Impact of aging and exercise on skeletal muscle mitochondrial capacity, energy metabolism, and physical function.

The relationship between the age-associated decline in mitochondrial function and its effect on skeletal muscle physiology and function remain unclear. In the current study, we examined to what extent physical activity contributes to the decline in mitochondrial function and muscle health during aging and compared mitochondrial function in young and older adults, with similar habitual physical activity levels. We also studied exercise-trained older adults and physically impaired older adults. Aging was associated with a decline in mitochondrial capacity, exercise capacity and efficiency, gait stability, muscle function, and insulin sensitivity, even when maintaining an adequate daily physical activity level. Our data also suggest that a further increase in physical activity level, achieved through regular exercise training, can largely negate the effects of aging. Finally, mitochondrial capacity correlated with exercise efficiency and insulin sensitivity. Together, our data support a link between mitochondrial function and age-associated deterioration of skeletal muscle.

Muscle fiber-type distribution, fiber-type-specific damage, and the Pompe disease phenotype.

Pompe disease is a lysosomal storage disorder caused by acid α-glucosidase deficiency and characterized by progressive muscle weakness. Enzyme replacement therapy (ERT) has ameliorated patients' perspectives, but reversal of skeletal muscle pathology remains a challenge. We studied pretreatment biopsies of 22 patients with different phenotypes to investigate to what extent fiber-type distribution and fiber-type-specific damage contribute to clinical diversity. Pompe patients have the same fiber-type distribution as healthy persons, but among nonclassic patients with the same GAA mutation (c.-32-13T>G), those with early onset of symptoms tend to have more type 2 muscle fibers than those with late-onset disease. Further, it seemed that the older, more severely affected classic infantile patients and the wheelchair-bound and ventilated nonclassic patients had a greater proportion of type 2x muscle fibers. However, as in other diseases, this may be caused by physical inactivity of those patients.

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice.

OBJECTIVE: In conditions of continuous high-fat (HF) intake, the degree of saturation of the fatty acids (FAs) in the diet might have a crucial role in the onset of obesity and its metabolic complications. In particular, the FA composition of the diet might influence the storage form of lipids inside skeletal muscle. The aim of the present study was to examine whether the FA composition of HF diets differentially affects weight gain and accumulation of myocellular triacylglycerol (TAG) and diacylglycerol (DAG). Furthermore, we examined whether the FA composition of the diet was reflected in the composition of the myocellular lipid intermediates. DESIGN: C57Bl6 mice were fed HF diets (45% energy) mainly containing palm oil (PO), cocoa butter (CB), olive oil (OO) or safflower oil (SO; n=6 per group) for 8 weeks. A low-fat diet (10% energy, PO) was used as control. Body weight was monitored weekly. At the end of the dietary intervention, myocellular TAG and DAG content and profiles were measured. RESULTS: We here show that HF_CB prevented weight gain after 8 weeks of HF feeding. Furthermore, the HF diet rich in SO prevented the accumulation of both myocellular TAG and DAG. Interestingly, the FA composition of DAG and TAG in skeletal muscle was a reflection of the dietary FA composition. CONCLUSION: Already after a relatively short period, the dietary FA intake relates to the FA composition of the lipid metabolites in the muscle. A diet rich in polyunsaturated FAs seems to prevent myocellular lipid accumulation.

Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

AIM: To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. METHODS: Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. RESULTS: The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or beta1-adrenoceptor (beta1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac beta-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. CONCLUSION: db/db mice show impaired cardiac functional reserve capacity during maximal beta-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism.

Regulation of mitochondrial biogenesis during myogenesis.

Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, C(2)C(12) myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and beta-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4-8-fold during differentiation. Additionally, an increase in the ratio of myosin heavy chain (MyHC) slow vs MyHC fast protein content was observed. PPAR transcriptional activity and transcript levels of PPAR-alpha, the PPAR co-activator PGC-1alpha, mitochondrial transcription factor A and nuclear respiratory factor 1 increased during differentiation while expression levels of PPAR-gamma decreased. In conclusion, expression and activity levels of genes known for their regulatory role in skeletal muscle oxidative capabilities parallel the increase in oxidative parameters during the myogenic program. In particular, PGC-1alpha and PPAR-alpha may be involved in the regulation of mitochondrial biogenesis during myogenesis.

Loss of 50% of excess weight using a very low energy diet improves insulin-stimulated glucose disposal and skeletal muscle insulin signalling in obese insulin-treated type 2 diabetic patients.

