| 1. |
- Kulkarni, Sameer S., et al.
(författare)
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Suppression of 5 '-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle
- 2011
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology, Inc.. - 0021-9258 .- 1083-351X. ; 286:40, s. 34567-34574
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Tidskriftsartikel (refereegranskat)abstract
- 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 (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (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-beta-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.
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| 2. |
- Sögård, Peter, et al.
(författare)
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Validation of the in vitro incubation of extensor digitorum longus muscle from mice with a mathematical model
- 2010
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Ingår i: Journal of biological systems. - : World Scientific. - 0218-3390. ; 18:3, s. 687-707
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Tidskriftsartikel (refereegranskat)abstract
- 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 method's 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.
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| 3. |
- Cansby, Emmelie, 1984, et al.
(författare)
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Increased expression of STK25 leads to impaired glucose utilization and insulin sensitivity in mice challenged with a high-fat diet.
- 2013
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Ingår i: FASEB journal : official publication of the Federation of American Societies for Experimental Biology. - : Wiley. - 1530-6860. ; 27:9, s. 3660-3671
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Tidskriftsartikel (refereegranskat)abstract
- Partial depletion of serine/threonine protein kinase 25 (STK25), a member of the Ste20 superfamily of kinases, increases lipid oxidation and glucose uptake in rodent myoblasts. Here we show that transgenic mice overexpressing STK25, when challenged with a high-fat diet, develop reduced glucose tolerance and insulin sensitivity compared to wild-type siblings, as evidenced by impairment in glucose and insulin tolerance tests as well as in euglycemic-hyperinsulinemic clamp studies. The fasting plasma insulin concentration was elevated in Stk25 transgenic mice compared to wild-type littermates (4.9±0.8 vs. 2.6±0.4 ng/ml after 17 wk on high-fat diet, P<0.05). Overexpression of STK25 decreased energy expenditure during the dark phase of observation (P<0.05), despite increased spontaneous activity. The oxidative capacity of skeletal muscle of transgenic carriers was reduced, as evidenced by altered expression of Cpt1, Acox1, and ACC. Hepatic triglycerides and glycogen were elevated (1.6- and 1.4-fold, respectively; P<0.05) and expression of key enzymes regulating lipogenesis (Fasn), glycogen synthesis (Gck), and gluconeogenesis (G6pc, Fbp1) was increased in the liver of the transgenic mice. Our findings suggest that overexpression of STK25 in conditions of excess dietary fuels associates with a shift in the metabolic balance in peripheral tissues from lipid oxidation to storage, leading to a systemic insulin resistance.-Cansby, E., Amrutkar, M., Mannerås Holm, L., Nerstedt, A., Reyahi, A., Stenfeldt, E., Borén, J., Carlsson, P., Smith, U., Zierath, J.R., Mahlapuu, M. Increased expression of STK25 leads to impaired glucose utilization and insulin sensitivity in mice challenged with a high-fat diet.
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| 4. |
- Ka, Sojeong, et al.
(författare)
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The expression of carnitine palmitoyl-CoA transferase-1B is influenced by a cis-acting eQTL in two chicken lines selected for high and low body weight
- 2013
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Ingår i: Physiological Genomics. - : American Physiological Society. - 1094-8341 .- 1531-2267. ; 45:9, s. 367-376
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Tidskriftsartikel (refereegranskat)abstract
- Carnitine palmitoyl-CoA transferase-1B is a mitochondrial enzyme in the fatty acid oxidation pathway. In a previous study, CPT1B was identified as differentially expressed in the hypothalamus of two lines of chickens established by long-term selection for high (HWS) or low (LWS) body weight. Mammals have three paralogs (CPT1a, b and c) while non-mammalian vertebrates only have two (CPT1A, B). CPT1A is expressed in liver and CPT1B in muscle. CPT1c is expressed in hypothalamus, where it regulates feeding and energy expenditure. We identified an intronic length polymorphism, fixed for different alleles in the two populations and mapped the hitherto missing CPT1B locus in the chicken genome assembly, to the distal tip of chromosome 1p. Based on molecular phylogeny and gene synteny we suggest that chicken CPT1B is pro-orthologous of the mammalian CPT1c. Chicken CPT1B was differentially expressed in both muscle and hypothalamus but in opposite directions: higher levels in hypothalamus but lower levels in muscle in the HWS than in the LWS line. Using an advanced inter-cross population of the lines, CPT1B expression was found to be influenced by a cis-acting expression quantitative trait locus in muscle. The increased expression in hypothalamus and reduced expression in muscle is consistent with an increased food intake in the HWS line and at the same time reduced fatty acid oxidation in muscle yielding a net accumulation of energy intake and storage. The altered expression of CPT1B in hypothalamus and peripheral tissue is likely to be a mechanism contributing to the remarkable difference between lines.
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| 5. |
- Russell, Aaron P., et al.
(författare)
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Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training
- 2013
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Ingår i: Journal of Physiology. - Chichester : Wiley-Blackwell. - 0022-3751 .- 1469-7793. ; 591:18, s. 4637-4653
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Tidskriftsartikel (refereegranskat)abstract
- The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r=-0.71; P= 0.04), miR-31 and HDAC4 protein (r =-0.87; P= 0.026) and miR-31 and NRF1 protein (r =-0.77; P= 0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health. © 2013 The Authors. The Journal of Physiology © 2013 The Physiological Society.
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| 6. |
- Sogaard, Peter, et al.
(författare)
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Spatial Insulin Signalling in Isolated Skeletal Muscle Preparations
- 2010
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Ingår i: Journal of Cellular Biochemistry. - : Wiley-Liss, Inc.. - 0730-2312 .- 1097-4644. ; 109:5, s. 943-949
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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