The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night1–3. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and while rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes4, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism, and insulin signaling is delayed in circadian mutant mice, and both Clock5,6 and Bmal17 mutants exhibit impaired glucose tolerance, reduced insulin secretion, and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival, and synaptic vesicle assembly. Remarkably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective β-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the β-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger onset of diabetes mellitus.

In muscle cells, insulin elicits recruitment of the glucose transporter GLUT4 to the plasma membrane. This process engages sequential signaling from insulin receptor substrate (IRS)-1 to phosphatidylinositol (PI) 3-kinase and the serine/threonine kinase Akt. GLUT4 translocation also requires an Akt-independent but PI 3-kinase-and Racdependent remodeling of filamentous actin. Although IRS-1 phosphorylation is often reduced in insulin-resistant states in vivo, several conditions eliciting insulin resistance in cell culture spare this early step. Here, we show that insulin-dependent Rac activation and its consequent actin remodeling were abolished upon exposure of L6 myotubes beginning at doses of C2-ceramide or oxidant-producing glucose oxidase as low as 12.5 mol/l and 12.5 mU/ml, respectively. At 25 mol/l and 25 mU/ml, glucose oxidase and C2-ceramide markedly reduced GLUT4 translocation and glucose uptake and lowered Akt phosphorylation on Ser473 and Thr308, yet they affected neither IRS-1 tyrosine phosphorylation nor its association with p85 and PI 3-kinase activity. Small interfering RNA-dependent Rac1 knockdown prevented actin remodeling and GLUT4 translocation but spared Akt phosphorylation, suggesting that Rac and actin remodeling do not contribute to overall Akt activation. We propose that ceramide and oxidative stress can each affect two independent arms of insulin signaling to GLUT4 at distinct steps, Rac-GTP loading and Akt phosphorylation. Diabetes 56:394 -403, 2007 I nsulin promotes dietary glucose disposal into skeletal muscle through recruitment of GLUT4-containing vesicles to the cell surface. Signaling to GLUT4 requires tyrosine phosphorylation of the insulin receptor substrate (IRS)-1 isoform, which recruits and activates phosphatidylinositol (PI) 3-kinase (1). The latter triggers activation of several serine/threonine kinases, notably Akt, which, through its substrate AS160, regulates GLUT4 vesicle mobilization to and/or fusion with the plasma membrane (2).Along with proper signaling, GLUT4 translocation and stimulation of glucose uptake require dynamic changes in the actin cytoskeleton. Insulin induces actin filament remodeling in mature skeletal muscle (3) and muscle cells in culture (4) that manifest as mesh-like structures beneath the plasma membrane. Actin filament-disrupting drugs (e.g., cytochalasin D and latrunculin B) and actin-stabilizing drugs (e.g., jasplakinolide) inhibit GLUT4 translocation and its consequent glucose uptake in muscle (4,5) and adipose (6,7) cells, as do toxins that inhibit Rho family GTPases that control actin dynamics (8). Interestingly, under these conditions, IRS-1 phosphorylation and PI 3-kinase activity remain unaffected (9 -11). In muscle cells, actin remodeling was also prevented by wortmannin or expression of a dominant-negative mutant of the p85 subunit of class I PI 3-kinase (5), but not by a dominantnegative Akt mutant (12). Hence, insulin signaling bifurcates downstream of PI 3-kinase, one arm leading to actin remodeling and another to Akt activation, bo...

Insulin causes distinct cortical actin remodeling in muscle and fat cells, and interfering with actin dynamics halts glucose transporter 4 (GLUT4) translocation to the membrane. Phosphatidylinositol 3-kinase (PI3-K) and the small G protein Rac govern myocyte actin remodeling, whereas TC10 alpha contributes to adipocyte actin dynamics downstream of Cbl-associated protein (CAP) and Cbl, independently of PI3-K. Given the importance of insulin action in both cell types, it is paramount to determine whether signaling pathways and actin manifestations are cell type specific. We found CAP expression and insulin-mediated Cbl phosphorylation in differentiated myotubes but not in myoblasts. Unlike adipocytes, Cbl is phosphorylated on Y774 and Y731 in myotubes. TC10 alpha and beta-transcripts are amplified by RT-PCR in muscle cells, but the endogenous proteins are barely detectable using two unrelated antibodies. TC10 alpha transfected into myoblasts is activated by insulin despite the lack of CAP expression and Cbl phosphorylation. Moreover, dominant-negative TC10 alpha mutants do not prevent insulin-induced actin remodeling in either myoblasts or myotubes and do not interfere with insulin-mediated recruitment of c-myc epitope-tagged GLUT4 to the cell surface. In contrast to TC10 alpha, endogenous Rac is readily detectable in both muscle cells and adipocytes and binds GTP after insulin in a PI3-K-dependent manner. These data suggest that whereas individual components of the CAP to TC10 pathway are regulated by insulin, a functional TC10-dependent signaling pathway leading to actin remodeling and GLUT4 translocation may not operate in myocytes, as it does in adipocytes.

Summary Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR-induced dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis and inflammation. Furthermore, our data identifies promising pathways for therapeutic targets to combat age-related diseases and promote health in humans.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 (CDKN2A) locus and mutations in NF-kB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-na€ ve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFb pathway activation signature.