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AMP-activated protein kinase (AMPK) is an enzyme crucial in cellular metabolism found to be inhibited in many metabolic diseases including type 2 diabetes. Thiazolidinediones (TZDs) are a class of anti-diabetic drug known to activate AMPK through increased phosphorylation at Thr172, however there has been no research to date on whether they have any effect on inhibition of AMPK's lesser known site of inhibition, Ser485/491. HepG2 cells were treated with troglitazone and phosphorylation of AMPK was found to increase at both Thr172 and Ser485 in a dose- and time-dependent manner. Treatment of HepG2 cells with insulin and PMA led to increases in p-AMPK Ser485 via Akt and PKD1 respectively; however these kinases were not found to be implicated in increases seen from troglitazone. Incubation with the other TZDs, rosiglitazone and pioglitazone, let to a minor increase in p-AMPK Ser485 phosphorylation as well as AMPK activity; however these findings were significantly less than those of troglitazone under equal conditions. These data suggest that the effects of troglitazone on AMPK are more complex than previously thought. Phosphorylation at sites of both activation and inhibition can occur in tandem, although the mechanism by which this occurs has not yet been elucidated.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.

Morbidly obese individuals are predisposed to a wide range of disorders, including type 2 diabetes, atherosclerotic cardiovascular disease, fatty liver disease, and certain cancers. Remarkably, all of these disorders can be improved or prevented by Roux-en-Y gastric bypass (RYGB) surgery. We have reported that decreased AMPK activity, together with increased oxidative stress and inflammation in adipose tissue, is associated with insulin resistance in morbidly obese bariatric surgery patients. In the current study, we assessed how these parameters are affected by RYGB surgery. Eleven patients (average age of 46 ± 4 years) were studied immediately prior to surgery and 3 months postoperatively. We measured subcutaneous adipose tissue AMPK phosphorylation (threonine 172, an index of its activation), malonyl-CoA content, protein carbonylation (a marker of oxidative stress), plasma adiponectin, and mRNA expression of several inflammatory cytokines. After surgery, AMPK activity increased 3.5-fold and oxidative stress decreased by 50% in subcutaneous adipose tissue. In addition, malonyl-CoA levels were reduced by 80%. Furthermore, patients had improvements in their BMI and insulin sensitivity (HOMA) and had increased circulating high–molecular weight adiponectin and decreased fasting plasma insulin levels. In contrast, the expression of inflammatory markers in subcutaneous adipose tissue was unchanged postoperatively, although plasma CRP was diminished by 50%.

Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.

A subpopulation of obese individuals remains insulin sensitive (ISO). They represent a unique human model to investigate factors underlying insulin resistance (IR) without the confounding effect of major differences in weight/adiposity. Altered fatty-acid (FA) metabolism in sc adipose tissue (SAT) contributes to obesity-associated IR.To test the hypothesis that ISO and body mass index-matched insulin-resistant obese (IRO) patients demonstrate differential SAT expression profiles of genes involved in glycerolipid-FA metabolism and that weight loss-induced improvement of IR ameliorates these changes.A cross-sectional and longitudinal study.Thirty-eight nondiabetic obese women were stratified into ISO (n = 25) or IRO (n = 13) groups based on hyperinsulinemic-euglycemic clamp results. Subjects were studied before and after a 6-month hypocaloric diet intervention.mRNA (quantitative RT-PCR) and protein (mass spectrometry and immunoblots) levels were measured in SAT biopsies.Despite having age, body mass index, and fat mass similar to ISO individuals, IRO patients had lower insulin sensitivity and glucose tolerance (P.05). Baseline SAT mRNA and protein levels of genes involved in both the synthesis and lipolysis of glycerolipid-FAs were higher in IRO individuals (P.05), even when groups were matched for visceral adipose tissue content. The dietary intervention resulted in approximately 6% weight loss in both the IRO and ISO groups (P.05) but only ameliorated insulin sensitivity in IRO individuals (P.05). Likewise, the intervention reduced the expression of most glycerolipid-FA metabolism genes (P.05), with expression levels in IRO individuals being restored to ISO levels.Increased SAT expression of genes involved in both the synthesis and hydrolysis of glycerolipid-FAs is closely associated with IR in obese women. The results suggest that enhanced glycerolipid-FA cycling in SAT contributes to obesity-associated IR.

Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser485/491, 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 Ser485/491 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 Ser485/491 p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr172. Akt inhibition both attenuated the increase in Ser485/491 p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser485/491 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 Ser485/491 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 Ser485/491 on its α-subunit.

