Oleanolic acid reduces hyperglycemia beyond treatment period with Akt/FoxO1-induced suppression of hepatic gluconeogenesis in type 2 diabetic mice

The present study investigated the chronic efficacy of oleanolic acid (OA), a triterpenoid selected from our recent screening, on hyperglycemia in type-2 diabetic mice. C57BL/6J mice were fed a high-fat diet followed by low doses of streptozotocin to generate a type-2 diabetic model. OA (100 mg/kg/day) was administered orally for 2 weeks with its effects monitored for 6 weeks. High-fat feeding and streptozotocin generated a steady hyperglycemia (21.261.1 mM) but OA administration reversed the hyperglycemia by ,60%

Thienopyridone Drugs Are Selective Activators of AMP-Activated Protein Kinase β1-Containing Complexes

SummaryThe AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the β subunit carbohydrate-binding module and residues from the γ subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the β1 subunit. Our findings highlight the regulatory role played by the β subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator

Differing Endoplasmic Reticulum Stress Response to Excess Lipogenesis versus Lipid Oversupply in Relation to Hepatic Steatosis and Insulin Resistance

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by ∼60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (∼40–60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (∼60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance

Structure and function of AMPK: subunit interactions of the AMPK heterotrimeric complex

© 2007 Dr. Tristan J. IseliAMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes

Non-viral causes of liver cancer: does obesity led inflammation play a role

Liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality. Hepatocellular carcinoma (HCC) accounts for around 90% of primary liver cancers. Chronic infection with hepatitis B and hepatitis C viruses are two of most common causes of liver cancer. However, there are non-viral factors that are associated with liver cancer development. Numerous population studies have revealed strong links between obesity and the development of liver cancer. Obesity can alter hepatic pathology, metabolism and promote inflammation, leading to nonalcoholic fatty liver disease (NAFLD) and the progression to the more severe form, non-alcoholic steatohepatitis (NASH). NASH is characterised by prominent steatosis and inflammation, and can lead to HCC. Here, we discuss the role of obesity in inflammation and the principal signalling mechanisms involved in HCC formation

Compound K modulates fatty acid-induced lipid droplet formation and expression of proteins involved in lipid metabolism in hepatocytes

Background & Aims: A key factor in the development of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) is hepatic steatosis. Incubation of human hepatic cells with free fatty acids (FFAs) causes accumulation of neutral lipids in lipid droplets (LDs) and serves as a model for hepatic steatosis. Ginsenosides, active constituents of ginsengs, have demonstrated beneficial effects in various pharmacological areas, including diabetes, however their effect on lipid accumulation in hepatocytes remains unclear. Here, we examine the effect of compound K (ComK), an active metabolite of ginsenosides, on the regulation of LD formation and on the expression of proteins involved in lipid homeostasis in hepatocytes. Methods: HuH7 cells were pretreated with ComK, followed by lipid loading with FFA. LDs were visualized using Oil Red O staining and immunohistochemistry for the LD-related protein PLIN2. Triglyceride levels were determined in isolated LDs. The expression of proteins involved in lipid homeostasis was examined by Western blotting. Results: Treatment with ComK significantly decreased LD formation in FFA-loaded HuH7 cells and increased phosphorylation levels of AMPK, and its substrate ACC. ComK also increased protein expression of peroxisome proliferator-activated receptor-α (PPAR-α) and acyl-CoA oxidase (ACOX1) together with elevated activity of a PPAR-α response element reporter construct. These effects were inhibited by the PPAR-α antagonist MK886. Conclusions: ComK reduced LD formation and TG accumulation in FFA-loaded hepatocytes, in part by up-regulating AMPK activity and PPAR-α related pathways. These results suggest that ComK may have efficacy for the treatment of hepatic steatosis and associated diseases

BMTs activate AMPK through CaMKK and not through intracellular Ca²⁺ changes.

<p>Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot (<b>A</b>) and quantified by densitometry (<b>B</b>). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK <i>in vitro</i> kinase assay (<b>C</b>). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p<0.05, **p<0.01 to V by one-way ANOVA.</p

Activation of AMPK involves CaMKKβ and not LKB1.

<p>Serum-starved HeLa cells (<b>A–C</b>) or L6 myotubes (<b>D–F</b>) were pre-treated ±1 µM STO-609 for 15 min before being incubated in either vehicle (0.1% DMSO), 2 mM AICAR, or 10 µM BMTs 17 or 1, for 30 min; or 1 µM ionomycin (Iono) for 10 min before lysis. Whole cell lysates (10 µg) were analysed by Western blot analysis (<b>A</b> and <b>D</b>) and quantified by densitometry (<b>B</b> and <b>E</b>). AMPK complexes were isolated by immunoprecipitation with a pan-AMPKβ antibody and assayed by <i>in vitro</i> AMPK kinase assay (<b>C</b> and <b>F</b>). Incorporation of ³²P into the AMARA substrate peptide was then assessed by β–scintillation counting. Data are means ± SEM (n = 4–8 per group). *p<0.05, **p<0.01 for compound effects by two-way ANOVA; ^##p<0.01 for STO-609 effects, ^†p<0.05, ^††p<0.01 for interactions between treatment groups.</p

BMTs do not directly activate AMPK in isolated AMPK complexes.

<p>AMPK complexes were isolated from untreated 18 hr serum-starved L6 myotube lysates by immunoprecipitation using a pan-AMPKβ antibody. The isolated AMPK was then incubated <i>in vitro</i> with either vehicle (V) alone, 10 µM BMT-17, BMT-1 or Abbott compound (A-769662) in the presence or absence of 200 µM AMP in AMPK assay buffer for 10 min at 30°C. The <i>in vitro</i> kinase assay was then initiated with the addition of 200 µM ³²P-Mg.ATP and assayed for a further 10 min at 30°C. Incorporation of the ³²P into the substrate peptide was then assessed by beta scintillation counting. Data are means ± SEM for 5 separate experiments. **p<0.01 for compound effect, ^##p<0.01 for AMP effects by two-way ANOVA. No interaction between treatment groups was apparent.</p

Changes in Akt and FoxO1 in the liver 4 weeks after the removal of OA.

<p>Four weeks after the cessation of OA treatment, mice were sacrificed following a 5–7 hour fast. Liver samples were freeze-clamped and stored at −80°C for subsequent Western blotting analysis. Representative Western blot images of phosphorylated and total Akt and FoxO1(<i>A</i>). Quantification of p-Akt/GAPDH (<i>B</i>), t-Akt/GAPDH (<i>C</i>), p-/t-Akt (<i>D</i>), p-FoxO1/GAPDH (<i>E</i>), t-FoxO1/GAPDH (<i>F</i>) and p-/t-FoxO1 (<i>G</i>). * p<0.05 vs. CH; † p<0.05, †† p<0.01 vs. T2D-Veh, n = 6–8 per group.</p