Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.     

Metabolomics Analysis Reveals that AICAR Affects Glycerolipid,Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells

AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using ¹³C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.     

Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes through AMP-activated Protein Kinase (AMPK)

Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60–80 μm). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy.     

Effects of Exercise on AMPK Signaling and Downstream Components to PI3K in Rat with Type 2 Diabetes

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr³⁰⁸) and (Ser⁴⁷³) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.     

Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPK^Ser-485, but not AMPK^Thr-172, phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPK^Ser-485, but not AMPK^Thr-172, hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPK^Ser-485 hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPK^S485A mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPK^Ser-485 hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPK^Ser-485 phosphorylation to the increased risk of AD in obesity and diabetes.     

Ptc1 Protein Phosphatase 2C Contributes to Glucose Regulation of SNF1/AMP-activated Protein Kinase (AMPK) in Saccharomyces cerevisiae

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.     

The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.     

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) Effect on Glucose Production,but Not Energy Metabolism,Is Independent of Hepatic AMPK in Vivo

Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 8 mg·kg⁻¹·min⁻¹)-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1α2^lox/lox) and liver-specific AMPK knock-out mice (α1α2^lox/lox + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1α2^lox/lox and α1α2^lox/lox + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ∼20% in α1α2^lox/lox, which was exacerbated by ∼2-fold in α1α2^lox/lox + Albcre. This corresponded to a ∼6-fold rise in AMP/ATP in α1α2^lox/lox + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ∼30% lower in α1α2^lox/lox + Albcre than α1α2^lox/lox livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.     

Identification and validation of the pathways and functions regulated by the orphan nuclear receptor,ROR alpha1, in skeletal muscle

The retinoic acid receptor-related orphan receptor (ROR) alpha has been demonstrated to regulate lipid metabolism. We were interested in the RORα1 dependent physiological functions in skeletal muscle. This major mass organ accounts for ∼40% of the total body mass and significant levels of lipid catabolism, glucose disposal and energy expenditure. We utilized the strategy of targeted muscle-specific expression of a truncated (dominant negative) RORα1ΔDE in transgenic mice to investigate RORα1 signaling in this tissue. Expression profiling and pathway analysis indicated that RORα influenced genes involved in: (i) lipid and carbohydrate metabolism, cardiovascular and metabolic disease; (ii) LXR nuclear receptor signaling and (iii) Akt and AMPK signaling. This analysis was validated by quantitative PCR analysis using TaqMan low-density arrays, coupled to statistical analysis (with Empirical Bayes and Benjamini–Hochberg). Moreover, westerns and metabolic profiling were utilized to validate the genes, proteins and pathways (lipogenic, Akt, AMPK and fatty acid oxidation) involved in the regulation of metabolism by RORα1. The identified genes and pathways were in concordance with the demonstration of hyperglycemia, glucose intolerance, attenuated insulin-stimulated phosphorylation of Akt and impaired glucose uptake in the transgenic heterozygous Tg-RORα1ΔDE animals. In conclusion, we propose that RORα1 is involved in regulating the Akt2-AMPK signaling pathways in the context of lipid homeostasis in skeletal muscle.     

PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle

Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance due to its role in the regulation of fatty acid oxidation. The 5’-AMP-activated protein kinase (AMPK) is an energy sensor in cells, but its role in PPARα regulation in vivo remains unknown. In this study, we examined PPARα expression in the skeletal muscle of AMPKα2 overexpression (OE), knockout (KO) and wild-type (WT) mice after four weeks of exercise under intermittent hypoxia. WT, OE and KO mice were used at 40 mice/strain and randomly subdivided into four subgroups: control (C), running (R), hypoxia (H), and running plus hypoxia (R+H) at 10 mice/group. The treadmill running was performed at the speed of 12 m/min, 60 min/day with a slope of 0 degree for four weeks. The hypoxia treatment was performed in daytime with normobaric hypoxia (11.20% oxygen, 8 hours/day). In the R+H group, the treadmill running was conducted in the hypoxic condition. AMPKα2, phosphor-AMPKα (p-AMPKα) (Thr172), nuclear PPARα proteins were measured by Western blot and the medium chain acyl coenzyme A dehydrogenase (MCAD) mRNA, the key enzyme for fatty acid oxidation and one of the PPARα target genes, was also measured in skeletal muscles after the interventions. The results showed that nuclear PPARα protein was significantly increased by R+H in WT muscles, the increase was enhanced by 41% (p<0.01) in OE mice, but was reduced by 33% (p<0.01) in KO mice. The MCAD mRNA expression was increased after four weeks of R+H intervention. The change in MCAD mRNA followed a similar trend as that of PPARα protein in OE and KO mice. Our data suggest that the increase in nuclear PPARα protein by four-week exercise training under the intermittent hypoxia was dependent on AMPK activation.     

Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.     

Sumoylation of AMPKβ2 subunit enhances AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is a heterotrimer composed of a catalytic α and two regulatory subunits (β and γ). AMPK activity is regulated allosterically by AMP and by the phosphorylation of residue Thr-172 within the catalytic domain of the AMPKα subunit by upstream kinases. We present evidence that the AMPKβ2 subunit may be posttranslationally modified by sumoylation. This process is carried out by the E3-small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT PIASy, which modifies the AMPKβ2 subunit by the attachment of SUMO2 but not SUMO1 moieties. Of interest, AMPKβ1 is not a substrate for this modification. We also demonstrate that sumoylation of AMPKβ2 enhances the activity of the trimeric α2β2γ1 AMPK complex. In addition, our results indicate that sumoylation is antagonist and competes with the ubiquitination of the AMPKβ2 subunit. This adds a new layer of complexity to the regulation of the activity of the AMPK complex, since conditions that promote ubiquitination result in inactivation, whereas those that promote sumoylation result in the activation of the AMPK complex.     

Combination of honokiol and magnolol inhibits hepatic steatosis through AMPK-SREBP-1?c pathway

Honokiol and magnolol, as pharmacological biphenolic compounds of Magnolia officinalis, have been reported to have antioxidant and anti-inflammatory properties. Sterol regulatory element binding protein-1 c (SREBP-1 c) plays an important role in the development and processing of steatosis in the liver. In the present study, we investigated the effects of a combination of honokiol and magnolol on SREBP-1 c-dependent lipogenesis in hepatocytes as well as in mice with fatty liver due to consumption of high-fat diet (HFD). Liver X receptor α (LXRα) agonists induced activation of SREBP-1 c and expression of lipogenic genes, which were blocked by co-treatment of honokiol and magnolol (HM). Moreover, a combination of HM potently increased mRNA of fatty acid oxidation genes. HM induced AMP-activated protein kinase (AMPK), an inhibitory kinase of the LXRα-SREBP-1 c pathway. The role of AMPK activation induced by HM was confirmed using an inhibitor of AMPK, Compound C, which reversed the ability of HM to both inhibit SREBP-1 c induction as well as induce genes for fatty acid oxidation. In mice, HM administration for four weeks ameliorated HFD-induced hepatic steatosis and liver dysfunction, as indicated by plasma parameters and Oil Red O staining. Taken together, our results demonstrated that a combination of HM has beneficial effects on inhibition of fatty liver and SREBP-1 c-mediated hepatic lipogenesis, and these events may be mediated by AMPK activation.     

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK,DNA Synthesis and Proliferation in Pancreatic Cancer Cells

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser⁷⁹. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr³⁸⁹ and S6 at Ser^240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α₁ and α₂ catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.     

AMPK α1 Activation Is Required for Stimulation of Glucose Uptake by Twitch Contraction,but Not by H2O2, in Mouse Skeletal Muscle

Background

AMPK is a promising pharmacological target in relation to metabolic disorders partly due to its non-insulin dependent glucose uptake promoting role in skeletal muscle. Of the 2 catalytic α-AMPK isoforms, α₂ AMPK is clearly required for stimulation of glucose transport into muscle by certain stimuli. In contrast, no clear function has yet been determined for α₁ AMPK in skeletal muscle, possibly due to α-AMPK isoform signaling redundancy. By applying low-intensity twitch-contraction and H₂O₂ stimulation to activate α₁ AMPK, but not α₂ AMPK, in wildtype and α-AMPK transgenic mouse muscles, this study aimed to define conditions where α₁ AMPK is required to increase muscle glucose uptake.

