Learning dependency model for AMP-activated protein kinase regulation

The AMP-activated protein kinase (AMPK) acts as a metabolic master switch regulating several intracellular systems. The effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. With increasingly available AMPK regulation data, it is critical to develop an efficient way to analyze the data since this assists in further understanding AMPK pathways. Bayesian networks can play an important role in expressing the dependency and causality in the data. This paper aims to analyse the regulation data using B-Course, a powerful analysis tool to exploit several theoretically elaborate results in the fields of Bayesian and causal modelling, and discover a certain type of multivariate probabilistic dependencies. The identified dependency models are easier to understand in comparison with the traditional frequent patterns.

AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle

AMP-activated protein kinase (AMPK) has recently emerged as a key signaling protein in skeletal muscle, coordinating the activation of both glucose and fatty acid metabolism in response to increased cellular energy demand. To determine whether AMPK signaling may also regulate gene transcription in muscle, rats were given a single subcutaneous injection (1 mg/g) of the AMP analog 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR). AICAR injection activated (P < 0.05) AMPK-α₂ (~2.5-fold) and transcription of the uncoupling protein-3 (UCP3, ~4-fold) and hexokinase II (HKII, ~10-fold) genes in both red and white skeletal muscle. However, AICAR injection also elicited (P < 0.05) an acute drop (60%) in blood glucose and a sustained (2-h) increase in blood lactate, prompting concern regarding the specificity of AICAR on transcription. To maximize AMPK activation in muscle while minimizing potential systemic counterregulatory responses, a single-leg arterial infusion technique was employed in fully conscious rats. Relative to saline-infused controls, single-leg arterial infusion of AICAR (0.125, 0.5, and 2.5 µg · g^-1 · min^-1 for 60 min) induced a dose-dependent increase (2- to 4-fold, P < 0.05) in UCP3 and HKII transcription in both red and white skeletal muscle. Importantly, AICAR infusion activated transcription only in muscle from the infused leg and had no effect on blood glucose or lactate levels. These data provide evidence that AMPK signaling is linked to the transcriptional regulation of select metabolic genes in skeletal muscle.

Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting

Fasting forces adaptive changes in whole body and skeletal muscle metabolism that increase fat oxidation and decrease the oxidation of carbohydrate. We tested the hypothesis that 40 h of fasting would decrease pyruvate dehydrogenase (PDH) activity and increase PDH kinase (PDK) isoform mRNA expression in human skeletal muscle. The putative transcriptional activators of PDK isozymes, peroxisome proliferator-activated receptor-α (PPAR-α) protein, and forkhead homolog in rhabdomyosarcoma (FKHR) mRNA were also measured. Eleven healthy adults fasted after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and skeletal muscle samples taken at 3, 15, and 40 h postprandial. Fasting increased plasma free fatty acid, glycerol, and β-hydroxybutyrate concentrations and decreased glucose and insulin concentrations. PDH activity decreased from 0.88 ± 0.11 mmol acetyl-CoA · min^-1 · kg wet muscle wt^-1 at 3 h to 0.62 ± 0.10 (P = not significant) and 0.39 ± 0.06 (P < 0.05) mmol · min^-1 · kg wet mass^-1 after 15 and 40 h of fasting. Although all four PDK isoforms were expressed in human skeletal muscle, PDK^-2 and -4 mRNA were the most abundant. PDK^-1 and ^-3 mRNA abundance was ~1 and 15% of the PDK^-2 and 4^- levels, respectively. The 40-h fast had no effect on PDK^-1, ^-2, and ^-3 mRNA expression. PDK-4 mRNA was significantly increased ~3-fold after 15 h and ~14-fold after 40 h of fasting. Skeletal muscle PPAR-α protein and FKHR mRNA abundance were unaffected by the fast. The results suggest that decreased PDH activation after 40 h of fasting may have been a function of the large increase in PDK-4 mRNA expression and possible subsequent increase in PDK protein and activity. The changes in PDK-4 expression and PDH activity did not coincide with increases in the transcriptional activators PPAR-α and FKHR.

Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle

This study examined the effects of short- and long-term aerobic training on the stable up-regulation of pyruvate dehydrogenase (PDH) and PDH kinase (PDK) in human skeletal muscle. We hypothesized that 8 weeks, but not 1 week, of aerobic training would increase total PDH (PDHt) and PDK activities compared to pretraining, and this would be detectable at the level of gene transcription (mRNA) and/or gene translation (protein). Resting muscle biopsies were taken before and after 1 and 8 weeks of aerobic cycle exercise training. PDHt and PDK activities, and their respective protein and mRNA expression, did not differ after 1 week of aerobic training. PDHt activity increased 31% after 8 weeks and this may be partially due to a 1.3-fold increase in PDH-E₁α protein expression. PDK activity approximately doubled after 8 weeks of aerobic training and this was attributed to a 1.3-fold increase in PDK2 isoform protein expression. Similar to 1 week, no changes were observed at the mRNA level after 8 weeks of training. These findings  suggest that aerobically trained human skeletal muscle has an increased maximal capacity to utilize carbohydrates, evident by increased PDHt, but increased metabolic control sensitivity to pyruvate through increased contribution of PDK2 to total PDK activity.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-β (NF-κβ). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-κβ before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

The suppressor of cytokine signaling 3 inhibits leptin activation of AMP-Kinase in cultured skeletal muscle of obese humans

Context: Leptin is thought to regulate whole-body adiposity and insulin sensitivity, at least in part, by stimulating fatty acid metabolism via activation of AMP-kinase (AMPK) in skeletal muscle. Human obesity is associated with leptin resistance, and recent studies have demonstrated that hypothalamic expression of the suppressors of cytokine signaling 3 (SOCS3) regulates leptin sensitivity in rodents.

Objective: The objective of the study was to investigate the effects of leptin on fatty acid oxidation and AMPK signaling in primary myotubes derived from lean and obese skeletal muscle and evaluate the contribution of SOCS3 to leptin resistance and AMPK signaling in obese humans.

Results: We demonstrate that leptin stimulates AMPK activity and increases AMPK Thr172 and acetyl-CoA carboxylase-ß Ser222 phosphorylation and fatty acid oxidation in lean myotubes but that in obese subjects leptin-dependent AMPK signaling and fatty acid oxidation are suppressed. Reduced activation of AMPK was associated with elevated expression of IL-6 (~3.5-fold) and SOCS3 mRNA (~2.5-fold) in myotubes of obese subjects. Overexpression of SOCS3 via adenovirus-mediated infection in lean myotubes to a similar degree as observed in obese myotubes prevented leptin but not AICAR (5-amino-imidazole-4-carboxamide-1-ß-D-ribofuranoside) activation of AMPK signaling.

Conclusions: These data demonstrate that SOCS3 inhibits leptin activation of AMPK. These data suggest that this impairment of leptin signaling in skeletal muscle may contribute to the aberrant regulation of fatty acid metabolism observed in obesity and that pharmacological activation of AMPK may be an effective therapy to bypass SOCS3-mediated skeletal muscle leptin resistance for the treatment of obesity-related disorders.

Thrifty metabolism that favors fat storage after caloric restriction: a role for skeletal muscle phosphatidylinositol-3-kinase activity and AMP-activated protein kinase

Energy conservation directed at accelerating body fat recovery (or catch-up fat) contributes to obesity relapse after slimming and to excess fat gain during catch-up growth after malnutrition. To investigate the mechanisms underlying such thrifty metabolism for catch-up fat, we tested whether during refeeding after caloric restriction rats exhibiting catch-up fat driven by suppressed thermogenesis have diminished skeletal muscle phosphatidylinositol-3-kinase (PI3K) activity or AMP-activated protein kinase (AMPK) signaling—two pathways required for hormone-induced thermogenesis in ex vivo muscle preparations. The results show that during isocaloric refeeding with a low-fat diet, at time points when body fat, circulating free fatty acids, and intramyocellular lipids in refed animals do not exceed those of controls, muscle insulin receptor substrate 1-associated PI3K activity (basal and in vivo insulin-stimulated) is lower than that in controls. Isocaloric refeeding with a high-fat diet, which exacerbates the suppression of thermogenesis, results in further reductions in muscle PI3K activity and in impaired AMPK phosphorylation (basal and in vivo leptin-stimulated). It is proposed that reduced skeletal muscle PI3K/AMPK signaling and suppressed thermogenesis are interdependent. Defective PI3K or AMPK signaling will reduce the rate of substrate cycling between de novo lipogenesis and lipid oxidation, leading to suppressed thermogenesis, which accelerates body fat recovery and furthermore sensitizes skeletal muscle to dietary fat-induced impairments in PI3K/AMPK signaling.—Summermatter, S., Mainieri, D., Russell, A. P., Seydoux, J., Montani, J. P., Buchala, A., Solinas, G., Dulloo, A. G. Thrifty metabolism that favors fat storage after caloric restriction: a role for skeletal muscle phosphatidylinositol-3-kinase activity and AMP-activated protein kinase.

