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1.
Am J Physiol Regul Integr Comp Physiol ; 307(9): R1115-23, 2014 Nov 01.
Article in English | MEDLINE | ID: mdl-25163918

ABSTRACT

In skeletal muscle the Rab-GTPase-activating protein TBC1D1 has been implicated in the regulation of fatty acid oxidation by an unknown mechanism. We determined whether TBC1D1 altered fatty acid utilization via changes in protein-mediated fatty acid transport and/or selected enzymes regulating mitochondrial fatty acid oxidation. We also determined the effects of TBC1D1 on glucose transport and oxidation. Electrotransfection of mouse soleus muscles with TBC1D1 cDNA increased TBC1D1 protein after 2 wk (P<0.05), without altering its paralog AS160. TBC1D1 overexpression decreased basal palmitate oxidation (-22%) while blunting 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated palmitate oxidation (-18%). There was a tendency to increase fatty acid esterification (+10 nmol·g(-1)·60 min(-1), P=0.07), which reflected the reduction in fatty acid oxidation (-12 nmol·g(-1)·60 min(-1)). Concomitantly, basal (+21%) and AICAR-stimulated glucose oxidation (+8%) were increased in TBC1D1-transfected muscles relative to their respective controls (P<0.05), independent of changes in GLUT4 and glucose transport. The reductions in TBC1D1-mediated fatty acid oxidation could not be attributed to changes in the transporter FAT/CD36, muscle mitochondrial content, CPT1 expression or the expression and phosphorylation of AS160, acetyl-CoA carboxylase, or AMPK. However, TBC1D1 overexpression reduced ß-HAD enzyme activity (-18%, P<0.05). In conclusion, TBC1D1-mediated reduction of muscle fatty acid oxidation appears to occur via inhibition of ß-HAD activity.


Subject(s)
3-Hydroxyacyl CoA Dehydrogenases/metabolism , Muscle, Skeletal/enzymology , Nuclear Proteins/metabolism , Palmitates/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Aminoimidazole Carboxamide/analogs & derivatives , Animals , Fatty Acids/metabolism , GTPase-Activating Proteins , Gene Expression Regulation/physiology , Glucose/metabolism , Lipid Peroxidation , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Oxidation-Reduction , Ribonucleotides
2.
Acta Physiol (Oxf) ; 205(1): 71-81, 2012 May.
Article in English | MEDLINE | ID: mdl-22463611

ABSTRACT

AIM: The increase in skeletal muscle fatty acid metabolism during exercise has been associated with the release of calcium. We examined whether this increase in fatty acid oxidation was attributable to a calcium-induced translocation of the fatty acid transporter CD36 to the sarcolemma, thereby providing an enhanced influx of fatty acids to increase their oxidation. METHODS: Calcium release was triggered by caffeine (3 mm) to examine fatty acid oxidation in intact soleus muscles of WT and CD36-KO mice, while fatty acid transport and mitochondrial fatty acid oxidation were examined in giant vesicles and isolated mitochondria, respectively, from caffeine-perfused hindlimb muscles of WT and CD36-KO mice. Western blotting was used to examine calcium-induced signalling. RESULTS: In WT, caffeine stimulated muscle palmitate oxidation (+136%), but this was blunted in CD36-KO mice (-70%). Dantrolene inhibited (WT) or abolished (CD36-KO) caffeine-induced palmitate oxidation. In muscle, caffeine-stimulated palmitate oxidation was not attributable to altered mitochondrial palmitate oxidation. Instead, in WT, caffeine increased palmitate transport (+55%) and the translocation of fatty acid transporters CD36, FABPpm, FATP1 and FATP4 (26-70%) to the sarcolemma. In CD36-KO mice, caffeine-stimulated FABPpm, and FATP1 and 4 translocations were normal, but palmitate transport was blunted (-70%), comparable to the reductions in muscle palmitate oxidation. Caffeine did not alter the calcium-/calmodulin-dependent protein kinase II phosphorylation but did increase the phosphorylation of AMPK and acetyl-CoA carboxylase comparably in WT and CD36-KO. CONCLUSION: These studies indicate that sarcolemmal CD36-mediated fatty acid transport is a primary mediator of the calcium-induced increase in muscle fatty acid oxidation.


Subject(s)
CD36 Antigens/metabolism , Caffeine/pharmacology , Lipid Metabolism/drug effects , Muscle, Skeletal/drug effects , Palmitic Acid/metabolism , Animals , CD36 Antigens/genetics , Calcium/metabolism , Mice , Mice, Knockout , Mitochondria, Muscle/drug effects , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Oxidation-Reduction/drug effects
3.
Diabetologia ; 55(2): 479-88, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22101973

