Ferenc Szekeres

The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose and insulin tolerance tests were normal in TBC1D1-deficient Nob1.10(SJL) mice, yet the 4-h-fasted insulin concentration was increased. Insulin-stimulated peripheral glucose utilization during a euglycemic hyperinsulinemic clamp was similar between genotypes, whereas the suppression of hepatic glucose production was increased in TBC1D1-deficient mice. In isolated extensor digitorum longus (EDL) but not soleus muscle, glucose transport in response to insulin, AICAR, or contraction was impaired by TBC1D1 deficiency. The reduction in glucose transport in EDL muscle from TBC1D1-deficient Nob1.10(SJL) mice may be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice. In conclusion, TBC1D1 plays a role in regulation of glucose metabolism in skeletal muscle. Moreover, functional TBC1D1 is required for AICAR- or contraction-induced metabolic responses, implicating a role in energy-sensing signals.

Suppression of 5′-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle

Background: The 5′-nucleotidase (NT5) family of enzymes dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. Results: NT5 silencing increases the intracellular availability of AMP/ATP and invokes AMP-activated protein kinase (AMPK) activation, glucose uptake, and lipid oxidation. Conclusion: NT5C enzymes inhibit basal lipid oxidation and glucose transport in skeletal muscle. Significance: Suppression of cytosolic NT5C expression or activity may bypass metabolic inflexibility in type 2 diabetes. The 5′-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr172), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser79) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5′-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.

Genetic Defects in Human Pericentrin Are Associated With Severe Insulin Resistance and Diabetes

OBJECTIVE Genetic defects in human pericentrin (PCNT), encoding the centrosomal protein pericentrin, cause a form of osteodysplastic primordial dwarfism that is sometimes reported to be associated with diabetes. We thus set out to determine the prevalence of diabetes and insulin resistance among patients with PCNT defects and examined the effects of pericentrin depletion on insulin action using 3T3-L1 adipocytes as a model system. RESEARCH DESIGN AND METHODS A cross-sectional metabolic assessment of 21 patients with PCNT mutations was undertaken. Pericentrin expression in human tissues was profiled using quantitative real-time PCR. The effect of pericentrin knockdown on insulin action and adipogenesis in 3T3-L1 adipocytes was determined using Oil red O staining, gene-expression analysis, immunoblotting, and glucose uptake assays. Pericentrin expression and localization also was determined in skeletal muscle. RESULTS Of 21 patients with genetic defects in PCNT, 18 had insulin resistance, which was severe in the majority of subjects. Ten subjects had confirmed diabetes (mean age of onset 15 years [range 5–28]), and 13 had metabolic dyslipidemia. All patients without insulin resistance were younger than 4 years old. Knockdown of pericentrin in adipocytes had no effect on proximal insulin signaling but produced a twofold impairment in insulin-stimulated glucose uptake, approximately commensurate with an associated defect in cell proliferation and adipogenesis. Pericentrin was highly expressed in human skeletal muscle, where it showed a perinuclear distribution. CONCLUSIONS Severe insulin resistance and premature diabetes are common features of PCNT deficiency but are not congenital. Partial failure of adipocyte differentiation may contribute to this, but pericentrin deficiency does not impair proximal insulin action in adipocytes.

Effects of Nordic walking on cardiovascular risk factors in overweight individuals with type 2 diabetes, impaired or normal glucose tolerance

Physical activity remains a valuable prevention for metabolic disease. The effects of Nordic walking on cardiovascular risk factors were determined in overweight individuals with normal or disturbed glucose regulation.

Blood pressure regulation by CD4+ lymphocytes expressing choline acetyltransferase

Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been determined. We previously showed that CD4+ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals. Here we show that these CD4+CD44hiCD62Llo T helper cells by gene expression are a distinct T-cell population defined by ChAT (CD4 TChAT). Mice lacking ChAT expression in CD4+ cells have elevated arterial blood pressure, compared to littermate controls. Jurkat T cells overexpressing ChAT (JTChAT) decreased blood pressure when infused into mice. Co-incubation of JTChAT and endothelial cells increased endothelial cell levels of phosphorylated endothelial nitric oxide synthase, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasorelaxant nitric oxide. The isolation and characterization of CD4 TChAT cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation.

