AMP-activated Protein Kinase Α1 Research Articles

Constitutive activation of AMPK α1 in vascular endothelium promotes high‐fat diet‐induced fatty liver injury: role of COX‐2 induction

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, comprises three (α, β and γ) subunits, each with a unique tissue distribution. As AMPK has a wide range of protein and gene targets, defining its role has been difficult. Here, we have studied a transgenic mouse model overexpressing the constitutively active α1 subunit of AMPK in endothelial cells (EC-AMPK) to elucidate its role in energy homeostasis. Wild-type and EC-AMPK mice were fed with a high fat diet for 16 weeks. Drugs (or vehicles) were given daily by oral gavage. Body weight, fat mass composition, glucose and lipid levels were monitored regularly. Tissues including aortae and liver were collected for quantitative RT-PCR, Western blotting, elisa, histological and biochemical evaluations. Compared with wild-type animals, high fat diet caused more severe metabolic defects in EC-AMPK mice, which exhibited increased body weight and fat mass, elevated blood pressure, augmented glucose and lipid levels, impaired glucose tolerance, hepatomegaly and steatohepatitis. Constitutive activation of AMPK α1 in endothelial cells induced COX-2 expression and arterial inflammation. Genes involved in lipid metabolism were down-regulated in aortae and livers of EC-AMPK mice. Chronic treatment with selective COX-2 inhibitors, celecoxib or nimesulide, significantly ameliorated arterial inflammation, steatohepatitis and hyperlipidaemia in EC-AMPK mice, without altering their blood pressure or clotting. Constitutive activation of endothelial AMPK α1 promotes vascular inflammation and the development of obesity-induced fatty livers largely via induction of COX-2.

Thymoquinone induces heme oxygenase-1 expression in HaCaT cells via Nrf2/ARE activation: Akt and AMPKα as upstream targets

Thymoquinone (TQ), an active constituent of Nigella sativa, possesses anti-inflammatory and anticancer properties. Multiple lines of evidence suggest that the induction of heme oxygenase-1 (HO-1) suppresses inflammation and carcinogenesis. In the present study, we examined the effect of TQ on HO-1 expression in human keratinocytes (HaCaT) and elucidated its underlying molecular mechanisms. TQ induced the expression of HO-1 in HaCaT cells in a concentration- and time-dependent manner. Treatment with TQ increased the localization of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) in the nucleus and elevated the antioxidant response element (ARE)-reporter gene activity. Knockdown of Nrf2 abrogated TQ-induced HO-1 expression and the ARE luciferase activity. TQ induced the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and cyclic AMP-activated protein kinase-α (AMPKα). Pharmacological inhibition of Akt or AMPKα, but not that of ERK, abrogated TQ-induced nuclear localization of Nrf2, the ARE-luciferase activity and the expression of HO-1. TQ also generated reactive oxygen species (ROS) and pretreatment with N-acetyl cysteine (NAC) abrogated TQ-induced ROS accumulation, Akt and AMPKα activation, Nrf2 nuclear localization, the ARE-luciferase activity, and HO-1 expression in HaCaT cells. Taken together, TQ induces HO-1 expression in HaCaT cells by activating Nrf2 through ROS-mediated phosphorylation of Akt and AMPKα.

Hepatic oxidative stress in ovariectomized transgenic mice expressing the hepatitis C virus polyprotein is augmented through suppression of adenosine monophosphate‐activated protein kinase/proliferator‐activated receptor gamma co‐activator 1 alpha signaling

Oxidative stress plays an important role in hepatocarcinogenesis of hepatitis C virus (HCV)-related chronic liver diseases. Despite the evidence of an increased proportion of females among elderly patients with HCV-related hepatocellular carcinoma (HCC), it remains unknown whether HCV augments hepatic oxidative stress in postmenopausal women. The aim of this study was to determine whether oxidative stress was augmented in ovariectomized (OVX) transgenic mice expressing the HCV polyprotein and to investigate its underlying mechanisms. OVX and sham-operated female transgenic mice expressing the HCV polyprotein and non-transgenic littermates were assessed for the production of reactive oxygen species (ROS), expression of inflammatory cytokines and antioxidant potential in the liver. Compared with OVX non-transgenic mice, OVX transgenic mice showed marked hepatic steatosis and ROS production without increased induction of inflammatory cytokines, but there was no increase in ROS-detoxifying enzymes such as superoxide dismutase 2 and glutathione peroxidase 1. In accordance with these results, OVX transgenic mice showed less activation of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which is required for the induction of ROS-detoxifying enzymes, and no activation of adenosine monophosphate-activated protein kinase-α (AMPKα), which regulates the activity of PGC-1α. Our study demonstrated that hepatic oxidative stress was augmented in OVX transgenic mice expressing the HCV polyprotein by attenuation of antioxidant potential through inhibition of AMPK/PGC-1α signaling. These results may account in part for the mechanisms by which HCV-infected women are at high risk for HCC development when some period has passed after menopause.

