Denis R. Joanisse

Fat content in individual muscle fibers of lean and obese subjects

OBJECTIVE: To examine skeletal muscle intracellular triglyceride concentration in different fiber types in relation to obesity.DESIGN: Skeletal muscle fiber type distribution and intracellular lipid content were measured in vastus lateralis samples obtained by needle biopsy from lean and obese individuals.SUBJECTS: Seven lean controls (body mass index (BMI) 23.0±3.3 kg/m2; mean±s.d.) and 14 obese (BMI 33.7±2.7 kg/m2) individuals; both groups included comparable proportions of men and women.MEASUREMENTS: Samples were histochemically stained for the identification of muscle fiber types (myosin ATPase) and intracellular lipid aggregates (oil red O dye). The number and size of fat aggregates as well as their concentration within type I, IIA and IIB muscle fiber types were measured. The cellular distribution of the lipid aggregates was also examined.RESULTS: The size of fat aggregates was not affected by obesity but the number of lipid droplets within muscle fibers was twice as abundant in obese compared to lean individuals. This was seen in type I (298±135 vs 129±75; obese vs lean, P<0.05), IIA (132±67 vs 79±29; P<0.05), and IIB (103±63 vs 51±13; P<0.05) muscle fibers. A more central distribution of lipid droplets was observed in muscle fibers of obese compared to lean subjects (27.2±5.7 vs 19.7±6.4%; P<0.05).CONCLUSION: The higher number of lipid aggregates and the disposition to a greater central distribution in all fiber types in obesity indicate important changes in lipid metabolism and/or storage that are fiber type-independent.

Mitochondrial Dysregulation in the Pathogenesis of Diabetes: Potential for Mitochondrial Biogenesis-Mediated Interventions

Muscle mitochondrial metabolism is a tightly controlled process that involves the coordination of signaling pathways and factors from both the nuclear and mitochondrial genomes. Perhaps the most important pathway regulating metabolism in muscle is mitochondrial biogenesis. In response to physiological stimuli such as exercise, retrograde signaling pathways are activated that allow crosstalk between the nucleus and mitochondria, upregulating hundreds of genes and leading to higher mitochondrial content and increased oxidation of substrates. With type 2 diabetes, these processes can become dysregulated and the ability of the cell to respond to nutrient and energy fluctuations is diminished. This, coupled with reduced mitochondrial content and altered mitochondrial morphology, has been directly linked to the pathogenesis of this disease. In this paper, we will discuss our current understanding of mitochondrial dysregulation in skeletal muscle as it relates to type 2 diabetes, placing particular emphasis on the pathways of mitochondrial biogenesis and mitochondrial dynamics, and the therapeutic value of exercise and other interventions.

FATTY ACID CONTENT AND ENZYMES OF FATTY ACID METABOLISM IN OVERWINTERING COLD-HARDY GALL INSECTS

Fatty acid content and enzymes of fatty acid metabolism were studied in overwintering larvae of two cold-hardy gall insects, the freeze-tolerant fly Eurosta solidaginis and the freeze-avoiding moth Epiblema scudderiana. Both species increased the proportion of unsaturated fatty acids during the winter. Whereas total lipid content did not change in Eurosta solidaginis, a decrease in total lipids over the winter in Epiblema scudderiana suggested the use of fat reserves to maintain basal metabolism. Changes in the activities of enzymes of fat oxidation correlated with these observations in Eurosta solidaginis: hydroxyacyl-CoA dehydrogenase, carnitine-palmitoyl transferase, and acetoacetyl-CoA thiolase activities all decreased during overwintering. In Epiblema scudderiana the same activities were constant, decreased, or increased. These activities were, however, higher in the fat-oxidizing, freeze-avoiding species than in the freeze-tolerant larvae. Lipid content in Epiblema scudderiana increased again by early spring, possibly indicating this pool as the fate of carbon derived from the spring clearance of the cryoprotectant glycerol pool. Decreased activities of malic enzyme and ATP-citrate lyase suggested decreased potential for fatty acid synthesis in both species over the winter, consistent with the cessation offeeding in the fall. The potential for ketone body metabolism, measured as the activity of P-hydroxybutyrate dehydrogenase, increased greatly in both species during overwintering; however, levels ofβ-hydroxybutyrate remained less than 0.35 μmol/g wet mass throughout the study period. These data indicate that changes to storage lipid profiles in order to maintain fluidity and to lipid-metabolizing enzyme activities may play important roles in insect cold hardiness.