Spatial insulin signalling in isolated skeletal muscle preparations

During in vitro incubation in the absence or presence of insulin, glycogen depletion occurs in the inner core of the muscle specimen, concomitant with increased staining of hypoxia‐induced‐factor‐1‐alpha and caspase‐3, markers of hypoxia and apoptosis, respectively. The aim of this study was to determine whether insulin is able to diffuse across the entire muscle specimen in sufficient amounts to activate signalling cascades to promote glucose uptake and glycogenesis within isolated mouse skeletal muscle. Phosphoprotein multiplex assay on lysates from muscle preparation was performed to detect phosphorylation of insulin‐receptor on Tyr1146, Akt on Ser473 and glycogen‐synthases‐kinase‐3 on Ser21/Ser9. To address the spatial resolution of insulin signalling, immunohistochemistry studies on cryosections were performed. Our results provide evidence to suggest that during the in vitro incubation, insulin sufficiently diffuses into the centre of tubular mouse muscles to promote phosphorylation of these signalling events. Interestingly, increased insulin signalling was observed in the core of the incubated muscle specimens, correlating with the location of oxidative fibres. In conclusion, insulin action was not restricted due to insufficient diffusion of the hormone during in vitro incubation in either extensor digitorum longus or soleus muscles from mouse under the specific experimental settings employed in this study. Hence, we suggest that the glycogen depleted core as earlier observed is not due to insufficient insulin action. J. Cell. Biochem. 109: 943–949, 2010.

Effects of fibre type and diffusion distance on mouse skeletal muscle glycogen content in vitro

In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin‐stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia‐induced‐factor‐1‐alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations. J. Cell. Biochem. 107: 1189–1197, 2009.

Signaling and metabolic properties of fast and slow smooth muscle types from mice

This study aims to improve the classification of smooth muscle types to better understand their normal and pathological functional phenotypes. Four different smooth muscle tissues (aorta, muscular arteries, intestine, urinary bladder) with a 5-fold difference in maximal shortening velocity were obtained from mice and classified according to expression of the inserted myosin heavy chain (SMHC-B). Western blotting and quantitative PCR analyses were used to determine 15 metabolic and 8 cell signaling key components in each tissue. The slow muscle type (aorta) with a 12 times lower SMHC-B had 6-fold lower expression of the phosphatase subunit MYPT1, a 7-fold higher expression of Rhokinase 1, and a 3-fold higher expression of the PKC target CPI17, compared to the faster (urinary bladder) smooth muscle. The slow muscle had higher expression of components involved in glucose uptake and glycolysis (type 1 glucose transporter, 3 times; hexokinase, 13 times) and in gluconeogenesis (phosphoenolpyruvate carboxykinase, 43 times), but lower expression of the metabolic sensing AMP-activated kinase, alpha 2 isoform (5 times). The slow type also had higher expression of enzymes involved in lipid metabolism (hormone-sensitive lipase, 10 times; lipoprotein lipase, 13 times; fatty acid synthase, 6 times; type 2 acetyl-coenzyme A carboxylase, 8 times). We present a refined division of smooth muscle into muscle types based on the analysis of contractile, metabolic, and signaling components. Slow compared to fast smooth muscle has a lower expression of the deactivating phosphatase and upregulated Ca2+ sensitizing pathways and is more adapted for sustained glucose and lipid metabolism.

VALIDATION OF THE IN VITRO INCUBATION OF EXTENSOR DIGITORUM LONGUS MUSCLE FROM MICE WITH A MATHEMATICAL MODEL

In vitro incubation of tissues; in particular, skeletal muscles from rodents, is a widely-used experimental method in diabetes research. This experimental method has previously been validated, both experimentally and theoretically. However, much of the methods experimental data remains unclear, including the high-rate of lactate production and the lack of an observable increase in glycogen content, within a given time. The predominant hypothesis explaining the high-rate of lactate production is that this phenomenon is dependent on a mechanism in glycolysis that works as a safety valve, producing lactate when glucose uptake is super-physiological. Another hypothesis is that existing anoxia forces more ATP to be produced from glycolysis, leading to an increased lactate concentration. The lack of an observable increase in glycogen content is assumed to be dependent on limitations in sensitivity of the measuring method used. We derived a mathematical model to investigate which of these hypotheses is most likely to be correct. Using our model, data analysis indicates that the in vitro incubated muscle specimens, most likely are sensing the presence of existing anoxia, rather than an overflow in glycolysis. The anoxic milieu causes the high lactate production. The model also predicts an increased glycogenolysis. After mathematical analyses, an estimation of the glycogen concentration could be made with a reduced model. In conclusion, central anoxia is likely to cause spatial differences in glycogen concentrations throughout the entire muscle. Thus, data regarding total glycogen levels in the incubated muscle do not accurately represent the entire organ. The presented model allows for an estimation of total glycogen, despite spatial differences present in the muscle specimen.