletal muscle. Uncoupled mice had decreased adiposity, increased temperature and metabolic rate, elevated muscle SIRT and AMP kinase, and serum characterized by increased adiponectin and decreased IGF-1 and fibrinogen. Accelerating metabolism in skeletal muscle does not appear to impact aging but may delay age-related disease.     

TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes

Lymphocytes lacking the TRPM7 (transient receptor potential cation channel, subfamily M, member 7) dual function ion channel/protein kinase exhibit a unique phenotype: they are unable to proliferate in regular media, but proliferate normally in media supplemented with 10–15 mM extracellular Mg²⁺. Here, we have analyzed the molecular mechanisms underlying this phenotype. We find that upon transition from proliferation-supporting Mg²⁺-supplemented media to regular media, TRPM7-deficient cells rapidly downregulate their rate of growth, resulting in a secondary arrest in proliferation. The downregulated growth rate of transitioning cells is associated with a deactivation of signaling downstream from phosphoinositide 3-kinase, and expression of constitutively active p110 phosphoinositide 3-kinase is sufficient to support growth and proliferation of TRPM7-deficient cells in regular media. Together, these observations indicate that TRPM7 channels are required for sustained phosphoinositide 3-kinase-dependent growth signaling and therefore, that TRPM7 is positioned alongside phosphoinositide 3-kinases as a central regulator of lymphocyte growth and proliferation.