UC San Diego

A calorie-restricted diet robustly improves skeletal muscle insulin sensitivity and is a cornerstone lifestyle intervention for preventing and treating clinical hyperglycemia. While studies demonstrate that insulin- stimulated activation of phosphatidylinositol 3-kinase (PI3K) and RAC-beta serine/threonine-protein kinase (Akt) are fundamental to enhanced muscle insulin sensitivity after calorie restriction (CR), the downstream signaling steps remain to be fully elucidated. We have previously demonstrated that the lysine acetyltransferases, E1A binding protein (p300) or cAMP response element binding protein binding protein (CBP) are required for insulin-stimulated glucose uptake by skeletal muscle, at both sub-maximal and maximal concentrations of insulin. Moreover, p300/CBP are phosphorylated by Akt, which increases their acetyltransferase activity. Considering these findings together, the objective of this project was to determine whether p300 or CBP are required for the ability of CR to enhance skeletal muscle insulin sensitivity. We hypothesized that combined inhibition of p300 and CBP acetyltransferase activity would prevent CR-induced enhancement of skeletal muscle insulin sensitivity. To address this objective, 8-week-old male C57BL/6NJ mice were either fed ad libitum (AL) or were fed a CR diet (60% of AL intake) for 21 days. Then, an ex vivo [3H]-2-deoxyglucose (2DOG) approach was used to assess basal and insulin-stimulated (60 μU/mL [0.36 nM]) 2DOG uptake in paired extensor digitorum longus (EDL) and soleus from fasted (4 h) mice. To define the role of p300/CBP acetyltransferase activity, paired muscles from AL- or CR-fed mice were pre-incubated for 60 minutes with either Vehicle (DMSO; [AL-Vehicle, CR-Vehicle]) or the p300/CBP acetyltransferase activity inhibitor, iP300w (25mM; [AL-iP300w, CR-iP300w]). As expected, insulin-stimulated 2DOG uptake (i.e. Insulin 2DOG minus basal 2DOG) in Vehicle incubated muscles was ~2.9-fold and ~2.0-fold higher in the soleus and EDL, respectively, from CR- vs. AL-fed mice. Remarkably, iP300w not only blocked insulin-stimulated 2DOG uptake in AL-fed mice, but it also abrogated the insulin-sensitizing effects of CR on muscle insulin sensitivity. To further define the importance of p300 and CBP to CR-induced enhancement of skeletal muscle insulin sensitivity, we studied mice with tamoxifen-inducible and skeletal muscle-specific knockout of CBP and heterozygous (HZ) loss of p300 (referred to as, i-mPZ/mCKO) or knockout of p300 and HZ loss of CBP (referred to as, i-mCZ/mPKO). In these mice, the AL diet or 21 days of CR was initiated 3 weeks after ~10 week-old mice were dosed with tamoxifen (5 consecutive days, 2 mg/day); Cre recombinase-negative littermates, who were also dosed with tamoxifen, were the experimental controls (referred to as wildtype [WT]). Interestingly, oral glucose tolerance and EDL insulin sensitivity were comparably enhanced by CR in WT and both i-mPZ/mCKO and i-mCZ/mPKO mice, thus suggesting that just one allele of p300 or CBP is sufficient for CR to enhance skeletal muscle insulin sensitivity. Taken together, these results demonstrate that p300 or CBP are essential for the ability of calorie restriction to enhance skeletal muscle insulin sensitivity.