Intramyocellular accumulation of fatty acid derivatives like ceramide plays a crucial role in altering the insulin message. If short-term action of ceramide inhibits the protein kinase B (PKB/Akt), long-term action of ceramide on insulin signaling is less documented. Short-term treatment of either the C2C12 cell line or human myotubes with palmitate (ceramide precursor, 16h) or directly with ceramide (2h) induces a loss of the insulin signal through the inhibition of PKB/Akt. Extended periods
... treatment with palmitate (48h) or ceramide (16h), however, shows an inhibition of insulin signaling through increased-IRS1 serine 307 phosphorylation. The double-stranded RNA-dependent protein kinase (PKR) could play a central role to mediate long-term ceramide effects on IRS1, as recent studies showed that PKR acts as a key modulator of metabolic infl ammation, insulin sensitivity and glucose homeostasis in obesity. Here, we show that both PKR mRNA and PKR phosphorylation are increased in muscle of high fat diet fed mice compared to control mice as well as in myotubes from diabetic patients compared to human control myotubes. These results are confi rmed in vitro in both human and C2C12 myotubes in response to either palmitate or ceramide. Pre-treatment of C2C12 or human myotubes with PKR inhibitors prevents the inhibitory effect of either palmitate or ceramide on IRS1. Finally, we show that c-Jun kinase (JNK) mediates ceramide-activated PKR inhibitory action on IRS1. Altogether, our data show that ceramide inhibits effi ciently insulin signalling by targeting in a time dependent manner two important actors, e.g. PKB/Akt and IRS1 in myotubes. Type A insulin resistance (IR) syndrome is caused by genetic mutations in the insulin receptor (INSR) gene. We aimed to identify the molecular mechanism of severe IR in a 35-years-old nonobese woman who was diagnosed as having type 2 diabetes at age 31 during pregnancy. She presented acanthosis nigricans, hyperglycemia (fasting plasma glucose-173mg/dl, HbA1c-10.3%), severe hyperinsulinemia (fasting insulin-66µU/ ml), high C-peptide (2.1ng/ml), normal lipid profi le and absence of polycystic ovary syndrome. Tests for insulin and INSR antibodies were both negative. To explore the underlying molecular defect of IR, we sequenced the INSR gene. We found a 3-basepair in-frame deletion in exon 17 of one allele of the INSR, which is predicted to substitute Glutamine for Arginine and Glutamic acid (ΔArg1027 Glu1028) in the kinase domain. The same mutation was found in father's INSR, who also showed severe IR with diabetes. To clarify how the mutation impairs the receptor function, we generated plasmid vectors expressing the wild-type (WT) and ΔArg1027 Glu1028 mutants under CMV promoter and transfected them to CHO-KI cells. Blasticidine was used for selecting stable transformants. Clones with comparable (expression levels) insulin binding levels (as demonstrated by Western blot analyses) revealed by Insulin binding assays were chosen for further experiments. Scatchard analyses of insulin binding assay using 125I-insulin showed that Bmax and Kd were comparable between the two receptors. Western blot analysis revealed that the expression levels and proteolytic processing of the INSR were comparable. Treatment with insulin robustly increased autophosphorylation of WT, but not of the mutant INSR. In conclusion, we found defective autophosphorylation of the INSR with ΔArg1027 Glu1028 mutation, which might cause severe IR. Women with Type 2 diabetes (T2D) have a 49% increase in breast cancer related mortality compared with women without T2D. Epidemiological studies report that increased endogenous insulin levels and increased insulin receptor (IR) expression are associated with poor survival in breast cancer patients. We used the non-obese female MKR mouse to study the effects of hyperinsulinemia on breast cancer progression. In the MKR mice a signifi cant increase in tumor size and pulmonary metastasis is observed, compared to wild type mice. In this study, we aimed to determine the mechanisms through which hyperinsulinemia and the canonical IR signaling pathway drive tumor growth and metastasis. 100,000 MVT-1 (c-myc/vegf overexpressing) cells were injected orthotopically into 8-10 week old MKR mice. MKR mice developed signifi cantly larger MVT-1 (353.29±44mm3) tumor volumes than control mice (183.21±47mm3), p<0.05 with more numerous pulmonary metastases. Western blot and immunofl uorescent staining of primary tumors showed an increase in vimentin, an intermediate fi lament, typically expressed in cells of mesenchymal origin, and c-myc, a known transcription factor. Both vimentin and c-myc are associated with cancer metastasis. To assess if insulin and IR signaling directly affects the expression these markers, in vitro studies were performed on MVT-1 and human MCF7 cells. 10nM of insulin signifi cantly increased the expression of c-myc at 60 minutes and vimentin at 48 hours in MVT-1 cells. Silencing the IR and inhibiting IR signaling decreased c-myc expression in both cell lines. These results imply that the hyperinsulinemia may drive tumor growth and metastasis through the IR by increasing vimentin and c-myc expression. These observations allow us to begin to understand the different key players that contribute to tumor progression in the setting of T2D. These downstream elements could open the fi eld to new targets for therapy to improve survival in women with breast cancer and hyperinsulinemia. Xenin-25 is a peptide co-secreted with glucose-dependent insulinotropic polypeptide (GIP) from intestinal K-cells following a meal. Xenin-25 is believed to play a role in glucose homeostasis and possibly potentiate the biological action of GIP. We have investigated the effects of sub-chronic administration of a longer-acting xenin-25 analogue, xenin-25[Lys 13 PAL], in mice fed a high fat (45%) diet. Initial acute studies confi rmed the superior persistent glucose lowering (p<0.05) and insulin releasing (p<0.05) actions of xenin-25[Lys 13 PAL] compared to native xenin-25. Twice daily intraperitoneal injection of xenin-25[Lys 13 PAL] for 14 days had no signifi cant effect on energy intake or body weight. Circulating plasma glucose and insulin levels were also unchanged. However, on day 14, overall plasma glucose levels during a glucose tolerance and oral nutrient challenge were signifi cantly (p<0.05) lowered by xenin-25[Lys 13 PAL] treatment. These changes were accompanied by signifi cant enhancement of intraperitoneal (p<0.05) and oral (p<0.001) nutrient-stimulated insulin concentrations when compared to controls. Moreover, xenin-25[Lys 13 PAL] treated high fat mice had markedly improved insulinotropic (p<0.01) and glucose-lowering (p<0.01) actions in response to exogenous GIP administration on day 14 when compared to saline controls. However, no appreciable change in insulin sensitivity was observed with xenin-25[Lys 13 PAL] treatment. Finally, ambulatory activity was signifi cantly (p<0.05 to p<0.001) increased during the dark phase in xenin-25[Lys 13 PAL] mice compared to controls. These data indicate that sustained administration of a stable analogue of xenin-25 exerts a spectrum of benefi cial metabolic effects in high fat fed mice. This demonstrates the utility of long-acting analogues of xenin-25 as novel treatments for type 2 diabetes.