Insulin resistance (IR) and hyperinsulinemia are hallmarks of the metabolic syndrome, as are central adiposity, dyslipidemia, and a predisposition to type 2 diabetes, atherosclerotic cardiovascular disease, hypertension, and certain cancers. Regular exercise and calorie restriction have long been known to increase insulin sensitivity and decrease the prevalence of these disorders. The subsequent identification of AMP-activated protein kinase (AMPK) and its activation by exercise and fuel deprivation have led to studies of the effects of AMPK on both IR and metabolic syndrome-related diseases. In this review, we evaluate this body of literature, with special emphasis on the hypothesis that dysregulation of AMPK is both a pathogenic factor for these disorders in humans and a target for their prevention and therapy.

Our objective was to determine if the insulin-sensitizing drug pioglitazone acutely reduces insulin secretion and causes metabolic deceleration in vivo independently of change in insulin sensitivity. We assessed glucose homeostasis by hyperinsulinemic-euglycemic and hyperglycemic clamp studies and energy expenditure by indirect calorimetry and biotelemetry in male Wistar and obese hyperinsulinemic Zucker diabetic fatty (ZDF) rats 45 min after a single oral dose of pioglitazone (30 mg/kg). In vivo insulin secretion during clamped hyperglycemia was reduced in both Wistar and ZDF rats after pioglitazone administration. Insulin clearance was slightly increased in Wistar but not in ZDF rats. Insulin sensitivity in Wistar rats assessed by the hyperinsulinemic-euglycemic clamp was minimally affected by pioglitazone at this early time point. Pioglitazone also reduced energy expenditure in Wistar rats without altering respiratory exchange ratio or core body temperature. Glucose-induced insulin secretion (GIIS) and oxygen consumption were reduced by pioglitazone in isolated islets and INS832/13 cells. In conclusion, pioglitazone acutely induces whole-body metabolic slowing down and reduces GIIS, the latter being largely independent of the insulin-sensitizing action of the drug. The results suggest that pioglitazone has direct metabolic deceleration effects on the β-cell that may contribute to its capacity to lower insulinemia and antidiabetic action.

Suppression of the enzyme glycogen synthase kinase 3β (GSK3β) increases both the turnover of damaged cellular material and the activity of the enzyme AMP-activated protein kinase (AMPK) to potentially attenuate the damage inflicted by excess sugar and fat on blood vessels., High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308, C249–C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.

We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4(+) mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific.

Exercise can prevent endothelial cell (EC) dysfunction and atherosclerosis even in the absence of improvements in plasma lipids. However, the mechanisms responsible for these effects are incompletely understood. In this study we examined in mice whether an acute bout of exercise activates enzymes that could prevent EC dysfunction, such as AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). We also examined whether exercise alters known regulators of these enzymes. C57BL/6 mice underwent a single bout of exhaustive treadmill exercise after which their aortas were analyzed for activation of AMPK, AMPK regulatory proteins, eNOS, and various enzymes that, like AMPK, activate eNOS. We found that such exercise acutely activates both AMPK and eNOS in the whole aorta and that the magnitude of these effects correlated with both the distance run and activation of the AMPK regulatory proteins silent information regulator-1 (SIRT1)-LKB1 and CaMKKβ. In contrast, Akt, PKA, PKG, and Src, other kinases known to activate eNOS, were unaffected. Immunohistochemical analysis revealed that AMPK and eNOS were both activated in the ECs of the aorta. This study provides the first evidence that an acute bout of exercise activates AMPK and eNOS in the endothelium of the aorta. The results also suggest that AMPK likely is the principal activator of eNOS in this setting and that its own activation may be mediated by both SIRT1-LKB1 and CaMKKβ.

Background Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. Methods and Results Mice harboring 1 functional allele of ABC-me (ABC-me +/− ) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me +/− mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me +/− hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me +/− hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. Conclusions Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

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.

