SUMMARY While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here, we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.

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-tissuespecific knockout of both the ␣1 and ␣2 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.A dipose tissue plays a key role in whole-body energy homeostasis by storing triacylglycerol (TAG) during excess energy intake, which is hydrolyzed (so-called lipolysis) to release fatty acids (FAs) into the circulation for use by other tissues during energy shortage. Thus, adipose TAG metabolism, especially the unique function of adipose lipolysis for FA release, must be exquisitely regulated according to nutritional conditions. For example, during fasting, lipolysis is stimulated upon the release of catecholamines to activate the ␤-adrenergic receptor-adenylyl cyclase-cyclic AMP (cAMP)-protein kinase A (PKA) pathway in adipocytes. In contrast, in the fed state, insulin released from pancreatic islet ␤ cells activates phosphodiesterase 3B for degradation of cAMP in adipocytes, resulting in suppression of lipolysis (1). In addition to hormonal regulation, adipose lipolysis may also be regulated by the intracellular energy state for the maintenance of cellular TAG homeostasis.AMP-activated protein kinase (AMPK) is a widely expressed multisubstrate serine/threonine kinase and a well-known sensor of the intracellular energy state that responds to metabolic stresses and other regulatory signals. AMPK is a heterotrimeric complex with a catalytic ␣ subunit and two regulatory subunits, ␤ and ␥ (2). There are several isoforms of each of the AMPK subunits, including two isoforms of the catalytic subunit, ␣1 and ␣2. AMPK is known to be activated allosterically by AMP but also is activated by phosphorylation of its catalytic subunit at T172 by the upstream kinases liver kinase B1 a...

Multiple signals have been shown to be involved in regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. This study aimed to determine whether a Ca(2+)-stimulated kinase, CaMKK, is involved in regulation of contraction-induced substrate metabolism and whether it does so in an AMP-activated protein kinase (AMPK)-dependent manner. Rat hindlimbs were perfused at rest (n = 16), with 3 mM caffeine (n = 15), with 2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR; n = 16), or during moderate-intensity muscle contraction (MC; n = 14) and with or without 5 microM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased (P < 0.05) 64% and 71% by caffeine, 42% and 93% by AICAR, and 65% and 143% by MC. STO-609 abolished (P < 0.05) caffeine- and MC-induced FA uptake and oxidation but had no effect with AICAR treatment. Glucose uptake increased (P < 0.05) 104% by caffeine, 85% by AICAR, and 130% by MC, and STO-609 prevented the increase in glucose uptake in caffeine and muscle contraction groups. CaMKKbeta activity increased (P < 0.05) 113% by caffeine treatment and 145% by MC but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKKbeta activity. Caffeine, AICAR, and MC increased (P < 0.05) AMPKalpha2 activity by 295%, 11-fold, and 7-fold but did not affect AMPKalpha1 activity. STO-609 decreased (P < 0.05) AMPKalpha2 activity induced by caffeine treatment and MC by 60% and 61% but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and glucose transporter 4 (GLUT4) increased (P < 0.05) with caffeine, AICAR, and MC, and STO-609 prevented caffeine- and MC-induced increases in protein content. These results show the importance of Ca(2+)-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle and agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.

Excessive caloric intake leading to obesity is associated with insulin resistance and dysfuntion of islet β cells. High fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (βKO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. βKO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPARδ target genes involved in mitochondrial oxidation. Furthermore, synthetic PPARδ, but not PPARα, agonist restores GSIS and expression of mitochondrial oxidative genes in βKO mice, revealing desnutrin-catalyzed lipolysis generates PPARδ ligands. Finally, adenoviral expression of desnutrin in βKO islets restores all defects of βKO islet phenotype and function including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPARδ-mitochondrial oxidation axis in regulating islet β cell GSIS.

