Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK [alpha]2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK [alpha]2 activity reduced the association of AMPK with glycogen and increased AMPK [alpha]2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% [VO.sub.2] peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK [alpha]2 activity and acetyl-CoA carboxylase [beta] phosphorylation, increased translocation of AMPK [alpha]2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK [alpha]2 under conditions of low muscle glycogen enhances AMPK [alpha]2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle. <br /

Evidence for a second phosphorylation site on eIF-2α from rabbit reticulocytes

AbstractSer 51 in the NH2-terminal sequence of the α-subunit of eukaryotic peptide initiation factor 2 (eIF-2) has been identified as a second phosphorylation site for the heme-controlled eIF-2α kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the α-subunit of spectrin. A synthetic peptide corresponding to eIF-2α(41–54) is phosphorylated only in Ser 51 by the eIF-2α kinase

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma

AbstractA series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gpl20 (2–13), (55–65), gp41 (582–596) (659–670) and tatIII (71–83) were recognized by 41–67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582–596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579–599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (52%) to the tatIII peptide

Disrupting AMPK-glycogen binding in mice increases carbohydrate utilization and reduces exercise capacity

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism and binds glycogen, the primary storage form of glucose in liver and skeletal muscle. The effects of disrupting whole-body AMPK-glycogen interactions on exercise capacity and substrate utilization during exercise in vivo remain unknown. We used male whole-body AMPK double knock-in (DKI) mice with chronic disruption of AMPK-glycogen binding to determine the effects of DKI mutation on exercise capacity, patterns of whole-body substrate utilization, and tissue metabolism during exercise. Maximal treadmill running speed and whole-body energy utilization during submaximal running were determined in wild type (WT) and DKI mice. Liver and skeletal muscle glycogen and skeletal muscle AMPK α and β2 subunit content and signaling were assessed in rested and maximally exercised WT and DKI mice. Despite a reduced maximal running speed and exercise time, DKI mice utilized similar absolute amounts of liver and skeletal muscle glycogen compared to WT. DKI skeletal muscle displayed reduced AMPK α and β2 content versus WT, but intact relative AMPK phosphorylation and downstream signaling at rest and following exercise. During submaximal running, DKI mice displayed an increased respiratory exchange ratio, indicative of greater reliance on carbohydrate-based fuels. In summary, whole-body disruption of AMPK-glycogen interactions reduces maximal running capacity and skeletal muscle AMPK α and β2 content and is associated with increased skeletal muscle glycogen utilization. These findings highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and expand AMPK’s known roles in exercise and metabolism

A cardiolipin-activated protein kinase from rat liver structurally distinct from the protein kinases C

A cardiolipin- and protease-activated protein kinase (PAK) has been isolated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phosphorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzyme and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK-1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphatidylserine, diacylglycerol, and phorbol ester do not activate PAK-1 toward the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activation by cardiolipin or limited proteolysis were kinetically similar, with K-m values of 3.6 and 3.4 mu M, respectively, for the S6-(229-239) peptide substrate. However, differences were observed in the catalytic activities with protamine sulfate and the glycogen synthase-(1-12) peptide analogue as substrates. It was concluded that PAK-1 is a phospholipid regulated protein kinase with a primary structure, substrate specificity, and mechanism of regulation in vitro distinct from those of any known member of the protein kinase C superfamily

Hypothalamic CaMKK2 Contributes to the Regulation of Energy Balance

SummaryDetailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKα kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKα and β. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention

Skeletal muscle ACC2 S212 phosphorylation is not required for the control of fatty acid oxidation during exercise