Inflammation and infiltration of immune cells in white adipose tissue have been implicated in the development of obesity-associated insulin resistance. Likewise, dysregulation of the fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been proposed as a pathogenetic factor for these abnormalities based on both its links to insulin action and its anti-inflammatory effects. In this study, we examined the relationships between AMPK activity, the expression of multiple inflammatory markers in visceral (mesenteric and omental) and abdominal subcutaneous adipose tissue, and whole-body insulin sensitivity in morbidly obese patients (BMI 48 ± 1.9 kg/m2) undergoing gastric bypass surgery. AMPK activity was assessed by western-blots (P-AMPK/T-AMPK) and mRNA levels of various markers of inflammation by qRT-PCR. Patients were stratified as insulin sensitive obese or insulin resistant obese according to their HOMA-IR values. The results indicate that AMPK activity is lower in visceral than in subcutaneous abdominal adipose tissue of these patients and that this is associated with an increased expression of multiple inflammatory genes. They also revealed that AMPK activity is lower in adipose tissue of obese patients who are insulin resistant (HOMA-IR > 2.3) than in BMI-matched insulin sensitive subjects. Furthermore, this difference was evident in all three fat depots. In conclusion, the data suggest that there are close links between reduced AMPK activity and inflammation in white adipose tissue, and whole-body insulin resistance in obese humans. Whether adipose tissue AMPK dysregulation is a causal factor for the development of the inflammation and insulin resistance remains to be determined.

OBJECTIVE 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). RESEARCH DESIGN AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.—Liu, Q., Gauthier, M.-L., Sun, L., Ruderman, N., Lodish, H. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

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.

Background: Peroxisome proliferator-activated receptor α (PPARα) regulates the expression of fatty acid metabolism genes and is thought to play a role in the regulation of insulin secretion and lipid detoxification. We have examined the mechanism whereby glucose decreases PPARα gene expression in the pancreatic β-cell. Methods: INS832/13 β-cell and isolated rat islets were incubated at 3 and 20 mM glucose for 18 h in the absence or presence of adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators and inhibitors, as well as p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. In another set of experiments, INS832/13 were infected with an adenovirus expressing a dominant-negative form of AMPK. PPARα expression levels were measured by reverse transcription polymerase chain reaction and Western blot. Results: Elevated glucose reduced the abundance of the PPARα transcript and protein, and its target genes acyl-coenzyme A (CoA) oxidase (ACO) and uncoupling protein 2 (UCP-2) in INS832/13 β-cell and isolated rat islets. Glucose reduced AMPK activity, while the AMPK activators 5-amino-4-imidazolecarboxamide riboside and metformin increased PPARα expression and suppressed the action of glucose. By contrast, the AMPK inhibitor compound C mimicked the glucose effect. A dominant negative form of AMPKα reduced the PPARα, ACO and UCP-2 transcripts to the same extent as elevated glucose. Pharmacological evidence indicated that glucose-regulated PPARα expression does not involve p38 MAPK, a target of AMPK in several cell types. Conclusions: The results indicate that glucose represses PPARα gene expression via AMPK, but not via p38 MAPK in the β-cell.

OBJECTIVE 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. RESEARCH DESIGN AND METHODS We examined this question in skeletal muscle using an incubated rat extensor digitorum longus (EDL) muscle preparation as a tool. RESULTS 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 β-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 β-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. CONCLUSIONS 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.

We have previously shown that expression of the transcription factor ARNT/HIF1beta is reduced in islets of humans with type 2 diabetes. We have now found that ARNT is also reduced in livers of diabetics. To study the functional effect of its reduction, we created mice with liver-specific ablation (L-ARNT KO) using ARNT loxP mice and adenoviral-mediated delivery of Cre. L-ARNT KO mice had normal blood glucose but increased fed insulin levels. These mice also exhibited features of type 2 diabetes with increased hepatic gluconeogenesis, increased lipogenic gene expression, and low serum beta-hydroxybutyrate. These effects appear to be secondary to increased expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha), farnesoid X receptor (FXR), and sterol response element-binding protein 1c (SREBP-1c) and a reduction in phosphorylation of AMPK without changes in the expression of enzymes in ketogenesis, fatty acid oxidation, or FGF21. These results demonstrate that a deficiency of ARNT action in the liver, coupled with that in beta cells, could contribute to the metabolic phenotype of human type 2 diabetes.

