Medicine (St Vincent's) - Research Publications
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ItemPPARδ activation attenuates hepatic steatosis in Ldlr-/- mice by enhanced fat oxidation, reduced lipogenesis, and improved insulin sensitivity(ELSEVIER, 2014-07)PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr(-/-) mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1(-/-) hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1(-/-) hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1(-/-) hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.
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ItemNo Preview AvailableMetformin inhibits gluconeogenesis via a redox-dependent mechanism in vivo(NATURE PUBLISHING GROUP, 2018-09)Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.
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ItemNo Preview AvailableSingle phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin(NATURE PUBLISHING GROUP, 2013-12)The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed type 2 diabetes drug metformin. Ampk phosphorylates mouse acetyl-CoA carboxylase 1 (Acc1; refs. 3,4) at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl-CoA to malonyl-CoA. The latter metabolite is a precursor in fatty acid synthesis and an allosteric inhibitor of fatty acid transport into mitochondria for oxidation. To test the physiological impact of these phosphorylation events, we generated mice with alanine knock-in mutations in both Acc1 (at Ser79) and Acc2 (at Ser212) (Acc double knock-in, AccDKI). Compared to wild-type mice, these mice have elevated lipogenesis and lower fatty acid oxidation, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Notably, AccDKI mice made obese by high-fat feeding are refractory to the lipid-lowering and insulin-sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism and, in the setting of obesity, for metformin-induced improvements in insulin action.
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ItemMechanism of Action of Compound-13: An α1-Selective Small Molecule Activator of AMPK(CELL PRESS, 2014-07-17)AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, β1, or β2.
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ItemSalicylate improves macrophage cholesterol homeostasis via activation of Ampk(ELSEVIER, 2015-05)Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk β1-deficient (β1(-/-)) mice. Macrophages from Ampk β1(-/-) mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk β1(-/-) macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk β1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk β1(-/-) macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis.
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ItemAMPK β1 reduces tumor progression and improves survival in p53 null mice(WILEY, 2017-09)The AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T-cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin-1β (IL1β), reductions in acetyl-CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T-cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53-mutant tumors.
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ItemSkeletal muscle AMPK is essential for the maintenance of FNDC5 expression(WILEY, 2015-05)Fibronectin type III domain-containing protein 5 (FNDC5) expression is controlled by the transcriptional co-activator, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α). FNDC5 expression has been shown to be increased in muscle in response to endurance exercise in some but not all studies, therefore a greater understanding of the mechanisms controlling this process are needed. The AMP-activated protein kinase (AMPK) is activated by exercise in an intensity dependent manner and is an important regulator of PGC1α activity; therefore, we explored the role of AMPK in the regulation of FNDC5 using AMPK β1β2 double muscle-null mice (AMPK DMKO), which lack skeletal muscle AMPK activity. We found that FNDC5 expression is dramatically reduced in resting muscles of AMPK DMKO mice compared to wild-type littermates. In wild-type mice, activating phosphorylation of AMPK was elevated immediately post contraction and was abolished in muscle from AMPK DMKO mice. In contrast, PGC1α was increased in both wild-type and AMPK DMKO mice 3 h post contraction but FNDC5 protein expression was not altered. Lastly, acute or chronic activation of AMPK with the pharmacological AMPK activator AICAR did not increase PGC1α or FNDC5 expression in muscle. These data indicate that skeletal muscle AMPK is required for the maintenance of basal FNDC5 expression.
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ItemThe autophagy initiator ULK1 sensitizes AMPK to allosteric drugs(NATURE PUBLISHING GROUP, 2017-09-18)AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates β1-Ser108 on the regulatory β1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop.
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ItemAMPK signaling to acetyl-CoA carboxylase is required for fasting- and cold-induced appetite but not thermogenesis(ELIFE SCIENCES PUBLICATIONS LTD, 2018-01-30)AMP-activated protein kinase (AMPK) is a known regulator of whole-body energy homeostasis, but the downstream AMPK substrates mediating these effects are not entirely clear. AMPK inhibits fatty acid synthesis and promotes fatty acid oxidation by phosphorylation of acetyl-CoA carboxylase (ACC) 1 at Ser79 and ACC2 at Ser212. Using mice with Ser79Ala/Ser212Ala knock-in mutations (ACC DKI) we find that inhibition of ACC phosphorylation leads to reduced appetite in response to fasting or cold exposure. At sub-thermoneutral temperatures, ACC DKI mice maintain normal energy expenditure and thermogenesis, but fail to increase appetite and lose weight. We demonstrate that the ACC DKI phenotype can be mimicked in wild type mice using a ghrelin receptor antagonist and that ACC DKI mice have impaired orexigenic responses to ghrelin, indicating ACC DKI mice have a ghrelin signaling defect. These data suggest that therapeutic strategies aimed at inhibiting ACC phosphorylation may suppress appetite following metabolic stress.
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ItemInhibition of Adenosine Monophosphate-Activated Protein Kinase-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Signaling Leads to Hypercholesterolemia and Promotes Hepatic Steatosis and Insulin Resistance(JOHN WILEY & SONS LTD, 2019-01)Adenosine monophosphate-activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate-limiting enzyme in the mevalonate pathway 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine-871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK-HMGCR signaling in vivo, we generated mice with a Ser871-alanine (Ala) knock-in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild-type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up-regulated in HMGCR KI mice. When fed a high-carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK-HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high-carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high-carbohydrate diet.