Olga Kotova

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism

The cardiotonic steroid, ouabain, a specific inhibitor of Na+,K+-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nm. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of α1- and α2-subunits of Na+,K+-ATPase and promoted interaction of α1- and α2-subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3α/β, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nm ouabain increased phosphorylation of theα-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of α2-subunit, whereas abundance of the α1-subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nm concentration, similarly to 100 nm ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.

Activation of AMP-activated Protein Kinase Stimulates Na+,K+-ATPase Activity in Skeletal Muscle Cells

Background: Contractions activate the sodium pump, Na+,K+-ATPase, and the energy sensor, AMP-activated protein kinase (AMPK), in skeletal muscle. Results: AMPK activation increases Na+,K+-ATPase activity in skeletal muscle cells. Conclusion: Activation of Na+,K+-ATPase in skeletal muscle cells is AMPK-dependent. Significance: AMPK activation of the sodium pump may be crucial during exercise to maintain ion homeostasis and delay muscle fatigue. Contraction stimulates Na+,K+-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na+,K+-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na+,K+-ATPase α1-subunit to the plasma membrane and increases Na+,K+-ATPase activity as assessed by ouabain-sensitive 86Rb+ uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na+,K+-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na+,K+-ATPase activity. The effect of AICAR on Na+,K+-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na+,K+-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na+,K+-ATPase α1-subunit dephosphorylation at Ser18, which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na+,K+-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na+,K+-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.

Altered expression and insulin-induced trafficking of Na+, K+-ATPase in rat skeletal muscle: effects of high fat diet and exercise

Skeletal muscle Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na(+)-K(+)-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na(+)-K(+)-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na(+)-K(+)-ATPase alpha(1)-subunit protein expression was increased 1.6-fold (P < 0.05), whereas alpha(2)- and beta(1)-subunits and protein expression were decreased twofold (P < 0.01) in parallel with decrease in plasma membrane Na(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P < 0.05). DNA binding activity of the alpha(1)-subunit-regulating transcription factor ZEB (AREB6) and alpha(1) mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of alpha(2)- and beta(1)-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na(+)-K(+)-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na(+)-K(+)-ATPase regulation, particularly the alpha(2)-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes.

Effects of exercise on muscle glycogen synthesis signalling and enzyme activities in pigs carrying the PRKAG3 mutation.

The dominant RN− mutation in pigs results in excessive glycogen storage in skeletal muscle. The mutation is situated in the PRKAG3 gene, which encodes a muscle‐specific isoform of the AMP‐activated protein kinase (AMPK) γ3 subunit. AMPK is an important regulator of carbohydrate and fat metabolism in mammalian cells. The aim of the present study was to examine the effect of exercise on glycogen synthesis signalling pathways in muscle and to study enzyme activities of importance in carbohydrate metabolism in pigs with or without the PRKAG3 mutation. Glycogen content, metabolic enzyme activities and expression or phosphorylation of signalling proteins were analysed in skeletal muscle specimens obtained at rest, after a single treadmill exercise bout and after 3 h recovery. The PRKAG3 mutation carriers had higher glycogen content, a tendency for lower expression of AMPK (P < 0.07) and higher hexokinase and phosphorylase activities, whereas citrate synthase, 3‐hydroxyacyl‐CoA dehydrogenase and glycogen synthase activities did not differ between genotypes. Carriers and non‐carriers of the RN− mutation showed a similar degradation of glycogen after exercise, whereas the rate of resynthesis was faster in the carriers. Acute exercise stimulated Akt phosphorylation on Ser473 in both genotypes, and the effect was greater in the carriers than in the non‐carriers. Acute exercise also stimulated phosphorylation of Akt substrate of 160 kDA and Glycogen synthase kinase 3 in the carriers and GSK3α in the non‐carriers. In conclusion, the increased rate of glycogen synthesis following exercise in pigs carrying the PRKAG3 mutation correlates with an increased signalling response of Akt and its substrate, AS160, and a higher activity of hexokinase, indicating an increased glucose influx and phosphorylation of glucose, directed towards glycogen synthesis.

Production and characterization of monoclonal anti-idiotypic antibodies to muramylpeptide

Hybridomas producing monoclonal anti‐idiotypic antibodies (anti‐id MAbs) to N‐acetylglucosaminyl‐β1‐4‐N‐acetylmuramyl‐alanyl‐d‐isoglutamine (GMDP) were developed. Three clones of hybridomas demonstrated the properties characteristic for the Ab2β type of anti‐id antibody: they bound to Fab‐fragments of high‐affinity MAb to GMDP; dose‐dependent inhibition of this binding by GMDP was observed; immunization of mice with these MAbs resulted in production of GMDP‐specific antibodies. When these antibodies were used to stain blots from SDS‐PAGE of macrophage lysate, the same receptor proteins were specifically stained as upon staining with 125I‐labelled GMDP derivative.