AICAR and metformin, but not exercise, increase muscle glucose transport through AMPK-, ERK-, and PDK1-dependent activation of atypical PKC.

Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.

Combined thiazolidinedione-metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle.

AIMS/HYPOTHESES: Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione-metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. METHODS: Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione-metformin therapy for activation of muscle aPKC and PKBbeta and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic-hyperinsulinaemic clamp studies conducted with sub-maximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. RESULTS: Following combined thiazolidinedione-metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBbeta, were observed. Increases in PKBbeta enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. CONCLUSIONS/INTERPRETATION: Combined thiazolidinedione-metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBbeta in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.

Phenotypic and genetic diversity of Bacillus thuringiensis strains isolated in India active against Spodoptera litura.

Bacillus thuringiensis strains isolated from different agroclimatic regions of India were found to harbor cry1 family genes. Of 831 strains 18 that were found to produce 130- and 68-kDa mol wt proteins in sodium dodecyl sulfate polyacry1amide gel electrophoresis were subjected to bioassay against second instar larvae of Spodoptera litura. According to the time response curve, while the highest toxic activity against S. litura was observed in PBT-782 with an LT50 of 25.46 h, strains PBT-372, PBT-574, PBT-801, and PBT-716 in descending order of merit had LT50 values of 36.81, 48.18, 50.35, and 73.53 h. The results of the field experiment testing the efficacy of different B. thuringiensis strains in controlling S. litura larvae infecting peanut plants showed that the chemical insecticide chlorpyriphos was the most effective in controlling S. litura throughout the study period. However, among B. thuringiensis strains, PBT-372 was superior. All the B. thuringiensis strains except PBT-689 were found to contain cry1Ac1-type gene. However, only nine strains contained cry1Aa1 gene. While cry1Ab1 was present only in PBT-372 and PBT-689, cry1Ca1 was present in PBT-574, PBT-688, PBT-689, and PBT-695. cry1Da1 was detected only in PBT-688 and PBT-692. None of the strains contained cry1Ba1 and cry1Ea1 genes. When polymerase chain reaction analysis using cry1Ca1 primer was performed, PBT-695 produced an unexpected 739-bp product, which showed 33% homology with cry1Ca1 gene between nucleotides 1819 and 2107. Our results indicated that among the field-collected B. thuringiensis strains, PBT-372 harbors multiple cry-type genes and could be employed for biological control of insects.