Changes in Energy-Regulated Molecules in the Trophocytes and Fat Cells of Young and Old Worker Honeybees (Apis mellifera)

Trophocytes and fat cells of honeybees (Apis mellifera) have been used for cellular senescence studies, but the changes in the expression, concentration, and activity of cellular energy-regulated molecules that occur with aging in worker bees is unknown. In this study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old workers. The results showed that (i) adenosine monophosphate-activated protein kinase-α2 (AMPK-α2) expression increased with aging, whereas phosphorylated AMPK-α2 expression, the phosphorylated AMPK/AMPK ratio, and AMPK activity decreased with aging; (ii) adenosine diphosphate and adenosine triphosphate concentrations decreased with aging, the AMP concentration was unchanged, the adenosine diphosphate/adenosine triphosphate ratio did not change with aging, and the AMP/adenosine triphosphate ratio increased with aging; (iii) the cyclic AMP concentration decreased with aging, and cyclic AMP-specific phosphodiesterases activity increased with aging; (iv) silent information regulator 2 (Sir2) expression increased with aging, whereas its activity decreased with aging; and (v) peroxisome proliferator-activated receptor-α expression decreased with aging. These results show that the trophocytes and fat cells of young workers have higher cellular energy status and express higher levels of energy-regulated molecules than those of old workers and that aging results in a decline in the energy status of trophocytes and fat cells in worker honeybees.

Effects of Roux-en-Y gastric bypass and ileal transposition surgeries on glucose and lipid metabolism in skeletal muscle and liver

BackgroundRoux-en Y gastric bypass (RYGB) and ileal transposition (IT) surgeries produce weight loss and improve diabetic control; however, the mechanisms of glycemic improvements are largely unknown. Because skeletal muscle and liver play a key role in glucose homeostasis, we compared the effects of RYGB and IT surgeries on key molecules of glucose and lipid metabolism in muscle and liver. MethodsSprague-Dawley rats were subjected to RYGB, IT, or sham surgeries; sham-animals were ad-lib fed or pair-fed to RYGB rats (n = 7-9/group). At 8 weeks postoperatively, blood samples were collected for glucagon-like peptide-1 (GLP-1) and insulin analyses by ELISA. Leg muscle and liver tissues were analyzed for mRNA (RT-qPCR) and/or protein abundance (immuno blotting) of important molecules of glucose and lipid metabolism [glucose transporter-4 (GLUT-4), hexokinase, phosphofructokinase (PFK), adenosine monophosphate activated protein kinase-α (AMPKα), cytochrome C oxidase-IV (COX-IV), citrate synthase, carnitine palmitoyl transferase-1 (CPT-1), medium-chain acyl-CoA dehydrogenase (MCAD), peroxisome proliferator-activated receptor gamma co-activator 1 α (PGC-1 α), PGC-1-related coactivator (PRC), uncoupling protein-3 (UCP-3)]. ResultsPlasma GLP-1 concentrations were increased comparably with RYGB and IT. RYGB and IT increased muscle GLUT-4 protein content, muscle hexokinase mRNA, and liver PFK mRNA. IT increased muscle AMPKα and COX-IV protein content and liver citrate synthase activity. IT increased muscle CPT-1, MCAD and PRC mRNA, whereas RYGB increased UCP-3 mRNA in muscle and liver, and PGC-1 α mRNA in liver. ConclusionThe data suggest that RYGB and IT surgeries lead to enhanced GLP-1 secretion and produce similar stimulatory effects on important molecules of glucose metabolism but differential effects on key molecules of lipid oxidation in muscle and liver.

Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

AMP‐activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.