AIMS/HYPOTHESIS: Both energy restriction (ER) per se and weight loss improve glucose metabolism in obese insulin-treated type 2 diabetic patients. Short-term ER decreases basal endogenous glucose production (EGP) but not glucose disposal. In contrast the blood glucose-lowering mechanism of long-term ER with substantial weight loss has not been fully elucidated. The aim of this study was to investigate the effect of loss of 50% of excess weight [50% excess weight reduction (EWR)] on EGP, whole-body insulin sensitivity and the disturbed myocellular insulin-signalling pathway in ten obese insulin-treated type 2 diabetic patients. METHODS: A euglycaemic-hyperinsulinaemic clamp with stable isotopes ([6,6-(2)H2]glucose and [2H5]glycerol) combined with skeletal muscle biopsies was performed during a very low energy diet (VLED; 1,883 kJ/day) on day 2 and again after 50% EWR. Oral blood glucose-lowering agents and insulin were discontinued 3 weeks prior to the VLED and at the start of the VLED, respectively. RESULTS: Loss of 50% EWR (20.3+/-2.2 kg from day 2 to day of 50% EWR) normalised basal EGP and improved insulin sensitivity, especially insulin-stimulated glucose disposal (18.8+/-2.0 to 39.1+/-2.8 micromol kg fat-free mass(-1) min(-1), p=0.001). The latter was accompanied by improved insulin signalling at the level of the recently discovered protein kinase B/Akt substrates AS160 and PRAS40 along with a decrease in intramyocellular lipid (IMCL) content. CONCLUSIONS/INTERPRETATION: Considerable weight loss in obese, insulin-treated type 2 diabetic patients normalises basal EGP and improves insulin sensitivity resulting from an improvement in insulin signal transduction in skeletal muscle. The decrease in IMCL might contribute to this effect.

Skeletal muscle uncoupling protein-3 restores upon intervention in the prediabetic and diabetic state: implications for diabetes pathogenesis?

AIM: Skeletal muscle uncoupling protein-3 (UCP3) is reduced in type 2 diabetes, and in the pre-diabetic condition of impaired glucose tolerance (IGT). Here we examined whether intervention programs known to improve insulin sensitivity are paralleled by an increase in skeletal muscle UCP3 protein levels. METHODS: Skeletal muscle UCP3 protein content was measured before and after one year of an exercise intervention in muscle biopsies of eight diabetic subjects. In addition, UCP3 was measured in IGT subjects before and after 1 year of following a lifestyle-intervention program or serving as control. RESULTS: In the diabetic patients a significant increase of approximately 75% in UCP3 protein was found after 1 year of exercise training (P < 0.05). In IGT subjects UCP3 protein increased in the intervention group (P = 0.02), while UCP3 remained unaltered in the control group (P = 0.64). CONCLUSION: Both, exercise training and a lifestyle-intervention program increase UCP3 protein content in skeletal muscle of subjects with reduced glycaemic control, indicating a restoration towards normal UCP3 levels. These data support the idea that UCP3 has a role in the aetiology of type 2 diabetes mellitus.

Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1 alpha and PPAR beta/delta gene expression upon rosiglitazone treatment in obese patients with type 2 diabetes mellitus.

OBJECTIVE: To examine whether rosiglitazone alters gene expression of some key genes involved in mitochondrial biogenesis and oxidative capacity in skeletal muscle of type 2 diabetic patients, and whether this is associated with alterations in skeletal muscle oxidative capacity and lipid content. DESIGN: measured in muscle biopsies obtained from diabetic patients, before and after 8 weeks of rosiglitazone treatment, and matched controls. Furthermore, whole-body insulin sensitivity and substrate utilization were assessed. SUBJECTS: Ten obese type 2 diabetic patients and 10 obese normoglycemic controls matched for age and BMI. METHODS: Gene expression and mitochondrial protein content of complexes I-V of the respiratory chain were measured by quantitative polymerase chain reaction and Western blotting, respectively. Histochemical staining was used to quantify lipid accumulation and complex II succinate dehydrogenase (SDH) activity. Insulin sensitivity and substrate utilization were measured during a hyperinsulinemic-euglycemic clamp with indirect calorimetry. RESULTS: Skeletal-muscle mRNA of PGC-1 alpha and PPAR beta/delta--but not of other genes involved in glucose, fat and oxidative metabolism--was significantly lower in diabetic patients (P<0.01). Rosiglitazone significantly increased PGC-1 alpha ( approximately 2.2-fold, P<0.01) and PPAR beta/delta ( approximately 2.6-fold, P<0.01), in parallel with an increase in insulin sensitivity, SDH activity and metabolic flexibility (P<0.01). Surprisingly, none of the measured mitochondrial proteins was reduced in type 2 diabetic patients, nor affected by rosiglitazone treatment. No alterations were seen in muscular fat accumulation upon treatment. CONCLUSION: These results suggest that the insulin-sensitizing effect of rosiglitazone may involve an effect on muscular oxidative capacity, via PGC-1 alpha and PPAR beta/delta, independent of mitochondrial protein content and/or changes in intramyocellular lipid.