Methodology/Principal Findings

Following stimulation with H₂O₂ (3 mM, 20 min) or twitch-contraction (0.1 ms pulse, 2 Hz, 2 min), signaling and 2-deoxyglucose uptake were measured in incubated soleus muscles from wildtype and muscle-specific kinase-dead AMPK (KD), α₁ AMPK knockout or α₂ AMPK knockout mice. H₂O₂ increased the activity of both α₁ and α₂ AMPK in addition to Akt phosphorylation, and H₂O₂-stimulated glucose uptake was not reduced in any of the AMPK transgenic mouse models compared with wild type. In contrast, twitch-contraction increased the activity of α₁ AMPK, but not α₂ AMPK activity nor Akt or AS160 phosphorylation. Glucose uptake was markedly lower in α₁ AMPK knockout and KD AMPK muscles, but not in α₂ AMPK knockout muscles, following twitch stimulation.

Conclusions/Significance

These results provide strong genetic evidence that α₁ AMPK, but not α₂ AMPK, Akt or AS160, is necessary for regulation of twitch-contraction stimulated glucose uptake. To our knowledge, this is the first report to show a major and essential role of α₁ AMPK in regulating a physiological endpoint in skeletal muscle. In contrast, AMPK is not essential for H₂O₂-stimulated muscle glucose uptake, as proposed by recent studies.     

Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform α1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside or constitutively active α1AMPK (CA-α1) significantly inhibits; although inhibiting α1AMPK by short hairpin RNA knock-down or dominant-negative α1AMPK (DN-α1) increases LPS- and FFA-induced tumor necrosis factor α expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-α1 in macrophages significantly inhibits LPS- or FFA-induced NF-κB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-α1 increases the SIRT1 activator NAD⁺ content and SIRT1 expression in macrophages. Furthermore, α1AMPK activation mimics the effect of SIRT1 on deacetylating NF-κB, and the full capacity of AMPK to deacetylate NF-κB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.     

Mechanism of Butyrate Stimulation of Triglyceride Storage and Adipokine Expression during Adipogenic Differentiation of Porcine Stromovascular Cells

Short chain fatty acids (SCFA), products of microbial fermentation of dietary fiber, exert multiple metabolic effects in cells. Previously, we had demonstrated that soluble fiber influenced fat mass accumulation, gut microbial community structure and SCFA production in pigs. The current study was designed to identify effects of SCFA treatment during adipogenic differentiation of porcine stromovascular cells on lipid metabolism and adipokine expression. Differentiating cells were treated with varying concentrations of butyrate. Results show that butyrate treatment enhanced adipogenesis and lipid accumulation, perhaps through upregulation of glucose uptake and de novo lipogenesis and other mechanisms that include induction of SREBP-1c, C/EBPα/β, GLUT4, LPL, PPARγ, GPAT4, DGAT1 and DGAT2 expression. In addition, butyrate induced adiponectin expression, resulting in activation of downstream target genes, such as AMPK and AKT. Activation of AMPK by butyrate led to phosphorylation of ACC. Although increased ACO gene expression was seen with butyrate treatment, experiments with the peroxisomal fatty acid inhibitor, thioridazine, suggest that butyrate may have an inhibitory effect on peroxisomal fatty acid oxidation. Our studies also provide evidence that butyrate may inhibit lipolysis, perhaps in an FFAR3-dependent manner. Therefore, this study presents a novel paradigm for butyrate action in adipocytes and shows that adipocytes are capable of utilizing butyrate, leading to increased expression of adiponectin for enhanced glucose uptake and improved insulin sensitivity.