Thiazolidinediones (TZDs) have beneficial effects on glucose homeostasis via enhancement of insulin sensitivity and preservation of beta-cell function. How TZDs preserve beta-cells is uncertain, but it might involve direct effects via both peroxisome proliferator-activated receptor-gamma-dependent and -independent pathways. To gain insight into the independent pathway(s), we assessed the effects of short-term (or=90 min) exposure to pioglitazone (Pio) (10 to 50 microM) on glucose-induced insulin secretion (GIIS), AMP-activated protein kinase (AMPK) activation, and beta-cell metabolism in INS 832/13 beta-cells and rat islets. Pio caused a right shift in the dose-dependence of GIIS, such that insulin release was reduced at intermediate glucose but unaffected at either basal or maximal glucose concentrations. This was associated in INS 832/13 cells with alterations in energy metabolism, characterized by reduced glucose oxidation, mitochondrial membrane polarization, and ATP levels. Pio caused AMPK phosphorylation and its action on GIIS was reversed by the AMPK inhibitor compound C. Pio also reduced palmitate esterification into complex lipids and inhibited lipolysis. As for insulin secretion, the alterations in beta-cell metabolic processes were mostly alleviated at elevated glucose. Similarly, the antidiabetic agents and AMPK activators metformin and berberine caused a right shift in the dose dependence of GIIS. In conclusion, Pio acutely reduces glucose oxidation, energy metabolism, and glycerolipid/fatty acid cycling of the beta-cell at intermediate glucose concentrations. We suggest that AMPK activation and the metabolic deceleration of the beta-cell caused by Pio contribute to its known effects to reduce hyperinsulinemia and preserve beta-cell function and act as an antidiabetic agent.

AMPK (AMP-activated protein kinase) is a phylogenetically conserved fuel-sensing enzyme that is present in all mammalian cells. During exercise, it is activated in skeletal muscle in humans, and at least in rodents, also in adipose tissue, liver and perhaps other organs by events that increase the AMP/ATP ratio. When activated, AMPK stimulates energy-generating processes such as glucose uptake and fatty acid oxidation and decreases energy-consuming processes such as protein and lipid synthesis. Exercise is perhaps the most powerful physiological activator of AMPK and a unique model for studying its many physiological roles. In addition, it improves the metabolic status of rodents with a metabolic syndrome phenotype, as does treatment with AMPK-activating agents; it is therefore tempting to attribute the therapeutic benefits of regular physical activity to activation of AMPK. Here we review the acute and chronic effects of exercise on AMPK activity in skeletal muscle and other tissues. We also discuss the potential role of AMPK activation in mediating the prevention and treatment by exercise of specific disorders associated with the metabolic syndrome, including Type 2 diabetes and Alzheimer's disease.

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.

SIRT1, a histone/protein deacetylase, and AMP-activated protein kinase (AMPK) are key enzymes responsible for longevity and energy homeostasis. We examined whether a mechanistic connection exists between these molecules that involves the major AMPK kinase LKB1. Initial studies demonstrated that LKB1 is acetylated in cultured (HEK293T) cells, mouse white adipose tissue, and rat liver. In the 293T cells, SIRT1 overexpression diminished lysine acetylation of LKB1 and concurrently increased its activity, cytoplasmic/nuclear ratio, and association with the LKB1 activator STRAD. In contrast, short hairpin RNA for SIRT1, where studied, had opposite effects on these parameters. Mass spectrometric analysis established that acetylation of LKB1 occurs on multiple, but specific, lysine residues; however, only mutation of lysine 48 to arginine, which mimics deacetylation, reproduced all of the effects of activated SIRT1. SIRT1 also affected downstream targets of LKB1. Thus its overexpression increased AMPK and acetyl-CoA carboxylase phosphorylation, and conversely, RNA interference-mediated SIRT1 knockdown reduced AMPK phosphorylation and that of another LKB1 target MARK1. Consistent with the results in cultured cells, total LKB1 lysine acetylation was decreased by 60% in the liver of 48-h starved rats compared with starved-refed rats, and this was associated with modest but significant increases in both LKB1 and AMPK activities. These results suggest that LKB1 deacetylation is regulated by SIRT1 and that this in turn influences its intracellular localization, association with STRAD, kinase activity, and ability to activate AMPK.

AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (isoproterenol, forskolin, and isobutylmethylxanthine), which stimulate lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activation by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stimulated lipolysis and AMPK activation but not that of PKA. Forskolin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isoproterenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activation of AMPK in adipocytes by cAMP-inducing agents is a consequence of lipolysis and not of PKA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acylation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis.