Short-term training is accompanied by a down regulation of ACC2 mRNA in skeletal muscle.

Recently, we showed that short-term training induced a rapid increase in IMCL whilst insulin sensitivity tended to improve. Here we investigate molecular adaptations accompanying this physiological training-induced accumulation of IMCL. Nine untrained men (age: 23.3 +/- 3.2 y; maximal power output: 3.8 +/- 0.6 W/kg body weight) trained for two weeks. Before and after training, subjects cycled for three hours and biopsies were taken before and after exercise. mRNA concentrations of ACC2, HSL, LPL, Glut4 and HKII were quantified by RT-PCR and association of Glut4 with the membrane was quantified by immunohistochemical method. Endurance training resulted in a decrease of 29.1 % in ACC2 mRNA (p = 0.02). After training, ACC2 mRNA tended to decrease with acute exercise (- 24.4 % [p = 0.06]). HSL mRNA decreased with acute exercise after training (- 37.3 % [p = 0.002]). LPL mRNA concentrations increased with acute exercise before training (+ 42.4 % [p = 0.05]) and HKII mRNA increased with acute exercise before (+ 72.5 % [p = 0.025]) and after training (+ 99.3 % [p = 0.05]). After acute exercise, more Glut4 was associated with the membrane than before exercise, but it was not affected by training. We conclude that the training-induced increase in IMCL was accompanied by molecular adaptations in muscle to improve fat oxidative capacity, while markers of glucose metabolism were not yet changed. The present data are in line with the hypothesis that the fat oxidative capacity might be more important than the IMCL content in determining insulin sensitivity.

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers.

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.

Myopathological features in skeletal muscle of patients with chronic obstructive pulmonary disease.

Despite the fact that muscle weakness is a major problem in chronic obstructive pulmonary disease (COPD), detailed information on myopathological changes at the microscopic level in these patients is scarce, if indeed available at all. Vastus lateralis biopsies of 15 COPD weight-stable patients (body mass index (BMI) 23.9+/-1.0 kg x m(-2); fat-free mass index (FFMI) 17.2+/-1.7 kg x m(-2)) and 16 healthy age-matched controls (BMI 26.3+/-0.8 kg x m(-2); FFMI 19.6+/-2.2 kg x m(-2)) were evaluated. Histochemistry was used to evaluate myopathological features. Immunohistochemistry was used for the detection of macrophages and leukocytes, and active caspase 3 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) as markers of apoptosis. Fatty cell replacement and fibrosis were observed in both groups, the latter being slightly, but significantly, more pronounced in COPD. No differences between COPD and controls were found with respect to central nuclei, necrosis, regeneration, or fibre splitting. Signs of mitochondrial abnormalities were absent and normal numbers of inflammatory cells were found. Active caspase 3 positive myocytes were not observed and no difference was found in the number of TUNEL-positive myonuclei between controls and COPD patients (1.1% versus 1.0%, respectively). The cross-sectional area of type-IIX muscle fibres was smaller in COPD than in controls (2,566 versus 4,248 microm2). Except for the I to IIX shift in fibre types, the selective type-IIX atrophy and a slight accompanying increase in fibrosis and fat cell replacement in chronic obstructive pulmonary disease relative to age-matched controls, no other morphological abnormalities were observed in the muscle biopsies of chronic obstructive pulmonary disease patients. Also, in this group of clinically and weight stable chronic obstructive pulmonary disease patients, apoptosis appeared not to be involved in muscle pathology.

Uncoupling protein-3 content is decreased in peripheral skeletal muscle of patients with COPD.

