[Show abstract][Hide abstract] ABSTRACT: We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100μM) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of αAMPK at Ser 485/491 , and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser 485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.
PLoS ONE 05/2015; 10(5):e0127388. DOI:10.1371/journal.pone.0127388 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During gestation, hyperphagia is necessary to cope with the metabolic demands of embryonic development. There were three main aims of this study: Firstly, to investigate the impact of pregnancy on hypothalamic fatty acid metabolism, a key pathway for the regulation of energy balance. Secondly, to study whether pregnancy induces resistance to the anorectic effect of fatty acid synthase (FAS) inhibition and accumulation of malonyl-CoA in the hypothalamus, and, thirdly, whether changes in hypothalamic AMPK signaling are associated to BAT thermogenesis during pregnancy. Our data indicate that in pregnant rats, the hypothalamic fatty acid pathway shows an overall state that should lead to anorexia and elevated brown adipose tissue (BAT) thermogenesis: decreased activities of AMP-activated protein kinase (AMPK), FAS and carnitine palmitoyltransferase 1 (CPT1), coupled to increased acetyl-CoA carboxylase (ACC) function with subsequent elevation of malonyl-CoA levels. This profile seems dependent of estradiol levels, but not prolactin or progesterone. Despite the apparent anorexic and thermogenic signaling in the hypothalamus, pregnant rats remain hyperphagic and display reduced temperature and BAT function. Actually, pregnant rats develop resistance to the anorectic effects of central FAS inhibition, which is associated with a reduction of proopiomelanocortin (POMC) expression and its transcription factors phospho-signal transducer and activator of transcription 3 (pSTAT3) and phospho-forkhead box O1 (pFoxO1). This evidence demonstrates that pregnancy induces a state of resistance to the anorectic and thermogenic actions of hypothalamic cellular signals of energy surplus, which, in parallel to the already known refractoriness to leptin effects, likely contributes to gestational hyperphagia and adiposity.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40-70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit.
Archives of Biochemistry and Biophysics 08/2014; 562. DOI:10.1016/j.abb.2014.08.013 · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Estrogens play a major role in the modulation of energy balance through central and peripheral actions. Here, we demonstrate that central action of estradiol (E2) inhibits AMP-activated protein kinase (AMPK) through estrogen receptor alpha (ERα) selectively in the ventromedial nucleus of the hypothalamus (VMH), leading to activation of thermogenesis in brown adipose tissue (BAT) through the sympathetic nervous system (SNS) in a feeding-independent manner. Genetic activation of AMPK in the VMH prevented E2-induced increase in BAT-mediated thermogenesis and weight loss. Notably, fluctuations in E2 levels during estrous cycle also modulate this integrated physiological network. Together, these findings demonstrate that E2 regulation of the VMH AMPK-SNS-BAT axis is an important determinant of energy balance and suggest that dysregulation in this axis may account for the common changes in energy homeostasis and obesity linked to dysfunction of the female gonadal axis
[Show abstract][Hide abstract] ABSTRACT: Type 2 diabetes (T2D) is a metabolic disease characterized by insulin resistance, β-cell dysfunction, and elevated hepatic glucose output. Over 350 million people worldwide have T2D, and the International Diabetes Federation projects that this number will increase to nearly 600 million by 2035. There is a great need for more effective treatments for maintaining glucose homeostasis and improving insulin sensitivity. AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase whose activation elicits insulin-sensitizing effects, making it an ideal therapeutic target for T2D. AMPK is an energy-sensing enzyme that is activated when cellular energy levels are low, and it signals to stimulate glucose uptake in skeletal muscles, fatty acid oxidation in adipose (and other) tissues, and reduces hepatic glucose production. There is substantial evidence suggesting that AMPK is dysregulated in animals and humans with metabolic syndrome or T2D, and that AMPK activation (physiological or pharmacological) can improve insulin sensitivity and metabolic health. Numerous pharmacological agents, natural compounds, and hormones are known to activate AMPK, either directly or indirectly - some of which (for example, metformin and thiazolidinediones) are currently used to treat T2D. This paper will review the regulation of the AMPK pathway and its role in T2D, some of the known AMPK activators and their mechanisms of action, and the potential for future improvements in targeting AMPK for the treatment of T2D.
