The AMP-activated protein kinase (AMPK) is activated by exercise, and both pharmacological activation of AMPK and exercise robustly stimulate glucose uptake by skeletal muscle. Nevertheless, the molecular mechanisms underlying the increase in glucose uptake in response to these stimuli remain to be elucidated. Addressing this gap in knowledge, the goal of this study was to investigate the importance of the acetyltransferases, EA1 binding protein p300 (p300) and c-AMP response element-binding binding protein (CBP), to AMPK-mediated glucose uptake by skeletal muscle. Specifically, we used a radioactive 2-deoxyglucose (2DOG) uptake (2DOGU) approach to measure ex vivo glucose uptake in unstimulated and AMPK-stimulated (using the pharmacological activators, 5-Aminoimidazole-4-carboxamide ribonucleotide [AICAR] and MK-8772) in the extensor digitorum longus (EDL) and soleus from ~12-16 week old male and female mice (n=261) with skeletal muscle-specific knockout of p300 and/or CBP and their wildtype (WT) littermates. As expected in WT mice, AICAR and MK-8772 significantly increased 2DOGU in soleus and EDL, regardless of sex. Remarkably, in mice with knockout of p300 and CBP (i-mPCKO), this effect of AICAR and MK-8722 was blocked in male and female mice. Equally remarkable, AICAR-stimulated 2DOGU in mice with individual knockout of p300 or CBP, or AICAR- and MK8772-stimulated 2DOGU in mice with just one allele of p300 or CBP, was normal and not different from WT littermates. Taken together, we highlight a critical role for p300 and CBP in the regulation of AMPK-stimulated glucose transport in skeletal muscle.

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.

Impaired insulin-stimulated glucose uptake is a common metabolic disorder in aged and obese skeletal muscle, with this “insulin resistance” being the primary metabolic defect of type 2 diabetes. At a molecular level, skeletal muscle insulin signaling to glucose uptake is regulated by a phosphorylation-based, phosphoinositide 3-kinase (PI3K)/Akt-dependent signaling pathway. However, recent proteomic-based analysis in various tissues, including skeletal muscle, have identified more than 2000 acetylated, non-nuclear, proteins that impact a broad array of cellular processes including insulin signaling. The acetyltransferases p300 (E1A binding protein p300) and CBP (cAMP response element binding protein binding protein) are phosphorylated and activated by Akt, and p300/CBP can acetylate downstream insulin signaling and GLUT4 trafficking proteins, thus giving rise to a putative Akt-p300/CBP axis. Thus, the objective of this Dissertation was to determine the non-transcriptional importance of p300, CBP, and acetylation to skeletal muscle insulin sensitivity, for which we utilized small molecule inhibitors and muscle-specific knockout models. In Study 1, acute inhibition of deacetylases was sufficient to increase acetylation in L6 myotubes and skeletal muscle, however it did not alter insulin-stimulated glucose uptake or signaling. In Study 2, single knockout of either p300 or CBP in skeletal muscle did not impact glucose tolerance or skeletal muscle insulin action under either control, calorie restriction, or high-fat diet conditions. In Study 3, mice with combined double knockout of p300/CBP in skeletal muscle were severely glucose intolerant and skeletal muscle insulin resistant. Remarkably, this glucose intolerance and inability of skeletal muscle to respond to insulin was reversed in mice with just a single allele of either p300 or CBP. In the p300/CBP double-knockout mice, skeletal muscle insulin resistance was accompanied by significant downregulation of both mRNA and protein networks critical for insulin signaling, GLUT4 trafficking, and glucose metabolism. In summary, acutely increasing acetylation in skeletal muscle does not impact insulin stimulated glucose uptake or signaling. However, p300/CBP together are critical regulators of skeletal muscle insulin sensitivity, at least in part, by transcriptional regulation of the insulin signaling and GLUT4 trafficking pathways.