Mechanical efficiency is reduced in patients with chronic obstructive pulmonary disease (COPD). Furthermore, altered fibre-type distribution and metabolic profile has been observed in peripheral skeletal muscle of COPD patients. Since skeletal muscular uncoupling protein-3 (UCP3) has been implicated in the regulation of energy metabolism, the aim of this study was to assess UCP3 in peripheral skeletal muscle of COPD patients and healthy controls. A total of 16 COPD patients and 11 healthy age-matched control subjects were studied. Mechanical efficiency was measured by means of cycle ergometry. Biopsies were taken from the vastus lateralis, and UCP3 and cytochrome c (as a marker for mitochondrial content) levels were assessed by Western blotting. Muscle fibre types and metabolic profile were examined histochemically. UCP3 levels were markedly decreased in COPD compared to controls. In COPD patients, there was a positive correlation between UCP3 content and the forced expiratory volume in one second. UCP3 content was not related to mechanical efficiency, or other muscular data such as fibre types, markers of oxidative/glycolytic energy metabolism or cytochrome c. The authors of this study conclude that uncoupling protein-3 content is decreased in peripheral skeletal muscle of patients with chronic obstructive pulmonary disease and is related to disease severity, but not to mechanical efficiency. The low uncoupling protein-3 content is independent of the loss of oxidative capacity observed in these patients.

UCP3 protein expression is lower in type I, IIa and IIx muscle fiber types of endurance-trained compared to untrained subjects.

Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower ( P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups ( P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects ( P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.

The effect of mild cold exposure on UCP3 mRNA expression and UCP3 protein content in humans.

OBJECTIVE: In rodents, adaptive thermogenesis in response to cold exposure and high-fat feeding is accomplished by the activation of the brown adipose tissue specific mitochondrial uncoupling protein, UCP1. The recently discovered human uncoupling protein 3 is a possible candidate for adaptive thermogenesis in humans. In the present study we examined the effect of mild cold exposure on the mRNA and protein expression of UCP3. SUBJECTS: Ten healthy male volunteers (age 24.4 +/- 1.6 y; height 1.83 +/- 0.02 m; weight 77.3 +/- 3.0 kg; percentage body fat 19 +/- 2). DESIGN: Subjects stayed twice in the respiration chamber for 60 h (20.00-8.00 h); once at 22 degrees C (72 degrees F), and once at 16 degrees C (61 degrees F). After leaving the respiration chamber, muscle biopsies were taken and RT-competitive-PCR and Western blotting was used to measure UCP3 mRNA and protein expression respectively. RESULTS: Twenty-four-hour energy expenditure was significantly increased at 16 degrees C compared to 22 degrees C (P<0.05). At 16 degrees C, UCP3T (4.6 +/- 1.0 vs 7.7 +/- 1.5 amol/microg RNA, P=0.07), UCP3L (2.0 +/- 0.5 vs 3.5 +/- 0.9 amol/microg RNA, P=0.1) and UCP3S (2.6 +/- 0.6 vs 4.2 +/- 0.7 amol/microg RNA, P=0.07) mRNA expression tended to be lower compared with at 22 degrees C, whereas UCP3 protein content was, on average, not different. However, the individual differences in UCP3 protein content (16-22 degrees C) correlated positively with the differences in 24 h energy expenditure (r=0.86, P<0.05). CONCLUSION: The present study suggests that UCP3 protein content is related to energy metabolism in humans and might help in the metabolic adaptation to cold exposure. However, the down-regulation of UCP3 mRNA with mild cold exposure suggests that prolonged cold exposure will lead to lower UCP3 protein content. What the function of such down-regulation of UCP3 could be is presently unknown.

Increased muscle fatigability in GLUT-4-deficient mice.

GLUT-4 plays a predominant role in glucose uptake during muscle contraction. In the present study, we have investigated in mice whether disruption of the GLUT-4 gene affects isometric and shortening contractile performance of the dorsal flexor muscle complex in situ. Moreover, we have explored the hypothesis that lack of GLUT-4 enhances muscle fatigability. Isometric performance normalized to muscle mass during a single tetanic contraction did not differ between wild-type (WT) and GLUT-4-deficient [GLUT-4(-/-)] mice. Shortening contractions, however, revealed a significant 1.4-fold decrease in peak power per unit mass, most likely caused by the fiber-type transition from fast-glycolytic fibers (IIB) to fast-oxidative fibers (IIA) in GLUT-4(-/-) dorsal flexors. In addition, the resting glycogen content was significantly lower (34%) in the dorsal flexor complex of GLUT-4(-/-) mice than in WT mice. Moreover, the muscle complex of GLUT-4(-/-) mice showed enhanced susceptibility to fatigue, which may be related to the decline in the muscle carbohydrate store. The significant decrease in relative work output during the steady-state phase of the fatigue protocol suggests that energy supply via alternative routes is not capable to compensate fully for the lack of GLUT-4.

Uncoupling protein 3 content is decreased in skeletal muscle of patients with type 2 diabetes.