Objectives— Sedentary lifestyle increases the risk of cardiovascular disease and diabetes. Vascular dysfunction contributes to atherogenesis and has been linked to insulin resistance. Methods and Results— We measured insulin sensitivity by glucose tolerance test and vascular function by ultrasound and venous occlusion plethysmography in 20 healthy subjects (14 men, 6 women) at baseline and during 5 days of bed rest. Bed rest led to a 67% increase in the insulin response to glucose loading ( P P =0.01) and the calf (28.5±7.0 to 22.2±8.7 mL/min/dL, P =0.003) indicating impaired microvascular function. Bed rest decreased brachial artery diameter and increased systolic blood pressure suggesting increased basal arterial tone. There were no changes in circulating inflammatory markers arguing against systemic inflammation as a mechanism for vascular dysfunction in this setting. Conclusions— Physical inactivity was associated with the development of insulin resistance, dyslipidemia, increased blood pressure, and impaired microvascular function in healthy volunteers. Our findings may provide insight into the pathogenesis of vascular disease in sedentary individuals and emphasize that even short-term physical inactivity may have adverse metabolic and vascular consequences.

AMP-activated protein kinase (AMPK) plays a critical role both in sensing and regulating cellular energy state. In experimental animals, its activation has been shown to reduce the risk of obesity and diabetes-related co-morbidities such as insulin resistance, the metabolic syndrome and atherosclerotic cardiovascular disease. However, in humans, AMPK activation alone often does not completely resolve these conditions. Thus, an improved understanding of AMPK action and regulation in metabolic and other diseases is needed. Herein, we provide a brief description of the enzymatic regulation of AMPK and review its role in maintaining energy homeostasis. We then discuss tissue-specific actions of AMPK that become distorted during such conditions as obesity, type 2 diabetes and certain cancers. Finally, we explore recent findings regarding the interactions of AMPK with mammalian target of rapamycin complex 1 and the lysosome and discuss how changes in these relationships during overnutrition may lead to AMPK dysfunction. A more thorough understanding of AMPK's molecular interactions during diseases of overnutrition may provide key insights for the development of AMPK-based combinatorial treatments for metabolic disease.

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.

Stratifying the management of type 2 diabetes (T2D) has to take into account marked variability in patient phenotype due to heterogeneity in its pathophysiology, different stages of the disease process, and multiple other patient factors including comorbidities. The focus here is on the very challenging subgroup of patients with T2D who are overweight or obese with insulin resistance (IR) and the most refractory hyperglycemia due to an inability to change lifestyle to reverse positive energy balance. For this subgroup of patients with T2D, we question the dogma that IR is primarily harmful to the body and should be counteracted at any cost. Instead we propose that IR, particularly in this high-risk subgroup, is a defense mechanism that protects critical tissues of the cardiovascular system from nutrient-induced injury. Overriding IR in an effort to lower plasma glucose levels, particularly with intensive insulin therapy, could therefore be harmful. Treatments that nutrient off-load to lower glucose are more likely to be beneficial. The concepts of “IR as an adaptive defense mechanism” and “insulin-induced metabolic stress” may provide explanation for some of the unexpected outcomes of recent major clinical trials in T2D. Potential molecular mechanisms underlying these concepts; their clinical implications for stratification of T2D management, particularly in overweight and obese patients with difficult glycemic control; and future research requirements are discussed.

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished asmore » they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.« less

The study was designed to evaluate whether changes in malonyl-CoA and the enzymes that govern its concentration occur in human muscle as a result of physical training. Healthy, middle-aged subjects were studied before and after a 12-wk training program that significantly increased V̇o2 maxby 13% and decreased intra-abdominal fat by 17%. Significant decreases (25–30%) in the concentration of malonyl-CoA were observed after training, 24–36 h after the last bout of exercise. They were accompanied by increases in both the activity (88%) and mRNA (51%) of malonyl-CoA decarboxylase (MCD) in muscle but no changes in the phosphorylation of AMP kinase (AMPK, Thr172) or of acetyl-CoA carboxylase. The abundance of peroxisome proliferator-activated receptor (PPAR)γ coactivator-1α (PGC-1α), a regulator of transcription that has been linked to the mediation of MCD expression by PPARα, was also increased (3-fold). In studies also conducted 24–36 h after the last bout of exercise, no evidence of increased whole body insulin sensitivity or fatty acid oxidation was observed during an euglycemic hyperinsulinemic clamp. In conclusion, the concentration of malonyl-CoA is diminished in muscle after physical training, most likely because of PGC-1α-mediated increases in MCD expression and activity. These changes persist after the increases in AMPK activity and whole body insulin sensitivity and fatty acid oxidation, typically caused by an acute bout of exercise in healthy individuals, have dissipated.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2-4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3-kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C(2)-ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia-induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP-activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.

