GSK137647 chemical information metabolite concentrations were calculated as the ratio among every single metabolite in the sample and the normal compounds.Final results are expressed as the imply common mistake. Statistical significance was assessed with ANOVA and, when acceptable, Student’s 
t test. A P value of < 0.05 was considered statistically significant.The effect of ER stress on AMPK activity was investigated. When MIN6 cells were exposed to tunicamycin, AMPK activity, indicated by levels of p-AMPK and p-ACC, was significantly inhibited (Fig 1A). As mentioned previously, C/EBP expression in ordinary conditions is minimal, but was significantly enhanced by ER stress in pancreatic beta cells concomitant with increased levels of p-eIF2 and CHOP (Fig 1A) [5]. We examined the possibility that C/EBP, the expression of which is enhanced by ER stress, affects AMPK activity. Overexpression of empty vector in MIN6 cells did not affect the expression of C/EBP and ER stress markers (Fig 1B upper left) and the efficiency of transient transfection was dependent on the amount of C/ EBP DNA used for transfection (Fig 1B, lower left). Overexpression of C/EBP in MIN6 cells also caused a significant reduction of AMPK activity, as indicated by the decreased phosphorylation of AMPK and ACC (Fig 1B, middle and right). We established pancreatic beta cell-specific C/EBP TG mice, as previously described [5]. The TG mice did not exceed glucose levels of about 200 mg/dL, indicating that the effect of hyperglycemia remained minimal [16]. Pancreatic islets isolated from TG mice showed significantly decreased AMPK phosphorylation. Taken together, we believe this findings indicate that the reduced AMPK phosphorylation was caused not by hyperglycemia, but rather by accumulation of C/EBP (Fig 1C). To elucidate the link between AMPK activity and C/EBP, we evaluated AMP and ATP levels by metabolomic analysis. Compared with wild-type, islets from the TG group had significantly lower AMP levels, but no significant change in ATP levels (Fig 1D). As a result, the AMP/ATP ratio was significantly lowered. Thus, when pancreatic beta cells are exposed to ER stress, C/EBP accumulates, lowering the AMP/ATP ratio and AMPK activity.We next examined the effect of AMPK activity on C/EBP expression levels in pancreatic beta cells. MIN6 cells were transfected with a C/EBP expression vector and subsequently stimulated with 5-amino-1--D-ribofuranosyl-imidazole-4-carboxamide (AICAR). The elevated AMPK activity was accompanied by a decrease in C/EBP expression (Fig 2A). Similarly, metformin also reduced C/EBP expression (Fig 2B). Next, the effect of AMPK on C/EBP Fig 1. Accumulation of C/EBP due to ER stress lowers AMPK activity. A: MIN6 cells were treated with 1 g/mL tunicamycin (Tm) for 24 h and analyzed with the indicated antibodies (left). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). B: MIN6 cells were transfected with C/ EBP expression vectors or empty vector. The cells were analyzed with the indicated antibodies (left and middle). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). C: Isolated islets from wild-type (WT) and TG mice at 12 weeks of age were analyzed with the indicated antibodies(left). Quantitation of the16885432 AMPK phosphorylation level is normalized to total AMPK (right).