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The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The postweaning fast of northern elephant seal pups is characterized by a lipid-dependent metabolism and associated with a decrease in plasma glucagon-like peptide-1 (GLP-1), insulin, and glucose and increased gluconeogenesis (GNG) and ketogenesis. We have also demonstrated that exogenous GLP-1 infusion increased plasma insulin despite simultaneous increases in cortisol and glucagon, which collectively present contradictory regulatory stimuli of GNG, ketogenesis, and glycolysis. To assess the effects of GLP-1 on metabolism using primary carbon metabolite profiles in late-fasted seal pups, we dose-dependently infused late-fasted seals with low (LDG; 10 pM/kg; n = 3) or high (HDG; 100 pM/kg; n = 4) GLP-1 immediately following a glucose bolus (0.5 g/kg), using glucose without GLP-1 as control (n = 5). Infusions were performed in similarly aged animals 6-8 wk into their postweaning fast. The plasma metabolome was measured from samples collected at five time points just prior to and during the infusions, and network maps constructed to robustly evaluate the effects of GLP-1 on primary carbon metabolism. HDG increased key tricarboxylic acid (TCA) cycle metabolites, and decreased phosphoenolpyruvate and acetoacetate (P < 0.05) suggesting that elevated levels of GLP-1 promote glycolysis and suppress GNG and ketogenesis, which collectively increase glucose clearance. These GLP-1-mediated effects on cellular metabolism help to explain why plasma GLP-1 concentrations decrease naturally in fasting pups as an evolved mechanism to help conserve glucose during the late-fasting period.

Prolonged fasting in northern elephant seals (NES) is characterized by a reliance on lipid metabolism, conservation of protein, and reduced plasma insulin. During early fasting, glucose infusion previously reduced plasma free fatty acids (FFA); however, during late-fasting, it induced an atypical elevation in FFA despite comparable increases in insulin during both periods suggestive of a dynamic shift in tissue responsiveness to glucose-stimulated insulin secretion. To better assess the contribution of insulin to this fasting-associated shift in substrate metabolism. We compared the responses of plasma metabolites (amino acids (AA), FFA, endocannabinoids (EC), and primary carbon metabolites (PCM)) to an insulin infusion (65 mU/kg) in early- and late-fasted NES pups (n = 5/group). Plasma samples were collected prior to infusion (T0) and at 10, 30, 60, and 120 min post-infusion, and underwent untargeted and targeted metabolomics analyses utilizing a variety of GC-MS and LC-MS technologies. In early fasting, the majority (72%) of metabolite trajectories return to baseline levels within 2 h, but not in late fasting indicative of an increase in tissue sensitivity to insulin. In late-fasting, increases in FFA and ketone pools, coupled with decreases in AA and PCM, indicate a shift toward lipolysis, beta-oxidation, ketone metabolism, and decreased protein catabolism. Conversely, insulin increased PCM AUC in late fasting suggesting that gluconeogenic pathways are activated. Insulin also decreased FFA AUC between early and late fasting suggesting that insulin suppresses triglyceride hydrolysis. Naturally adapted tolerance to prolonged fasting in these mammals is likely accomplished by suppressing insulin levels and activity, providing novel insight on the evolution of insulin during a condition of temporary, reversible insulin resistance.

De novo lipogenesis is precisely regulated by nutritional and hormonal conditions. The genes encoding various enzymes involved in this process, such as fatty acid synthase (FASN), are transcriptionally activated in response to insulin. We showed that USF1, a key transcription factor for FASN activation, directly interacted with the Mediator subunit MED17 at the FASN promoter. This interaction recruited Mediator, which can bring POL II and other general transcription machinery to the complex. Moreover, we showed that MED17 was phosphorylated at Ser53 by casein kinase 2 (CK2) in the livers of fed mice or insulin-stimulated hepatocytes, but not in the livers of fasted mice or untreated hepatocytes. Furthermore, activation of the FASN promoter in response to insulin required this CK2-mediated phosphorylation event, which occurred only in the absence of p38 MAPK-mediated phosphorylation at Thr570 Overexpression of a nonphosphorylatable S53A MED17 mutant or knockdown of MED17, as well as CK2 knockdown or inhibition, impaired hepatic de novo fatty acid synthesis and decreased triglyceride content in mice. These results demonstrate that CK2-mediated phosphorylation of Ser53 in MED17 is required for the transcriptional activation of lipogenic genes in response to insulin.