ABSTRACT

AIMS/HYPOTHESIS: Little is known about the subcellular distribution of lipids in insulin-resistant skeletal muscle. However, it has recently been suggested that lipid accumulation in the subsarcolemmal region directly contributes to insulin resistance. Therefore we hypothesised that regional differences in lipid distribution in insulin-resistant muscle may be mediated by: (1) a reduction in fatty acid trafficking into mitochondria; and/or (2) a regional increase in the enzymes regulating lipid synthesis. METHODS: Transmission electron microscopy was used to quantify lipid droplet and mitochondrial abundance in the subsarcolemmal and intermyofibrillar compartments in red and white muscles from lean and obese Zucker rats. To estimate rates of lipid trafficking into mitochondria, the metabolic fate of radiolabelled palmitate was determined. Key enzymes of triacylglycerol synthesis were also determined in each subcellular region. RESULTS: Subsarcolemmal-compartmentalised lipids represented a small absolute fraction of the overall lipid content in muscle, as regardless of fibre composition (red/white) or phenotype (lean/obese), lipid droplets were more prevalent in the intermyofibrillar region, whereas insulin-resistant white muscles were devoid of subsarcolemmal-compartmentalised lipid droplets. While, in obese animals, lipid droplets accumulated in both subcellular regions, in red muscle of these animals lipids only appeared to be trafficked away from intermyofibrillar mitochondria, a process that cannot be explained by regional differences in the abundance of triacylglycerol esterification enzymes. CONCLUSIONS/INTERPRETATION: Lipid accumulation in the subsarcolemmal region is not necessary for insulin resistance. In the intermyofibrillar compartment, the diversion of lipids away from mitochondria in insulin-resistant animals probably contributes to lipid accumulation in this subcellular area.


Subject(s)
Lipids/chemistry , Obesity/genetics , Subcellular Fractions/metabolism , Animals , DNA, Mitochondrial/metabolism , Disease Models, Animal , Fatty Acids/chemistry , Female , Glucose/metabolism , Insulin/metabolism , Microscopy, Electron, Transmission/methods , Mitochondria/metabolism , Obesity/metabolism , Oxygen/chemistry , Palmitic Acid/metabolism , Rats , Rats, Zucker , Triglycerides/chemistry
4.
Diabetologia ; 53(9): 2008-19, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20490453

ABSTRACT

AIMS/HYPOTHESIS: Reductions in peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) levels have been associated with the skeletal muscle insulin resistance. However, in vivo, the therapeutic potential of PGC-1alpha has met with failure, as supra-physiological overexpression of PGC-1alpha induced insulin resistance, due to fatty acid translocase (FAT)-mediated lipid accumulation. Based on physiological and metabolic considerations, we hypothesised that a modest increase in PGC-1alpha levels would limit FAT upregulation and improve lipid metabolism and insulin sensitivity, although these effects may differ in lean and insulin-resistant muscle. METHODS: Pgc-1alpha was transfected into lean and obese Zucker rat muscles. Two weeks later we examined mitochondrial biogenesis, intramuscular lipids (triacylglycerol, diacylglycerol, ceramide), GLUT4 and FAT levels, insulin-stimulated glucose transport and signalling protein phosphorylation (thymoma viral proto-oncogene 2 [Akt2], Akt substrate of 160 kDa [AS160]), and fatty acid oxidation in subsarcolemmal and intermyofibrillar mitochondria. RESULTS: Electrotransfection yielded physiologically relevant increases in Pgc-1alpha (also known as Ppargc1a) mRNA and protein ( approximately 25%) in lean and obese muscle. This induced mitochondrial biogenesis, and increased FAT and GLUT4 levels, insulin-stimulated glucose transport, and Akt2 and AS160 phosphorylation in lean and obese animals, while bioactive intramuscular lipids were only reduced in obese muscle. Concurrently, PGC-1alpha increased palmitate oxidation in subsarcolemmal, but not in intermyofibrillar mitochondria, in both groups. In obese compared with lean animals, the PGC-1alpha-induced improvement in insulin-stimulated glucose transport was smaller, but intramuscular lipid reduction was greater. CONCLUSIONS/INTERPRETATIONS: Increases in PGC-1alpha levels, similar to those that can be induced by physiological stimuli, altered intramuscular lipids and improved fatty acid oxidation, insulin signalling and insulin-stimulated glucose transport, albeit to different extents in lean and insulin-resistant muscle. These positive effects are probably attributable to limiting the PGC-1alpha-induced increase in FAT, thereby preventing bioactive lipid accumulation as has occurred in transgenic PGC-1alpha animals.


Subject(s)
Glucose/metabolism , Insulin/pharmacology , Muscle, Skeletal/metabolism , Obesity/metabolism , RNA-Binding Proteins/metabolism , Thinness/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Fatty Acids/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Lipid Metabolism/drug effects , Male , Muscle, Skeletal/drug effects , Oxidation-Reduction/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation/drug effects , Proto-Oncogene Mas , RNA-Binding Proteins/genetics , Rats , Rats, Zucker , Signal Transduction/drug effects , Transcription Factors/genetics
5.
J Neurochem ; 105(3): 865-73, 2008 May.
Article in English | MEDLINE | ID: mdl-18182056

ABSTRACT

Mu and delta opioid receptors (MORs and DORs) were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galpha(i/o) protein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galpha(i/o) proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a MOR fused to a non-functional Galpha subunit was co-expressed with the DOR-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Galpha subunit fused the DOR.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Opioid, delta/metabolism , Receptors, Opioid, mu/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Analgesics, Opioid/pharmacology , Cell Line , Central Nervous System/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Narcotic Antagonists/pharmacology , Neurons/metabolism , Nociceptors/metabolism , Opioid Peptides/metabolism , Pain/genetics , Pain/metabolism , Pain/physiopathology , Protein Binding/physiology , Protein Conformation , Receptors, G-Protein-Coupled/genetics , Receptors, Opioid, delta/drug effects , Receptors, Opioid, delta/genetics , Receptors, Opioid, mu/drug effects , Receptors, Opioid, mu/genetics , Recombinant Fusion Proteins/genetics
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