C1q/Tumor Necrosis Factor–Related Protein-9, a Novel Adipocyte-Derived Cytokine, Attenuates Adverse Remodeling in the Ischemic Mouse Heart via Protein Kinase A Activation

C1q/tumor necrosis factor-related protein-9 (CTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. However, the role of CTRP9 in postmyocardial infarction remodeling remains completely unknown. This study determined whether CTRP9 may regulate cardiac remodeling after acute myocardial infarction (AMI) and elucidated the underlying mechanisms. Male adult mice were subject to AMI by left anterior descending coronary artery ligation or sham surgery and treated with saline (vehicle) or globular CTRP9 via peritoneal implant osmotic pumps for 6 weeks. H9C2 cardiac cell lines were used in vitro for determining underlying mechanisms. Adipocyte CTRP9 expression and plasma CTRP9 levels were both significantly reduced after AMI. Compared with vehicle, CTRP9 treatment improved animal survival rate (P<0.05), restored cardiac function (P<0.05), attenuated adverse remodeling (P<0.01), and ameliorated cardiomyocyte apoptosis and fibrosis after AMI (P<0.01). Among the multiple antiremodeling molecules determined, AMP-activated protein kinase, protein kinase A (PKA), and Akt were significantly activated in CTRP9-treated heart. Surprisingly, CTRP9 remains cardioprotective in mice with cardiomyocyte-specific overexpression of a mutant AMP-activated protein kinase α2 subunit (AMPK-DN). Additional in vitro experiments demonstrated that administration of either PKA inhibitor or PKA-specific small interfering RNA virtually abolished the antiapoptotic effect of CTRP9 (P<0.05), whereas inhibition of Akt is less effective in blocking CTRP9 cardioprotection. Finally, CTRP9 phosphorylates BCL-2-associated agonist of cell death at its multiple antiapoptotic sites, an effect blocked by PKA inhibitor. We demonstrate that adipokine CTRP9 attenuates adverse cardiac remodeling after AMI, largely via a PKA-dependent pathway.

Light-to-Moderate Ethanol Feeding Augments AMPK-α Phosphorylation and Attenuates SREBP-1 Expression in the Liver of Rats

Fatty liver disease, a hepatic manifestation of metabolic syndrome, is one of the major causes of chronic liver diseases. Epidemiological studies suggest that regular light-to-moderate ethanol consumption lowers the risk of developing metabolic disorders including dislipidemia, insulin resistance, type 2 diabetes and fatty liver disease. However, the mechanism(s) of the protective effect of light-to-moderate ethanol consumption on the liver remains unknown. In the present study, we investigated the effects of light (6%, 0.94 g/kg/day) and moderate (12%, 1.88 g/kg/day) ethanol feeding in rats for 3 weeks on the circulating and hepatic biochemical profiles and on the hepatic protein expression and phosphorylation status of adenosine monophosphate-activated protein kinase-α (AMPK-α) and other down-stream targets of this enzyme including sterol regulatory element-binding protein-1 (SREBP-1), SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase). Despite no significant difference in food-intake among the groups, light ethanol treatment significantly increased the body weight compared to control rats. Serum glucose, insulin, total cholesterol, triglycerides, phospholipids and hepatic cholesterol and triglycerides were not significantly different among the groups. However, serum free fatty acids were significantly reduced with light ethanol treatment. Both light and moderate ethanol treatment significantly increased the hepatic levels of phosphorylated AMPK-α protein and this was associated with significant reduction of SREBP-1 protein expression, suggesting an enhanced fatty acid oxidation. In addition, light ethanol treatment significantly decreased the SCAP protein expression in the liver. However, liver HMG-CoA protein expression was not significantly different with ethanol consumption. Chronic light-to-moderate ethanol consumption increased AMPK activation which was associated with decreased expression of SREBP-1 and SCAP in the liver. Thus, our studies provide mechanistic evidence for the earlier epidemiological studies that indicate light-to-moderate ethanol intake lowers the risk of development of fatty liver disease and other metabolic disorders. Our studies demonstrate that the protective effects of light-to-moderate ethanol arise at least in part by increased phosphorylation of AMPK-α and decreased SREBP-1 expression in the liver. Further studies are warranted to determine the effects of light-to-moderate ethanol on intracellular up-stream and down-stream targets of AMPK and also on the implications of light-to-moderate ethanol in protecting non-alcoholic fatty liver disease.