Recently, a role for uncoupling protein-3 (UCP3) in carbohydrate metabolism and in type 2 diabetes has been suggested. Mice overexpressing UCP3 in skeletal muscle showed reduced fasting plasma glucose levels, improved glucose tolerance after an oral glucose load, and reduced fasting plasma insulin levels. However, data regarding the expression of UCP3 in patients with type 2 diabetes is inconsistent, and so far, there have been no reports of UCP3 protein content. Here we compared, for the first time, the protein levels of UCP3 in vastus lateralis muscle in 14 male type 2 diabetic patients (age 49.8 +/- 2.1 years; BMI 27.2 +/- 1.2 kg/m(2); mean +/- SE) with 16 male control subjects (age 48.0 +/- 1.9 years; BMI 23.4 +/- 0.6 kg/m(2)). We found that UCP3 protein levels were twice as low in patients with type 2 diabetes compared with control subjects (117 +/- 16 vs. 58 +/- 12 AU; P = 0.007). There was no correlation between UCP3 content and BMI. In conclusion, UCP3 content is lower in type 2 diabetic patients compared with healthy control subjects. These results are consistent with a role for UCP3 in glucose homeostasis and suggest a role for UCP3 in type 2 diabetes.

Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids.

The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.

The effect of weight reduction on skeletal muscle UCP2 and UCP3 mRNA expression and UCP3 protein content in Type II diabetic subjects.

AIMS/HYPOTHESIS: The aim of this study was to examine the effect of weight loss on UCP2/UCP3 mRNA expression and UCP3 protein content in subjects with Type II (non-insulin-dependent) diabetes mellitus. METHODS: We studied seven Type II diabetic subjects who followed a 10-week very low calorie diet. Expression of skeletal muscle UCP2 and UCP3 mRNA was measured using RT-competitive PCR and UCP3 protein content by western blotting, before and after the diet. Total and plasma fatty acid oxidation was measured using infusion of 13C labelled palmitate. RESULTS: Body weight decreased from 105.5 +/- 8.2 kg to 91.6 +/- 7.2 kg (p < 0.001), after 10 weeks of diet intervention. Expression of UCP2 and UCP3 mRNA were significantly reduced after 10 weeks of diet (p < 0.05) but UCP3 protein contents were not significantly altered. Notably, the change in UCP3L mRNA expression and UCP3 protein content after the very low calorie diet were negatively associated with changes in body weight (r = -0.97, p = 0.006 and r = -0.83, p = 0.043, respectively) and BMI (r = -0.99, p = 0.0007 and r = -0.9, p = 0.016, respectively). Furthermore, changes in UCP3L mRNA expression and UCP3 protein content induced by the diet were positively correlated with changes in cytosolic fatty acid-binding protein content (r = 0.93, p = 0.023 and r = 0.84, p = 0.039, respectively). No correlation between diet-induced changes in UCP3 protein and resting energy expenditure or plasma non-esterified fatty acid concentrations were found. CONCLUSION/INTERPRETATION: The negative correlation between the change in UCP3 protein content after weight loss and the change in BMI, suggests that the decrease in UCP3 during weight loss could prevent further weight loss. The finding that the change in UCP3 protein content correlates with the change in skeletal muscle fatty acid-binding protein content, suggests a role for UCPs in the handling of lipids as a fuel.

GLUT-4 expression is not consistently higher in type-1 than in type-2 fibres of rat and human vastus lateralis muscles; an immunohistochemical study.

In whole muscle homogenates, the glucose transporter-4 (GLUT-4) content is reported to be higher in muscles consisting predominantly of oxidative (type-1) muscle fibres than in muscles consisting predominantly of glycolytic (type-2) fibres. From these findings, it has been deduced that in rat muscle, oxidative fibres have an intrinsically higher level of GLUT-4 protein than glycolytic fibres. No data is available concerning human muscle. Moreover, the fibre-type-specific expression of GLUT-4 has not yet been examined directly. In this study, the relative abundance of GLUT-4 protein expression in individual fibres of different types within a muscle was compared directly in immunohistochemical assays. The human vastus lateralis muscle and a selection of rat muscles were studied using a novel GLUT-4 antiserum. It is concluded that the pattern of fibre-type-specific GLUT-4 expression differs between human and rats and varies between the different muscles studied, indicating that non-fibre-type-specific factor(s) affect expression of GLUT-4. The observation that within a muscle a fibre-type-specific expression of GLUT-4 was observed indicates that fibre-type-specific factors contribute to GLUT-4 expression as well. Thus, it can be postulated that both fibre-type-dependent and fibre-type-independent factors affect GLUT-4 expression.