No abstract

The obesity epidemic has led to an increased incidence of non–alcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP–activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed anti–type 2 diabetic drug metformin1,2. Ampk phosphorylates murine acetyl–CoA carboxylase3,4 (Acc) 1 at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl–CoA to malonyl–CoA, a precursor in fatty acid synthesis5 as well as an allosteric inhibitor of fatty acid transport into mitochondria for oxidation6. To test the physiological impact of these phosphorylation events we generated mice with alanine knock–in mutations in both Acc1 (Ser79) and Acc2 (Ser212) (Acc double knock–in, AccDKI). These mice have elevated lipogenesis and lower fatty acid oxidation compared to wild–type (WT) mice, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Remarkably, AccDKI mice made obese by high–fat feeding, are refractory to the lipid–lowering and insulin–sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism, and in the setting of obesity, for metformin–induced improvements in insulin action.

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes1. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity2. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor3,4. Nicotinamide is a precursor of NAD+, an important cofactor linking cellular redox states with energy metabolism5. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation6. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine–spermine N1-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism7,8. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD+ levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD+-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Rationale: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown. Objective: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice. Methods and Results: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Key Words: chronobiology Ⅲ ischemia/reperfusion Ⅲ myocardium N umerous aspects of cardiovascular physiology and pathophysiology demonstrate circadian rhythms. 1 In humans, heart rate, blood pressure, and cardiac output all increase in the early hours of the morning, as does the onset of adverse cardiac events, such as myocardial infarction. 2,3 These rhythms have been attributed primarily to time-of-day oscillations in neurohumoral influences, such as sympathetic or autonomic stimulation. 3,4 Although extracardiac factors undoubtedly play critical roles in modulation of cardiovascular function/dysfunction, increasing evidence suggests that intrinsic factors, such as cellautonomous circadian clocks, likely contribute. 1 Circadian clocks are transcriptionally based molecular mechanisms, composed of positive-and negative-feedback loops, with a free-running period of Ϸ24 hours. 5 This mechanism allows the cell to anticipate alterations in environmental stimuli, through time-of-day-dependent modulation of cellular responsiveness to extrinsic factors. 5 Circadian clocks have been identified/characterized in multiple cardiovascular-relevant cell types, including cardiomyocytes, vascular smooth muscle cells, and endothelial cells. 6 -8 Ubiquitous genetic ablation of circadian clock function markedly influences multiple cardiovascular parameters, including heart rate and blood pressure. 9 We have recently used a CCM (cardiomyocyte-specific circadian clock mutant) mouse to reveal regulation of myocardial gene expression, ␤-adrenergic responsiveness, metabolism, heart rate, and cardiac power by this mechanism. 10,11 Although circadian rhythms in myocardial infarction onset are well established, time-of-day oscillations in myocardial ischemia/reperfusion (I/R) tolerance have not been reported. Given that the cardiomyocyte circadian clock influences Original