Notch1 Receptor Regulates AKT Protein Activation Loop (Thr308) Dephosphorylation through Modulation of the PP2A Phosphatase in Phosphatase and Tensin Homolog (PTEN)-null T-cell Acute Lymphoblastic Leukemia Cells

Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

Anti-neuroinflammatory Effect of a Novel Caffeamide Derivative, KS370G, in Microglial cells

Accumulating evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play important roles in several neurodegenerative disorders. Therefore, development of methods for microglial inhibition is considered an important strategy in the search for neuroprotective agents. Caffeic acid phenethyl ester (CAPE) is distributed wildly in nature, but rapid decomposition by esterase leads to its low bioavailability. In this study, we investigated the effects of KS370G, a novel caffeic acid phenylethyl amide, on microglial activation. KS370G significantly inhibited the release of nitric oxide (NO) and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with KS370G also induced heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS)-3 expression in the microglia. Furthermore, the anti-inflammatory effects of KS370G were found to be regulated by phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) translocated to the nucleus. Moreover, KS370G showed significant anti-neuroinflammatory effects on microglial activation in vivo and on motor behavior as well. The protective effect of KS370G was weakened by an AMPK inhibitor Compound C. This study focuses on the importance of key molecular determinants of inflammatory homeostasis, AMPK, HO-1, and SOCS-3, and their possible involvement in anti-neuroinflammatory responses.

AMP-activated protein kinase (AMPK) α2 subunit mediates glycolysis in postmortem skeletal muscle

Postmortem glycolysis is directly linked to the incidences of PSE (pale, soft and exudative) and DFD (dark, firm and dry) meats which cause significant loss to meat industry. AMP-activated protein kinase (AMPK) is a major regulator of postmortem glycolysis. However, there are two isoforms of the AMPKα catalytic subunit, and their roles in glycolysis of postmortem muscle remain unclear. The objective was to identify the isoform specific roles of AMPK in postmortem glycolysis. Wild type, AMPKα1, and AMPKα2 knockout (KO) mice were used in the current study. AMPK in Longissimus muscle was activated shortly after death. AMPKα2 but not AMPKα1 KO abolished the activity of AMPK in postmortem muscle. In addition, AMPKα2 KO reduced postmortem pH decline and the generation of lactate, while AMPKα1 KO had no significant effect. Finally, the glycogen content of skeletal muscle was reduced in AMPKα2 KO but not AMPKα1 KO mice. Data clearly demonstrate that AMPKα2 catalytic subunit mainly regulates postmortem glycolysis in muscle.

LKB1 Regulates Lipid Oxidation During Exercise Independently of AMPK

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD+ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.

AMPK regulates contraction‐induced glucose uptake in situ but not ex vivo

The use of transgenic models studying the role of 5′AMP‐activated protein kinase (AMPK) in contraction‐induced glucose uptake (CTGU) has led to inconclusive results. Drawbacks of using less appropriate models displaying incomplete abrogation of AMPKα catalytic activity may explain these differences. The purpose of this study was to determine whether a total lack of both AMPK α1 and α2 catalytic subunits abolishes CTGU in muscle under ex vivo and in situ conditions.We generated a skeletal muscle specific AMPK‐α1 and‐α2 double knock‐out (dKO) mouse model. Electrical stimulation was applied directly to the muscle (ex vivo, 10 min (either 1 or 2 second trains/15 seconds)) or to the sciatic nerve (in situ, 15 min (1 train/second) +30 min recovery) to stimulate contraction. Muscle glucose uptake was measured using radioactively labeled isotopes. Ex vivo CTGU in AMPKα WT and dKO soleus and extensor digitorum longus (EDL) muscles was similar between genotypes. Equal force production was registered under all of the above conditions. Contrary to the ex vivo data, the CTGU in situ was impaired by ~50% in the tibialis anterior, soleus and EDL muscles of AMPKα dKO mice compared to WT mice (p&lt;0.05). Basal non stimulated glucose uptake was similar between genotypes, both ex vivo and in situ.The results suggest that AMPK's role in CTGU may be modulated by a factor(s) related to muscle perfusion and/or stimulation methodology.

Dietary wolfberry up‐regulates BCDO2 and enhances mitochondrial biogenesis in the retina of db/db type 2 diabetic mice

ObjectiveTo investigate whether wolfberry regulated β,β ‐ carotene 9′,10′‐dioxygenase 2 (BCDO2) expression and enhanced mitochondrial biogenesis in the diabetic retina.MethodsMale db/db and wild‐type mice at 6 weeks of age were fed the control or wolfberry diets for 8 weeks. At study termination, the retina tissues were collected for analysis by transmission electron microscopy, real‐time PCR, immunoprecipitation, and Western blot.ResultsWolfberry up‐regulated expression of retinal BCDO2 transcript and protein. Wolfberry induced activation and nuclear enrichment of retinal AMP‐activated protein kinase α2. Wolfberry attenuated hypoxia and mitochondrial stress by declined expression of hypoxia‐inducible factor‐1α, vascular endothelial growth factor, and heat shock protein 60. Wolfberry reversed mitochondrial dispersion in the retinal pigment epithelium, increased mitochondrial copy number, elevated citrate synthase activity, and upregulated the expression of peroxisome proliferator‐activated receptor γ co‐activator 1 α, nuclear respiratory factor 1, and mitochondrial transcription factor A.ConclusionsAnimal data suggest that increased consumption of dietary wolfberry enhances mitochondrial biogenesis leading to retinoprotection in diabetes. Grant Funding Source: National Institutes of Health COBRE Grant P20‐RR‐017686