A key metabolic action of insulin is to stimulate skeletal muscle to take up glucose from the systemic circulation. This “insulin-stimulated” glucose uptake is critical during the post-prandial period (i.e. after meal ingestion) as it reduces large excursions in blood glucose concentration; in fact, skeletal muscle accounts for as much as 85% of peripheral glucose uptake after a meal, thereby making it a fundamental organ to not just glycemic control, but also to overall health. Nevertheless, despite its obvious importance, there is still much unknown about the regulation of insulin-stimulated glucose uptake by skeletal muscle. To study this regulation, the field commonly uses mouse models, as insulin signaling and action in mouse skeletal muscle closely replicates that seen in human skeletal muscle. Remarkably, however, the physiological action of insulin on the dynamics of glucose uptake in mouse skeletal muscle, especially as it relates to the contributions of sex, fiber type, and insulin concentration have not been systematically analyzed. Thus, the objective of this Thesis was to investigate the interactive effect of time, sex, fiber type, and insulin concentration on basal and insulin-stimulated glucose uptake in mouse skeletal muscle. Specifically, we used an ex vivo radioactive 2-deoxyglucose uptake (2DOGU) approach and measured basal and insulin-stimulated (insulin concentration: 0.36 nM and 6 nM) glucose uptake for 5, 10, 15, 20, or 30 minutes in paired extensor digitorum longus (EDL) and soleus muscles from 12-15 week old female and male (6nM insulin only) mice. In both the soleus and EDL of female mice, 2DOGU in response to physiological insulin (0.36nM; i.e the circulating insulin concentration typically seen after a meal) was statistically greater than basal at 15 and beyond, but not at 5 or 10 min. In contrast, a supraphysiological insulin concentration (6nM) 2DOGU more rapidly increased 2DOGU above basal, such that it was greater than basal at by 5 minutes in soleus and 10 minutes in EDL, regardless of sex. As it relates to fiber type, the soleus (i.e. primarily slow twitch) had greater insulin-stimulated glucose uptake than the EDL (i.e. primarily fast-twitch), regardless of sex or insulin concentration. Finally, as it relates to sex differences, female mice had greater insulin-stimulated 2DOGU as compared to male, regardless of fiber type or time point. Taken together, this work demonstrates that in response to physiological insulin there is a delay of ~15 minutes from insulin exposure to when there is a robust increase in insulin-stimulated glucose uptake, and that increasing insulin concentration shortens this biological delay. Overall, this work demonstrates the importance sex, fiber type, insulin concentration and time to the regulation of insulin-stimulated glucose uptake by mouse skeletal muscle.

Exercise (i.e. contraction) robustly stimulates skeletal muscle glucose uptake, making it a cornerstone non-pharmaceutical intervention for treating clinical hyperglycemia and insulin-resistance. Remarkably, however, the molecular steps that regulate contraction-stimulated glucose uptake by skeletal muscle remain incompletely defined. The mammalian ortholog of Sir2, sirtuin 1 (SIRT1), is a protein deacetylase that is thought to link perturbations in energy flux associated with exercise, particularly at the level of NAD+ and NAD+/NADH, to subsequent cellular adaptations. Nevertheless, its role in contraction-stimulated glucose uptake has not been defined. The objective of this study was to determine the contribution of SIRT1 to contraction-stimulated glucose uptake in mouse skeletal muscle. Using radioactive 2-deoxyglucose uptake (2DOGU) approach, we measured ex vivo glucose uptake in unstimulated and electrically-stimulated (100 Hz contraction [2s train, 0.2ms pulse, 35V] every 15s for 10 min) extensor digitorum longus (EDL) and soleus from ~15 week old male and female mice with muscle-specific knockout of SIRT1 (mKO) and their wildtype (WT) littermates. As expected, force output decreased over the contraction protocol, although there were no differences in the rate of fatigue between genotypes. In EDL and soleus from WT mice, 2DOGU was ~2.5-fold higher in contracted vs. rested muscle, regardless of sex, and this effect was not impaired in mKO mice. Interestingly, the absolute rate of contraction-stimulated 2DOGU was ~1.3-fold higher in both the soleus and EDL of female compared to male mice, regardless of genotype. Taken together, our findings demonstrate that SIRT1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle.

The NAD+ dependent protein deacetylase sirtuin 1 (SIRT1) and the acetyltransferase general control of amino acid synthesis 5 (GCN5) are proposed to regulate glucose homeostasis and mitochondrial biogenesis through their reciprocal regulation of the acetylation status of peroxisome proliferator activated receptor-γ coactivator-α (PGC1α). However, the precise contribution of these two enzymes to glucose homeostasis and mitochondrial biogenesis in skeletal muscle, remains to be fully defined. To address this gap in knowledge, this

Thesis investigated whether overexpression of SIRT1 and knockout of GCN5 in skeletal muscle would enhance glucose homeostasis and mitochondrial biogenesis. To do this, we used

Cre-LoxP methodology to generate double transgenic (dTG) mice with inducible, skeletal muscle-specific overexpression of SIRT1 and knockout of GCN5. We assessed skeletal muscle insulin sensitivity by 2-deoxy-glucose uptake, glucose tolerance via an oral glucose tolerance test, exercise capacity, and muscle maximal respiratory function by high-resolution respirometry and compared findings in dTG mice to those in wildtype littermate controls (WT). As expected, there was robust overexpression of SIRT1 and knockout of GCN5 in skeletal muscle of dTG compared to WT mice, and this was accompanied by increased gene expression of some mitochondrial proteins and citrate synthase activity in dTG versus WT mice. Despite these changes, there was no genotype difference in maximal respiratory function of skeletal muscle. Moreover, oral glucose tolerance and skeletal muscle insulin sensitivity were comparable between genotypes. Taken together, these results demonstrate that concurrent overexpression of SIRT1 and knockout of GCN5 does not impact skeletal muscle glucose homeostasis or mitochondrial function.

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