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 06/2014; 7:241-53. DOI:10.2147/DMSO.S43731
[Show abstract][Hide abstract] ABSTRACT: Efficient coupling of cellular energy production to metabolic demand is crucial to maintain organismal homeostasis. Here, we report that the mitochondrial Sirtuin Sirt4 regulates mitochondrial ATP homeostasis. We find that Sirt4 affects mitochondrial uncoupling via the adenine nucleotide translocator 2 (ANT2). Loss of Sirt4 expression leads to decreased cellular ATP levelsin vitro and in vivo while Sirt4 overexpression is associated with increased ATP levels. Further, we provide evidence that lack of Sirt4 activates a retrograde signaling response from the mitochondria to the nucleus that includes AMPK, PGC1α, key regulators of β-oxidation such as Acetyl-CoA carboxylase, and components of the mitochondrial respiratory machinery. This study highlights the ability of Sirt4 to regulate ATP levels via ANT2 and a feedback loop involving AMPK.
[Show abstract][Hide abstract] ABSTRACT: It is well established that chronic exposure to excess nutrients leads to insulin resistance (IR) in skeletal muscle. Since skeletal muscle is responsible for 70-80% of insulin-stimulated glucose uptake, skeletal muscle IR is a key pathological component of type 2 diabetes (T2D). Recent evidence suggests that inhibition of the nutrient-sensing enzyme AMP-activated protein kinase (AMPK) is an early event in the development of IR in response to high glucose, branched chain amino acids (BCAA), or fatty acids (FA). Whether the decrease in AMPK activity is causal to the events leading to insulin resistance (increased mTOR/p70S6K signaling) remains to be determined. Interestingly, pharmacological activation of AMPK can prevent activation of mTOR/p70S6K and insulin resistance, while inhibition of mTOR with rapamycin prevents insulin resistance, but not AMPK downregulation. AMPK can be inhibited by increased energy state (reduced AMP/ATP ratio), decreased phosphorylation of its activation site (αThr172) (by decreased upstream kinase activity or increased phosphatase activity), increased inhibitory phosphorylation at αSer485/491, changes in redox state or hormone levels, or other yet to be identified mechanisms. Excess nutrients also lead to an accumulation of the toxic lipid intermediates diacylglycerol (DAG) and ceramides, both of which can activate various protein kinase C (PKC) isoforms, and contribute to IR. The mechanism responsible for the initial downregulation of AMPK in response to excess nutrients, and whether glucose, BCAA, and FA act through similar or different pathways requires further study. Identification of this mechanism and the relative importance of other events would be beneficial for designing novel pharmacological interventions to prevent and/or reverse IR. This review will focus on the some of the mechanisms responsible for AMPK downregulation and the relative sequence and importance of these events.
[Show abstract][Hide abstract] ABSTRACT: Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.
[Show abstract][Hide abstract] ABSTRACT: It has long been known that excesses of glucose and branched chain amino acids, such as leucine, lead to insulin resistance in skeletal muscle. A recent study in incubated rat muscle suggests that both molecules may do so by virtue of their ability to downregulate the fuel sensing and signaling enzyme AMP-activated protein kinase (AMPK) and activate mTOR/p70S6 kinase (p70S6K) signaling. The results also demonstrated that inhibition of mTOR/p70S6K with rapamycin prevented the development of insulin resistance but had no effect on AMPK activity (Thr172 phosphorylation of its catalytic subunit). In contrast, activation of AMPK by both AICAR and α-lipoic acid led to the phosphorylation of specific molecules that diminished both mTOR/p70S6K signaling and insulin resistance. These findings suggest that downregulation of AMPK precedes mTOR/p70S6K activation in mediating glucose and leucine-induced insulin resistance, although the mechanism by which it does so remains to be determined. Also requiring study is how an excess of the two nutrients leads to AMPK downregulation.