Supplementation with a Leucine Pulse during Continuous Feeding Stimulates Translation Initiation and Suppresses Protein Degradation Pathways in Muscle of Neonatal Pigs

Leucine acts as a nutrient signal to stimulate protein synthesis in skeletal muscle. Limited evidence suggests that leucine may suppress protein degradation. Twenty piglets (8‐d‐old) received formula by orogastric tube for 24 h continuously (CON) or by bolus (BOL), i.e., every 4 h for 15 min. For the CON+LEU group, leucine was administered parenterally (800 μmol·kg−1·h−1) every 4 h for 1 h. Protein synthesis in skeletal muscle was greater in CON+LEU and BOL than CON. Ribosomal protein S6 kinase 1 and 4E‐binding protein 1 phosphorylation and active eukaryotic initiation factor (eIF) eIF4E·eIF4G complex formation were higher in muscle of CON+LEU and BOL than CON suggesting increased translation initiation. Eukaryotic elongation factor 2 and AMP‐activated protein kinase‐α phosphorylation was unaffected by treatment. The LC3‐II to total LC3 ratio was lower in CON+LEU and BOL than CON suggesting reduced autophagy‐lysosome activation. There were no differences between groups in indices of the ubiquitin‐proteasome pathway, i.e., Atrogin‐1 and MURF‐1 abundance and FOX03 phosphorylation. In conclusion, administration of a leucine pulse during continuous orogastric feeding increases protein synthesis by stimulating translation initiation and suppresses autophagy‐lysosome, but not the ubiquitin‐proteasome, degradation pathways in skeletal muscle of neonates. (NIH AR444474 and USDA/ARS 6250–51000‐055)

Dietary wolfberry upregulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

Our aim was to investigate whether dietary wolfberry altered carotenoid metabolic gene expression and enhanced mitochondrial biogenesis in the retina of diabetic mice. Six-week-old male db/db and wild-type mice were fed the control or wolfberry diets for 8 weeks. At study termination, liver and retinal tissues were collected for analysis by transmission electron microscopy, real-time PCR, immunoprecipitation, Western blot, and HPLC. Wolfberry elevated zeaxanthin and lutein levels in the liver and retinal tissues and stimulated expression of retinal scavenger receptor class B type I, glutathione S-transferase Pi 1, and β,β-carotene 9',10'-oxygenase 2, and induced activation and nuclear enrichment of retinal AMP-activated protein kinase α2 (AMPK-α2). Furthermore, wolfberry attenuated hypoxia and mitochondrial stress as demonstrated by declined expression of hypoxia-inducible factor-1-α, vascular endothelial growth factor, and heat shock protein 60. Wolfberry enhanced retinal mitochondrial biogenesis in diabetic retinas as demonstrated by reversed mitochondrial dispersion in the retinal pigment epithelium, increased mitochondrial copy number, elevated citrate synthase activity, and upregulated expression of peroxisome proliferator-activated receptor γ co-activator 1α, nuclear respiratory factor 1, and mitochondrial transcription factorA. Consumption of dietary wolfberry could be beneficial to retinoprotection through reversal of mitochondrial function in diabetic mice.

AMP-activated protein kinase α1 protects against diet-induced insulin resistance and obesity. Diabetes 2012;61:3114–3125

Zhang W, Zhang X, Wang H, Guo X, Li H, Wang Y, Xu X, Tan L, Mashek MT, Zhang C, Chen Y, Mashek DG, Foretz M, Zhu C, Zhou H, …

Resveratrol Protects HUVECs from Oxidized-LDL Induced Oxidative Damage by Autophagy Upregulation via the AMPK/SIRT1 Pathway