Adaptation of the diaphragm and the vastus lateralis in mild-to-moderate COPD

The chronology of diaphragm and vastus lateralis adaptation in chronic obstructive pulmonary disease (COPD) has not been studied. The hypothesis of this study was that muscle changes would occur earlier in the diaphragm than in the vastus lateralis in COPD, a finding that would suggest that local factors would be more important than systemic factors in determining the muscle phenotypic expression, at least in mild-to-moderate disease. The adaptation of the vastus lateralis and diaphragm muscles was evaluated in patients with mild-to-moderate COPD and in subjects with normal pulmonary function. In both groups, the oxidative potential and the number of lipofuscin inclusions were higher in the diaphragm than in the vastus lateralis. Compared to control, the diaphragm in COPD had a higher oxidative capacity and a higher proportion of type I fibres, with a reciprocal decrease in type IIA fibres, while there was no difference in diaphragmatic cross sectional areas, capillarisation and lipofuscin inclusions. No significant differences were found in the vastus lateralis from both groups. In conclusion, these data indicate that the diaphragm in controls and in chronic obstructive pulmonary disease has a higher oxidative potential than the vastus lateralis, and that muscle adaptation occurs earlier in the diaphragm than in the vastus lateralis in mild-to-moderate chronic obstructive pulmonary disease.

Milk supplementation facilitates appetite control in obese women during weight loss: a randomised, single-blind, placebo-controlled trial.

Dairy products provide Ca and protein which may facilitate appetite control. Conversely, weight loss is known to increase the motivation to eat. This randomised controlled trial verified the influence of milk supplementation on appetite markers during weight loss. Low Ca consumer women participated in a 6-month energy-restricted programme (-2508 kJ/d or -600 kcal/d) and received either a milk supplementation (1000 mg Ca/d) or an isoenergetic placebo (n 13 and 12, respectively). Fasting appetite sensations were assessed by visual analogue scales. Anthropometric parameters and fasting plasma concentrations of glucose, insulin, leptin, ghrelin and cortisol were measured as well. Both groups showed a significant weight loss (P < 0·0001). In the milk-supplemented group, a time x treatment interaction effect showed that weight loss with milk supplementation induced a smaller increase in desire to eat and hunger (P < 0·05). Unlike the placebo group, the milk-supplemented group showed a lower than predicted decrease in fullness (-17·1 v. -8·8; -2·7 v. 3·3 mm, P < 0·05, measured v. predicted values, respectively). Even after adjustment for fat mass loss, changes in ghrelin concentration predicted those in desire to eat (r 0·56, P < 0·01), hunger (r 0·45, P < 0·05) and fullness (r -0·40, P < 0·05). However, the study did not show a between-group difference in the change in ghrelin concentration in response to the intervention. These results show that milk supplementation attenuates the orexigenic effect of body weight loss.

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly, Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate → glyceraldehyde → glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate → glycerol-3-phosphate → glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the α-glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.

Relation between in vivo and in vitro measurements of skeletal muscle oxidative metabolism

The relationships between in vivo 31P magnetic resonance spectroscopy (MRS) and in vitro markers of oxidative capacity (mitochondrial function) were determined in 27 women with varying levels of physical fitness. Following 90‐s isometric plantar flexion exercises, calf muscle mitochondrial function was determined from the phosphocreatine (PCr) recovery time constant, the adenosine diphosphate (ADP) recovery time constant, the rate of change of PCr during the initial 14 s of recovery, and the apparent maximum rate of oxidative adenosine triphosphate (ATP) synthesis (Qmax). Muscle fiber type distribution (I, IIa, IIx), citrate synthase (CS) activity, and cytochrome c oxidase (COX) activity were determined from a biopsy sample of lateral gastrocnemius. MRS markers of mitochondrial function correlated moderately (P < 0.05) with the percentage of type IIa oxidative fibers (r = 0.41 to 0.66) and CS activity (r = 0.48 to 0.64), but only weakly with COX activity (r = 0.03 to 0.26, P > 0.05). These results support the use of MRS to determine mitochondrial function in vivo.