[Show abstract][Hide abstract] ABSTRACT: Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser(473)) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser(9)) and AS160 (Thr(642)) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.
Archives of Biochemistry and Biophysics 03/2011; 509(2):133-41. DOI:10.1016/j.abb.2011.03.008 · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK).
Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model.
In the EDL, incubation with 100 or 200 μmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation.
The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormones have widespread cellular effects; however it is unclear whether their effects on the central nervous system (CNS) contribute to global energy balance. Here we demonstrate that either whole-body hyperthyroidism or central administration of triiodothyronine (T3) decreases the activity of hypothalamic AMP-activated protein kinase (AMPK), increases sympathetic nervous system (SNS) activity and upregulates thermogenic markers in brown adipose tissue (BAT). Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus (VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism. This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and increases expression of thermogenic markers in BAT. These effects are reversed by pharmacological blockade of the SNS. Thus, thyroid hormone-induced modulation of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of whole-body energy homeostasis.
Nature medicine 09/2010; 16(9):1001-1008. DOI:10.1038/nm.2207 · 28.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1,25-Dihydroxyvitamin D(3)-3-bromoacetate (1,25(OH)(2)D(3)-3-BE) is a vitamin D receptor-alkylating derivative of 1,25(OH)(2)D(3). The strong dose-dependent antiproliferative and apoptotic effects of this compound in androgen-sensitive and androgen-insensitive prostate cancer cells have been reported. In this communication, it is reported that 1,25(OH)(2)D(3)-3-BE strongly inhibits the growth of several pancreatic cancer cell lines. This effect is further accentuated by combination with 5-amino-imidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK)/acetyl-Co-enzyme A carboxylase (ACC) phosphorylation pathways and an inhibitor of Akt phosphorylation. It was observed that the anti-growth property of 1,25(OH)(2)D(3)-3-BE, either alone or in combination with AICAR resulted in the inhibition of Akt phosphorylation in BxPC-3 cells. In conclusion, 1,25(OH)(2)D(3)-3-BE displays a strong therapeutic potential, alone and in combination with AICAR, in pancreatic cancer.
Anticancer research 06/2010; 30(6):1875-80. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) and the histone/protein deacetylase SIRT1 are fuel-sensing molecules that have coexisted in cells throughout evolution. When a cell's energy state is diminished, AMPK activation restores energy balance by stimulating catabolic processes that generate ATP and downregulating anabolic processes that consume ATP but are not acutely needed for survival. SIRT1 in turn is best known historically for producing genetic changes that mediate the increase in longevity caused by calorie restriction. Although the two molecules have been studied intensively for many years, only recently has it become apparent that they have similar effects on diverse processes such as cellular fuel metabolism, inflammation, and mitochondrial function. In this review we will examine the evidence that these similarities occur because AMPK and SIRT1 both regulate each other and share many common target molecules. In addition, we will discuss the clinical relevance of these interactions and in particular the possibility that their dysregulation predisposes to disorders such as type 2 diabetes and atherosclerotic cardiovascular disease and is a target for their therapy.
[Show abstract][Hide abstract] ABSTRACT: Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.