Resveratrol could induce basal autophagy through the activation of sirtuin. In this study, we investigated the effect of resveratrol on oxidative injury of human umbilical endothelial vein cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and the role of autophagy in this effect. HUVECs were exposed to 100 mg/L ox-LDL for 24 h to cause oxidative injury. The effect of different concentrations of resveratrol on oxidative damage in HUVECs treated with ox-LDL was evaluated by MTT assay and superoxide dismutase (SOD) activity test. The autophagic level in different groups was measured by the protein expression of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (SQSTM1/P62). Autophagosomes were observed under electron microscope and fluorescence microscope (by MDC staining). The expression of silencing information regulator1 (Sirt1) and AMP activated protein kinaseα1 (AMPK) was investigated by Western blot. Autophagy inhibitor 3-methyladenine (3-MA) and Sirt1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527) were used to confirm the role of autophagy in this effect of resveratrol and the pathway involved. Resveratrol reversed the decreases in cell viability (72.9 ± 1.7 % of the control group) and SOD activity (14.37 ± 0.21 U/ml) caused by ox-LDL at 83.4 ± 1.4 % of the control group and 16.41 ± 0.27 U/ml respectively. This effect accompanied by upregulation of autophagy and increased protein expression of Sirt1 and AMPK phosphorylation on threonine 172 (p-AMPK). Both 3-MA and EX527 abolished the protective effect of resveratrol in cell viability, at 80.4 ± 2.7 % and 73.9 ± 1.1 % of the control group respectively. 3-MA inhibited autophagy activation without any change of Sirt1 expression at both the mRNA and protein level. EX527 suppressed the expression of Sirt1 and diminished the upregulation of autophagy. Addition of 3-MA or EX527 could not affect the protein level of p-AMPK. Resveratrol protected HUVECs from oxidative damage caused by ox-LDL. This effect was mediated by Sirt1-dependent autophagy via the AMPK/ Sirt1 pathway.

Metabolic adaptation of two pig muscles to cold rearing conditions1

Cold environment represents an external stress modulating animal growth and energy use. At muscle level, adaptation to cold conditions potentially involves energy homeostasis regulation gauged by the adenosine monophosphate (AMP)-activated protein kinase (AMPK). Our study aimed at evaluating the bare effects of short- and long-term cold exposure on growth performance, carcass traits, and metabolic characteristics of the oxidative semispinalis (SS) muscle and glycolytic LM and to evaluate the reversibility of short-term effects, with a special emphasis on AMPK activity. A total of 84 pigs fed ad libitum and individually housed were submitted after weaning to either Cold (from 23 ± 1 to 15 ± 3°C) or thermoneutral (T; from 28 ± 1 to 23 ± 1°C) temperature up to 24.7 ± 1.6 kg BW (25 BW). Twelve Cold and 12 T piglets were then slaughtered the same day. Eighteen remaining Cold piglets were reared at 12 ± 2°C (CC) whereas 18 Cold and 24 T piglets were reared at 23 ± 4°C (CT and TT, respectively) and slaughtered at 114.3 ± 5.9 kg (115 BW). The LM and SS samples were analyzed to determine glycolytic potential (GP), activities of lactate dehydrogenase (LDH), citrate synthase (CS), and β-hydroxy-acyl-CoA dehydrogenase (HAD) and to quantify AMPK phosphorylation Threonine 172 phosphorylated form of AMPK α1+α2 isoforms (pAMPK) / total AMPK α1+α2 isoforms (AMPK). Despite a greater ADFI (P < 0.001), Cold piglets exhibited less ADG (P < 0.001) and body fatness (P < 0.03) attesting an acute adaptation to a short-term cold exposure. A long-term cold adaptation evaluated on 115 BW pigs increased ADFI (P < 0.001) but did not influence ADG and carcass lean meat content. Cold environment influenced the dynamic of muscle metabolism in a muscle type dependent manner corresponding to an earlier and acute adaptation in SS from 8 kg onward and a belated adaptation in LM from 25 BW to 115 BW. Cold exposure was associated with a progressive increase of muscle oxidative capacity, first in the SS with greater HAD (P = 0.002) and CS activities (P = 0.03) at 25 BW and then both in SS and LM at 115 BW (P < 0.001). Conversely, in LM of CC pigs, increased GP (P < 0.001) and LDH activity (P = 0.03) were observed in addition to increased CS and HAD activities, highlighting the ability of LM to increase both its glycolytic and oxidative metabolism and to diversify its energy substrates. Pigs from CC group exhibited also less pAMPK/AMPK (P < 0.01) specifically in red SS muscle, denoting a reduced metabolic stress of this muscle after a long-term cold adaptation.