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

Insulin is important in the regulation of muscle metabolism. However, its role in the regulation of muscle long-chain fatty acid (LCFA) metabolism, independent of glucose, is not clear. To determine whether insulin regulates LCFA metabolism independent of glucose and if so, via which signaling pathway, L6 myotubes were incubated, in the presence or absence of insulin (100 nM) and with either an inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin (W), 50 nM), protein kinase B (PKB)/Akt (A, 10 mM), or atypical protein kinase C-z (aPKC-z) (mP, 100 mM). LCFA kinetic parameters were measured via incubation with [1-14 C]palmitate. Basal LCFA uptake was found to increase linearly with time (1-60 min) and concentration (50-750 mM). LCFA uptake increased in the presence of insulin and was maximum at 10 nM (P!0 . 05). Wortmannin prevented the insulininduced increase in LCFA uptake and decrease in LCFA oxidation. While mP abolished the insulin-induced increase in LCFA uptake, it did not prevent the insulin-induced decrease in LCFA oxidation. None of the variables were affected by Akt inhibition. These results suggest a direct effect of insulin on LCFA metabolism in muscle cells, and that downstream of PI3K, aPKC-z, but not PKB/Akt mediates the effects of insulin on LCFA uptake but not oxidation.

Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n = 10) and HFD mice (n = 10) were perfused with or without 450 μU ml −1 insulin. Muscles of CD (n = 8) and HFD mice (n = 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P < 0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and AktThr308 phosphorylation (P < 0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P < 0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P < 0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue proinflammatory markers. A precursor and unfavourable indicator of type 2 diabetes mellitus is insulin resistance in skeletal muscle (DeFronzo et al. 1992;Båvenholm et al. 2003). While the mechanisms leading to the development of insulin resistance have not been completely identified, there is evidence that its progression is linked to alterations in fatty acid (FA) metabolism (Laybutt et al. 1999;Chavez & Summers, 2003;Aas et al. 2004 (Winder & Hardie, 1999;Ruderman & Prentki, 2004).AMPK has been studied extensively for its role as an energy sensor during states of low energy balance, as exemplified by muscle contraction or hypoxia (Hutber et al. 1997;Mu et al. 2001). Additionally, multiple studies have linked FA intake and high-fat diets to alterations in AMPK activity in liver and skeletal muscle (Suchankova Fillmore et al. 2010). A recent study has linked the deletion of AMPKα2 activity to an exacerbation of high-fat diet-induced insulin resistance as it pertains to glucose metabolism (Fujii et al. 2008). However, in another study, it was shown that reduced AMPK activity did not affect the development of obesity-induced lipid accumulation and insulin resistance ). As such, the role of low muscle AMPK activ...

Abbott MJ, Bogachus LD, Turcotte LP. AMPK␣2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle. J Appl Physiol 111: 125-134, 2011. First published May 5, 2011 doi:10.1152/japplphysiol.00807.2010.-AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]). The purpose of this study was to determine whether increases in intracellular [Ca 2ϩ ] induced by caffeine act solely via AMPK␣ 2 and whether AMPK␣2 is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPK␣ 2 dominant-negative (DN) transgene mice were perfused during rest (n ϭ 11), treatment with 3 mM caffeine (n ϭ 10), or muscle contraction (n ϭ 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P Ͻ 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P Ͻ 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P Ͻ 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P Ͻ 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P Ͼ 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P Ͻ 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine-or contraction-mediated changes in the phosphorylation of Ca 2ϩ /calmodulin-dependent protein kinase I or ERK1/2 (P Ͼ 0.05). These data suggest that AMPK␣ 2 is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment. ERK1/2; muscle contraction; caffeine; calcium/calmodulin-dependent protein kinase kinase; acetyl-CoA carboxylase EVIDENCE SUGGESTS THAT MULTIPLE cellular signals regulate the changes in substrate metabolism with muscle contraction, including the AMP-activated protein kinase (AMPK) signaling cascade (2, 34, 35, 52). During states of low energy, such as muscle contraction or exercise, it is widely accepted that liver kinase B1 (LKB1) acts as an upstream AMPK kinase that phosphorylates and activates AMPK and initiates a multitude of signaling events to maintain energy homeostasis (39 -41). Although strong evidence indicates that the LKB1-AMPK cascade is a key signaling pathway that regulates changes in glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle (16,25,54,56), data also show that AMPK may not be the sole signal mediating contractioninduced changes in glucose uptake, FA uptake, and FA oxidation (35,36,56).Along with AMPK, mounting data suggest that increas...