[Show abstract][Hide abstract] ABSTRACT: The orexigenic effect of ghrelin is mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the hypothalamic arcuate nucleus (ARC). Recent evidence also indicates that ghrelin promotes feeding through a mechanism involving activation of hypothalamic AMP-activated protein kinase (AMPK) and inactivation of acetyl-CoA carboxylase and fatty acid synthase (FAS). This results in decreased hypothalamic levels of malonyl-CoA, increased carnitine palmitoyltransferase 1 (CPT1) activity, and mitochondrial production of reactive oxygen species. We evaluated whether these molecular events are part of a unique signaling cascade or whether they represent alternative pathways mediating the orexigenic effect of ghrelin. Moreover, we examined the gender dependency of these mechanisms, because recent evidence has proposed that ghrelin orexigenic effect is reduced in female rats. We studied in both genders the effect of ghrelin on the expression of AgRP and NPY, as well as their transcription factors: cAMP response-element binding protein (CREB and its phosphorylated form, pCREB), forkhead box O1 (FoxO1 and its phosphorylated form, pFoxO1), and brain-specific homeobox transcription factor (BSX). In addition, to establish a mechanistic link between ghrelin, fatty acid metabolism, and neuropeptides, we evaluated the effect of ghrelin after blockage of hypothalamic fatty acid beta oxidation, by using the CPT1 inhibitor etomoxir. Ghrelin-induced changes in the AMPK-CPT1 pathway are associated with increased levels of AgRP and NPY mRNA expression through modulation of BSX, pCREB, and FoxO1, as well as decreased expression of endoplasmic reticulum (ER) stress markers in a gender-independent manner. In addition, blockage of hypothalamic fatty acid beta oxidation prevents the ghrelin-promoting action on AgRP and NPY mRNA expression, also in a gender-independent manner. Notably, this effect is associated with decreased BSX expression and reduced food intake. Overall, our data suggest that BSX integrates changes in neuronal metabolic status with ARC-derived neuropeptides in a gender-independent manner.
The FASEB Journal 03/2010; 24(8):2670-9. DOI:10.1096/fj.09-150672 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GH plays a major role in the regulation of lipid metabolism and alterations in GH axis elicit major changes in fat distribution and mobilization. For example, in patients with GH deficiency (GHD) or in mice lacking the GH receptor, the percentage of fat is increased. In addition to the direct actions of GH on lipid metabolism, current evidence indicates that ghrelin, a stomach-derived peptide hormone with potent GH secretagogue action, increases lipogenesis in white adipose tissue (WAT) through a hypothalamic-mediated mechanism. Still, the mechanism by which GH tone modulates ghrelin actions on WAT remains unclear. Here we investigated the effect of central ghrelin administration on lipid metabolism in lipogenic tissues (liver and WAT) in the absence of GH, by using a model for the study of GHD, namely the spontaneous dwarf rat, which shows increased body fat. Our data demonstrate that central chronic ghrelin administration regulates adipose lipid metabolism, mainly in a GH-independent fashion, as a result of increased mRNA, protein expression, and activity levels of fatty acid metabolism enzymes. On the contrary, central ghrelin regulates hepatic lipogenesis de novo in a GH-independent fashion but lipid mobilization in a GH-dependent fashion because carnitine palmitoyltransferase 1 was decreased only in wild-type Lewis rats. These findings suggest the existence of a new central nervous system-based neuroendocrine circuit, regulating metabolic homeostasis of adipose tissue. Understanding the molecular mechanism underlying the interplay between GH and ghrelin and their effects on lipid metabolism will provide new strategies for the design and development of suitable drugs for the treatment of GHD, obesity, and its comorbidities.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-6 (IL-6) directly activates AMP-activated protein kinase (AMPK) in vivo and in vitro; however, the mechanism by which it does so is unknown.
We examined this question in skeletal muscle using an incubated rat extensor digitorum longus (EDL) muscle preparation as a tool.
AMPK activation by IL-6 coincided temporally with a nearly threefold increase in the AMP:ATP ratio in the EDL. The effects of IL-6 on both AMPK activity and energy state were inhibited by coincubation with propranolol, suggesting involvement of beta-adrenergic signaling. In keeping with this notion, IL-6 concurrently induced a transient increase in cAMP, and its ability to activate AMPK was blocked by the adenyl cyclase inhibitor 2'5'-dideoxyadenosine. In addition, like other beta-adrenergic stimuli, IL-6 increased glycogen breakdown and lipolysis in the EDL. Similar effects of IL-6 on AMPK, energy state, and cAMP content were observed in C2C12 myotubes and gastrocnemius muscle in vivo, indicating that they were not unique to the incubated EDL.
These studies demonstrate that IL-6 activates AMPK in skeletal muscle by increasing the concentration of cAMP and, secondarily, the AMP:ATP ratio. They also suggest that substantial increases in IL-6 concentrations, such as those that can result from its synthesis by muscles during exercise, may play a role in the mobilization of fuel stores within skeletal muscle as an added means of restoring energy balance.