Leptin Reduces the Expression and Increases the Phosphorylation of the Negative Regulators of GLUT4 Traffic TBC1D1 and TBC1D4 in Muscle of ob/ob Mice

Leptin improves insulin sensitivity in skeletal muscle. Our goal was to determine whether proteins controlling GLUT4 traffic are altered by leptin deficiency and in vivo leptin administration in skeletal muscle of wild type and ob/ob mice. Leptin-deficient ob/ob mice were divided in three groups: control, leptin-treated (1 mg/kg/d) and leptin pair-fed ob/ob mice. Microarray analysis revealed that 1,546 and 1,127 genes were regulated by leptin deficiency and leptin treatment, respectively. Among these, we identified 24 genes involved in intracellular vesicle-mediated transport in ob/ob mice. TBC1 domain family, member 1 (Tbc1d1), a negative regulator of GLUT4 translocation, was up-regulated (P = 0.001) in ob/ob mice as compared to wild types. Importantly, leptin treatment reduced the transcript levels of Tbc1d1 (P<0.001) and Tbc1d4 (P = 0.004) in the leptin-treated ob/ob as compared to pair-fed ob/ob animals. In addition, phosphorylation levels of TBC1D1 and TBC1D4 were enhanced in leptin-treated ob/ob as compared to control ob/ob (P = 0.015 and P = 0.023, respectively) and pair-fed ob/ob (P = 0.036 and P = 0.034, respectively) mice. Despite similar GLUT4 protein expression in wild type and ob/ob groups a different immunolocalization of this protein was evidenced in muscle sections. Leptin treatment increased GLUT4 immunoreactivity in gastrocnemius and extensor digitorum longus sections of leptin-treated ob/ob mice. Moreover, GLUT4 protein detected in immunoprecipitates from TBC1D4 was reduced by leptin replacement compared to control ob/ob (P = 0.013) and pair-fed ob/ob (P = 0.037) mice. Our findings suggest that leptin enhances the intracellular GLUT4 transport in skeletal muscle of ob/ob animals by reducing the expression and activity of the negative regulators of GLUT4 traffic TBC1D1 and TBC1D4

Expression and functional activity of PPARγ in pancreatic β cells

1. Rosiglitazone is an agonist of peroxisome proliferator activated receptor-γ (PPARγ) and ameliorates insulin resistance in type II diabetes. In addition, it may also promote increased pancreatic β-cell viability, although it is not known whether this effect is mediated by a direct action on the β cell. We have investigated this possibility. 2. Semiquantitative real-time reverse transcription–polymerase chain reaction analysis (Taqman®) revealed that freshly isolated rat islets and the clonal β-cell line, BRIN-BD11, express PPARγ, as well as PPARα and PPARδ. The levels of expression of PPARγ were estimated by reference to adipose tissue and were found to represent approximately 60% (islets) and 30% (BRIN-BD11) of that found in freshly isolated visceral adipose tissue. Western blotting confirmed the presence of immunoreactive PPARγ in rat (and human) islets and in BRIN-BD11 cells. 3. Transfection of BRIN-BD11 cells with a PPARγ-sensitive luciferase reporter construct was used to evaluate the functional competence of the endogenous PPARγ. Luciferase activity was modestly increased by the putative endogenous ligand, 15-deoxy-Δ(12,14) prostaglandin J(2) (15dPGJ(2)). Rosiglitazone also caused activation of the luciferase reporter construct but this effect required concentrations of the drug (50–100 μM) that are beyond the expected therapeutic range. This suggests that PPARγ is relatively insensitive to activation by rosiglitazone in BRIN-BD11 cells. 4. Exposure of BRIN-BD11 cells to the lipotoxic effector, palmitate, caused a marked loss of viability. This was attenuated by treatment of the cells with either actinomycin D or cycloheximide suggesting that a pathway of programmed cell death was involved. Rosiglitazone failed to protect BRIN-BD11 cells from the toxic actions of palmitate at concentrations up to 50 μM. Similar results were obtained with a range of other PPARγ agonists. 5. Taken together, the present data suggest that, at least under in vitro conditions, thiazolidinediones do not exert direct protective effects against fatty acid-mediated cytotoxicity in pancreatic β cells