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

Prolonged food deprivation increases lipid oxidation and utilization, which may contribute to the onset of the insulin resistance associated with fasting. Because insulin resistance promotes the preservation of glucose and oxidation of fat, it has been suggested to be an adaptive response to food deprivation. However, fasting mammals exhibit hypoinsulinemia, suggesting that the insulin resistance-like conditions they experience may actually result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. To determine whether fasting results in insulin resistance or in pancreatic dysfunction, we infused early- and late-fasted seals (naturally adapted to prolonged fasting) with insulin (0.065 U/kg), and a separate group of late-fasted seals with low (10 pM/kg) or high (100 pM/kg) dosages of glucagon-like peptide-1 (GLP-1) immediately following a glucose bolus (0.5g/kg), and measured the systemic and cellular responses. Because GLP-1 facilitates glucose-stimulated insulin secretion, these infusions provide a method to assess pancreatic insulin-secreting capacity. Insulin infusions increased the phosphorylation of insulin receptor and Akt in adipose and muscle of early and late fasted seals; however the timing of the signaling response was blunted in adipose of late fasted seals. Despite the dose-dependent increases in insulin and increased glucose clearance (high dose), both GLP-1 dosages produced increases in plasma cortisol and glucagon, which may have contributed to the glucogenic role of GLP-1. Results suggest that fasting induces adipose-specific insulin resistance in elephant seal pups, while maintaining skeletal muscle insulin sensitivity, and therefore suggests that the onset of insulin resistance in fasting mammals is an evolved response to cope with prolonged food deprivation.

Abstract: Food deprivation in mammals results in profound changes in fuel metabolism and substrate regulation. Among these changes are decreased reliance on the counter-regulatory dynamics by insulin-glucagon due to reduced glucose utilization, and increased concentrations of lipid substrates in plasma to meet the energetic demands of peripheral tissues. As the primary storage site of lipid substrates, adipose tissue must then be a primary contributor to the regulation of metabolism in food deprived states. Through its regulation of lipolysis, adipose tissue influences the availability of carbohydrate, lipid, and protein substrates. Additionally, lipid substrates can act as ligands to various nuclear receptors (retinoid x receptor (RXR), liver x receptor (LXR), and peroxisome proliferator-activated receptor (PPAR)) and exhibit prominent regulatory capabilities over the expression of genes involved in substrate metabolism within various tissues. Therefore, through its control of lipolysis, adipose tissue also indirectly regulates the utilization of metabolic substrates within peripheral tissues. In this review, these processes are described in greater detail and the extent to which adipose tissue and lipid substrates regulate metabolism in food deprived mammals is explored with comments on future directions to better assess the contribution of adipose tissue to metabolism. [Copyright &y& Elsevier]

The northern elephant seal pup (Mirounga angustirostris) undergoes a 2-3 month post-weaning fast, during which it depends primarily on the oxidation of fatty acids to meet its energetic demands. The concentration of non-esterified fatty acids (NEFAs) increases and is associated with the development of insulin resistance in late-fasted pups. Furthermore, plasma NEFA concentrations respond differentially to an intravenous glucose tolerance test (ivGTT) depending on fasting duration, suggesting that the effects of glucose on lipid metabolism are altered. However, elucidation of the lipolytic mechanisms including lipase activity during prolonged fasting in mammals is scarce. To assess the impact of fasting and glucose on the regulation of lipid metabolism, adipose tissue and plasma samples were collected before and after ivGTTs performed on early (2 weeks, N=5) and late (6-8weeks; N=8) fasted pups. Glucose administration increased plasma triglycerides and NEFA concentrations in late-fasted seals, but not plasma glycerol. Fasting decreased basal adipose lipase activity by 50%. Fasting also increased plasma lipase activity twofold and decreased the expressions of CD36, FAS, FATP1 and PEPCK-C by 22-43% in adipose tissue. Plasma acylcarnitine profiling indicated that late-fasted seals display higher incomplete LCFA (&bgr;-oxidation. Results suggest that long-term fasting induces shifts in the regulation of lipolysis and lipid metabolism associated with the onset of insulin resistance in northern elephant seal pups. Delineation of the mechanisms responsible for this shift in regulation during fasting can contribute to a more thorough understanding of the changes in lipid metabolism associated with dyslipidemia and insulin resistance in mammals. [ABSTRACT FROM AUTHOR]