Protein kinase C isozymes as potential therapeutic targets in immune disorders

Background: Members of the protein kinase C (PKC) family are key signalling mediators in immune responses, and pharmacological inhibition of PKCs may be useful for treating immune-mediated diseases. Objective: To review and discuss the insights gained so far into various PKC isozymes and the therapeutic potential and challenges of developing PKC inhibitors for immune disorder therapy. Methods: A literature review of the role of PKCs in immune cell signalling and recent studies describing immune functions associated with PKC isozyme deficiency in relevant mouse disease models, followed by specific case studies of current and potential therapeutic strategies targeting PKCs. Results/conclusion: There is vast amount of data supporting PKC isozymes as attractive drug targets for certain immune disorders. Although the development of specific PKC isozyme inhibitors has been challenging, some progress has been made. It remains to be seen if broad-scale or isozyme-selective inhibition of PKC will have clinical efficacy.

A pseudosymmetric cell adhesion regulatory domain in the β7 tail of the integrin α4β7 that interacts with focal adhesion kinase and src

The β7 integrins α4β7 and Eβ7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The α4β7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of α4β7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735-740 of the cytoplasmic tail of the β7 subunit inhibit the adhesion of T cells to β7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of α4β7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr⁷³⁵Phe and Tyr⁷⁴⁰Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of 4β7. The quasi-palindromic sequence YDRREY within the β7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of β7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease.

Adiponectin decreases pyruvate dehydrogenase kinase 4 gene expression in obese- and diabetic derived

Aim: To investigate the effects of globular adiponectin (gAd) on gene expression and whether these effects are mediated through 3',5'-cyclic monophosphate-activated protein kinase in skeletal muscle myotubes obtained from lean, obese and obese diabetic individuals.

Methods: Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Three distinct primary cell culture groups were established (lean, obese and obese diabetic; n = 7 in each group). Once differentiated, these cultures were then exposed to gAd or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 6 h.

Results: Stimulation with gAd decreased pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the obese and diabetic samples (p ≤ 0.05) and increased cytochrome c oxidase (COX) subunit 4 (COXIV) gene expression in the myotubes derived from lean individuals only (p < 0.05). AICAR treatment also decreased PDK4 gene expression in the obese- and diabetic-derived myotubes (p ≤ 0.05) and increased the gene expression of the mitochondrial gene, COXIII, in the lean-derived samples only (p < 0.05).

Conclusions: This study demonstrated distinct disparity between myotubes derived from lean compared with obese and obese diabetic individuals following gAd and AICAR treatment. Further understanding of the regulation of PDK4 in obese and diabetic skeletal muscle and its interaction with adiponectin signalling is required as this appears to be an important early molecular event in these disease states that may improve blood glucose control and metabolic flux.

AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5

Objective: Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms.

Research Design and Methods and Results: Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription.

Conclusions: These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin-dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86-fold) and CaMKKβ (10.1 ± 2.59-fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK-1 also increased 10-fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.

Growth restriction before and after birth increases kinase signaling pathways in the adult rat heart

To investigate the mechanisms for the previously reported development of adult cardiac hypertrophy in male rats following growth restriction, the levels of oxidative stress and activation of signaling kinases were measured in the left ventricle (LV) of adult rat offspring. In experiment one, bilateral uterine vessel ligation to induce uteroplacental insufficiency and growth restriction in the offspring (Restricted) or sham surgery was performed during pregnancy. Litters from sham mothers had litter size either reduced (Reduced Litter), which also restricted postnatal growth, or were left unaltered (Control). In males, Reduced Litter offspring had increased LV phosphorylation of AMPKa, p38 MAPK and Akt compared with Restricted and Controls (P,0.05). In females, both Restricted and Reduced Litter adult offspring had increased LV phosphorylation of p38 MAPK and Akt, however, only Restricted offspring had increased phosphorylation of AMPKa (P,0.05). In addition, only Restricted male offspring displayed LV oxidative stress (P,0.05). Experiment two investigated in mothers exposed to uteroplacental insufficiency or sham surgery the effects of cross-fostering offspring at birth, and therefore the effects of the postnatal lactational environment. Surprisingly, the cross-fostering itself resulted in increased LV phosphorylation of AMPKa and Akt in females and increased phosphorylation of Akt in males compared with Control non-cross-fostered offspring (P,0.05). In conclusion, kinase signaling in the adult LV can be programmed by uteroplacental insufficiency induced growth restriction in a gender-specific manner. In addition, the heart of adult rats is also sensitive to programming following the postnatal intervention of cross-fostering alone as well as by postnatal growth restriction.