Effects of weight loss and leptin on skeletal muscle in human subjects

Maintenance of a 10% or greater reduced body weight results in decreases in the energy cost of low levels of physical activity beyond those attributable to the altered body weight. These changes in nonresting energy expenditure are due mainly to increased skeletal muscle work efficiency following weight loss and are reversed by the administration of the adipocyte-derived hormone leptin. We have also shown previously that the maintenance of a reduced weight is accompanied by a decrease in ratio of glycolytic (phosphofructokinase) to oxidative (cytochrome c oxidase) activity in vastus lateralis muscle that would suggest an increase in the relative expression of the myosin heavy chain I (MHC I) isoform. We performed analyses of vastus lateralis muscle needle biopsy samples to determine whether maintenance of an altered body weight was associated with changes in skeletal muscle metabolic properties as well as mRNA expression of different isoforms of the MHC and sarcoplasmic endoplasmic reticular Ca(2+)-dependent ATPase (SERCA) in subjects studied before weight loss and then again after losing 10% of their initial weight and receiving twice daily injections of either placebo or replacement leptin in a single blind crossover design. We found that the maintenance of a reduced body weight was associated with significant increases in the relative gene expression of MHC I mRNA that was reversed by the administration of leptin as well as an increase in the expression of SERCA2 that was not significantly affected by leptin. Leptin administration also resulted in a significant increase in the expression of the less MHC IIx isoform compared with subjects receiving placebo. These findings are consistent with the leptin-reversible increase in skeletal muscle chemomechanical work efficiency and decrease in the ratio of glycolytic/oxidative enzyme activities observed in subjects following dietary weight loss.

Mitochondrial enzymes during overwintering in two species of cold-hardy gall insects

Larvae of two cold-hardy insects, a freeze tolerant Eurosta solidaginis and a freeze avoiding Epiblema scudderiana species, were sampled at different times over a winter season and the activities of three mitochondrial enzymes were measured. Citrate synthase (CS), glutamate dehydrogenase (GDH), and NAD-isocitrate dehydrogenase (NAD-IDH) activities all decreased by 50% over the mid-winter compared with autum values. In Eurosta solidaginis all activities returned to initial values by mid-April, but decreased again in late April when the larvae pupated. In Epiblema scudderiana, activities of GDH and NAD-IDH increased in the spring, but CS activity remained relatively unchanged from mid-winter on. To determine the influence of temperature alone on enzyme activities, mid-October larvae acclimated to −4 or 15°C were compared; results showed greater activity of NAD-IDH at 15°C than at −4°C in Eurosta solidaginis (P < 0.05 by the Students t-test), but a lower activity of GDH in Epiblema scudderiana at 15°C (P < 0.05). Other enzyme activities were unchanged by acclimation. Reduced activities of mitochondrial enzymes in mid-winter are consistent with winter dormancy of these species, and could reflect lower mitochondrial numbers at this time. The regulation of these seasonal changes appears to be largely a response to cues other than short-term temperature acclimation.

Cardioprotective Effects of Glucose and Insulin Administration While Maintaining Normoglycemia (GIN Therapy) in Patients Undergoing Coronary Artery Bypass Grafting

CONTEXT Coronary artery bypass grafting (CABG) is complicated by ischemia-reperfusion injury jeopardizing myocyte survival. OBJECTIVE The aim of the study was to investigate whether glucose and insulin administration, while maintaining normoglycemia (GIN therapy) using a hyperinsulinemic-normoglycemic clamp technique, is cardioprotective in patients undergoing CABG. DESIGN AND SETTING We conducted a randomized controlled trial at a tertiary care university teaching hospital. PATIENTS We studied 99 patients undergoing elective CABG. INTERVENTION Patients were randomly assigned to receive either GIN from the beginning of surgery until 24 h after CABG (GIN, n = 49) or standard metabolic care (control, n = 50). MAIN OUTCOME MEASURES We measured plasma concentrations of cardiac troponin I and free fatty acids, cardiac function as assessed by transesophageal echocardiography, glycogen content, glycogen synthase activity, and the expression of AMP-activated protein kinase (AMPK) and protein kinase B (AKT) in cardiomyocytes. RESULTS Patients receiving GIN therapy showed an attenuated release of cardiac troponin I (P < 0.05) and improved myocardial function (P < 0.05). Systemic free fatty acid concentrations were suppressed (P < 0.05), whereas intracellular glycogen content and glycogen synthase activity were not altered. The AMPK activity remained unchanged during ischemia in the GIN group, whereas it increased in the control group (P < 0.05). Enhanced AKT phosphorylation before ischemia was observed (P < 0.05) in the presence of GIN. However, there was no evidence for AKT-dependent AMPK inhibition. CONCLUSIONS GIN therapy protects the myocardium and inhibits ischemia-induced AMPK activation.