During submaximal exercise fatty acids are a predominant energy source for muscle contractions. An important regulator of fatty acid oxidation is acetyl‐CoA carboxylase (ACC), which exists as two isoforms (ACC1 and ACC2) with ACC2 predominating in skeletal muscle. Both ACC isoforms regulate malonyl‐CoA production, an allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT‐1); the primary enzyme controlling fatty acyl‐CoA flux into mitochondria for oxidation. AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that is activated during exercise or by pharmacological agents such as metformin and AICAR. In resting muscle the activation of AMPK with AICAR leads to increased phosphorylation of ACC (S79 on ACC1 and S221 on ACC2), which reduces ACC activity and malonyl‐CoA; effects associated with increased fatty acid oxidation. However, whether this pathway is vital for regulating skeletal muscle fatty acid oxidation during conditions of increased metabolic flux such as exercise/muscle contractions remains unknown. To examine this we characterized mice lacking AMPK phosphorylation sites on ACC2 (S212 in mice/S221 in humans‐ACC2‐knock‐in [ACC2‐KI]) or both ACC1 (S79) and ACC2 (S212) (ACC double knock‐in [ACCD‐KI]) during submaximal treadmill exercise and/or ex vivo muscle contractions. We find that surprisingly, ACC2‐KI mice had normal exercise capacity and whole‐body fatty acid oxidation during treadmill running despite elevated muscle ACC2 activity and malonyl‐CoA. Similar results were observed in ACCD‐KI mice. Fatty acid oxidation was also maintained in muscles from ACC2‐KI mice contracted ex vivo. These findings indicate that pathways independent of ACC phosphorylation are important for regulating skeletal muscle fatty acid oxidation during exercise/muscle contractions

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

Economic evaluation of an Australian nurse home visiting programme : a randomised trial at 3 years

Objectives To investigate the additional programme cost and cost-effectiveness of ‘right@home’ Nurse Home Visiting (NHV) programme in relation to improving maternal and child outcomes at child age 3 years compared with usual care. Design A cost–utility analysis from a government-as-payer perspective alongside a randomised trial of NHV over 3-year period. Costs and quality-adjusted lifeyears (QALYs) were discounted at 5%. Analysis used an intention-to-treat approach with multiple imputation. Setting The right@home was implemented from 2013 in Victoria and Tasmania states of Australia, as a primary care service for pregnant women, delivered until child age 2 years. Participants 722 pregnant Australian women experiencing adversity received NHV (n=363) or usual care (clinic visits) (n=359). Primary and secondary outcome measures First, a cost–consequences analysis to compare the additional costs of NHV over usual care, accounting for any reduced costs of service use, and impacts on all maternal and child outcomes assessed at 3 years. Second, cost–utility analysis from a government-as-payer perspective compared additional costs to maternal QALYs to express cost-effectiveness in terms of additional cost per additional QALY gained. Results When compared with usual care at child age 3 years, the right@home intervention cost $A7685 extra per woman (95$A7006 to $A8364) and generated 0.01 more QALYs (95$A50 000 per QALY. Conclusions Benefits of NHV to parenting at 2 years and maternal health and well-being at 3 years translate into marginal maternal QALY gains. Like previous cost-effectiveness results for NHV programmes, right@home is not cost-effective at 3 years. Given the relatively high up-front costs of NHV, long-term follow-up is needed to assess the accrual of health and economic benefits over time

Calcium/calmodulin-dependent protein kinase kinase 2 regulates hepatic fuel metabolism

Objective The liver is the primary internal metabolic organ that coordinates whole body energy homeostasis in response to feeding and fasting. Genetic ablation or pharmacological inhibition of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been shown to significantly improve hepatic health and peripheral insulin sensitivity upon overnutrition with high fat diet. However, the precise molecular underpinnings that explain this metabolic protection have remained largely undefined. Methods To characterize the role of CaMKK2 in hepatic metabolism, we developed and challenged liver-specific CaMKK2 knockout (CaMKK2LKO) mice with high fat diet and performed glucose and insulin tolerance tests to evaluate peripheral insulin sensitivity. We used a combination of RNA-Sequencing, glucose and fatty acid istotopic tracer studies, a newly developed Seahorse assay for measuring the oxidative capacity of purified peroxisomes, and a degenerate peptide libarary to identify putative CaMKK2 substrates that mechanistically explain the protective effects of hepatic CaMKK2 ablation. Results Consistent with previous findings, we show that hepatic CaMKK2 ablation significantly improves indices of peripheral insulin sensitivity. Mechanistically, we found that CaMKK2 phosphorylates and regulates GAPDH to promote glucose metabolism and PEX3 to blunt peroxisomal fatty acid catabolism in the liver. Conclusion CaMKK2 is a central metabolic fuel sensor in the liver that significantly contributes to whole body systems metabolism