Acute increases in the concentration of malonyl-CoA play a pivotal role in mediating the decrease in fatty acid oxidation that occurs in many tissues during refeeding after a fast. In this study, we assess whether such increases in malonyl-CoA in liver could be mediated by malonyl-CoA decarboxylase (MCD), as well as acetyl-CoA carboxylase (ACC). In addition, we examine how changes in the activity of ACC, MCD, and other enzymes that govern fatty acid and glycerolipid synthesis relate temporally to alterations in the activities of the fuel-sensing enzyme AMP-activated protein kinase (AMPK). Rats starved for 48 h and refed a carbohydrate chow diet for 1, 3, 12, and 24 h were studied. Refeeding caused a 40% decrease in the activity of the α1-isoform of AMPK within 1 h, with additional decreases in AMPKα1 activity and a decrease in AMPKα2 occurring between 1 and 24 h. At 1 h, the decrease in AMPK activity was associated with an eightfold increase in the activity of the α1-isoform of ACC and a 30% decrease in the activity of MCD, two enzymes thought to be regulated by AMPK. Also, the concentration of malonyl-CoA was increased by 50%. Between 1 and 3 h of refeeding, additional increases in the activity of ACC and decreases in MCD were observed, as was a further twofold increase in malonyl-CoA. Increases in the activity (60%) and abundance (12-fold) of fatty acid synthase occurred predominantly between 3 and 24 h and increases in the activity of mitochondrial sn-glycerol-3-phosphate acyltransferase (GPAT) and acyl-CoA:diaclyglycerol acyltransferase (DGAT) at 12 and 24 h. The results strongly suggest that early changes in the activity of MCD, as well as ACC, contribute to the increase in hepatic malonyl-CoA in the starved-refed rat. They also suggest that the changes in these enzymes, and later occurring increases in enzymes regulating fatty acid and glycerolipid synthesis, could be coordinated by AMPK.

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18h and induced apoptosis in approximately 20% of cells after 24h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.

Apoptosis of pericytes (PCs) is an early event in diabetic retinopathy. It is generally thought to be a consequence of sustained hyperglycemia. In keeping with this, long-term (>7 days) incubation of cultured PCs in a high-glucose media has been shown to increase apoptosis. We examine here whether the saturated free fatty acid palmitate, the concentration of which is often elevated in diabetes, has similar effects on cultured PCs. Incubation with 0.4 mmol/l palmitate for 24 h induced both oxidant stress and apoptosis, as evidenced by a sixfold increase in DCF fluorescence and a twofold increase in caspase-3 activation, respectively. NAD(P)H oxidase appeared to be involved in these responses, since overexpression of dominant-negative subunits of NAD(P)H oxidase, such as phox47(DN), diminished oxidant stress, and phox67(DN) and N-17 RAC1(DN) prevented the increase in caspase-3 activity. Likewise, overexpression of vRAC, a constitutively active RAC1, increased caspase-3 activity to the same extent as palmitate alone. The effects of vRAC and palmitate were not additive. In parallel with the increases in oxidative stress, the redox-sensitive transcription factor nuclear factor-κB (NF-κB) was activated in cells incubated with 0.4 mmol/l palmitate. Furthermore, inhibition of NF-κB activation by various means inhibited caspase-3 activation. Finally, incubation with palmitate increased the cellular content of ceramide, a molecule linked to apoptosis and increases in oxidative stress and NF-κB activation in other cells. In keeping with such a role, in PCs both coincubation with fumonisin B1 (a ceramide synthase inhibitor) and overexpression of ceramidase I reversed the proapoptotic effect of palmitate. On the other hand, they increased rather than decreased DCF fluorescence. In conclusion, the results suggest that palmitate-induced apoptosis in PCs is associated with activation of NAD(P)H oxidase and NF-κB and an increase in ceramide. The precise interactions between these molecules in causing apoptosis and the importance of oxidant stress as a contributory factor remain to be determined.

Insulin resistance contributes to type 2 diabetes and the metabolic syndrome. Activation of AMP-activated protein kinase (AMPK) in muscle and liver increases fatty acid oxidation and diminishes the synthesis of malonyl-CoA, glycerolipids, ceramide and other molecules associated with insulin resistance. In experimental animals, decreases in AMPK activity and increases in malonyl-CoA are linked to insulin resistance and might be targets for its therapy. Whether this is true for humans remains to be determined.