Ariane Minet

Identification of plant extracts with potential antidiabetic properties: Effect on human peroxisome proliferator-activated receptor (PPAR), adipocyte differentiation and insulin-stimulated glucose uptake

Thiazolidinediones (TZDs) are insulin sensitizing drugs used to treat type 2 diabetes. The primary target of the TZDs is the peroxisome proliferator‐activated receptor (PPAR) γ, a key regulator of adipogenesis and glucose homeostasis. Currently prescribed TZDs are full PPARγ agonists, and their use is associated with several side effects. Partial PPARγ agonists appear to be associated with fewer side effects but may still confer the desired insulin sensitizing action. Extracts from common medicinal/food plants were tested in a screening platform comprising a series of bioassays, including tests for PPARγ, α and δ transactivation, adipocyte differentiation and insulin‐stimulated glucose uptake, allowing identification of plants containing potentially interesting PPAR agonists. Twenty‐two plant extracts out of 133 were found to increase insulin‐stimulated glucose uptake and 18 extracts were found to activate PPARγ, 3 to activate PPARα and γ, 6 to activate PPARδ and γ, and 9 to activate PPARγ, α and δ. Among the 24 different plant species tested in the platform, 50% were shown to contain compounds capable of activating PPARγ and stimulating insulin‐dependent glucose uptake with no or little effect on adipocyte differentiation warranting further studies and characterization. Copyright

ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects.

To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eight obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase. Mitochondrial mass and the ATP synthesis rate, neither at baseline nor during acute insulin stimulation, were not different between groups. The ratio of ATP synthesis rate at hexokinase versus ATP synthesis rate at baseline was lower in diabetic mitochondria compared to lean mitochondria. Thus the lower content of muscle mitochondria in type 2 diabetes in vivo is an adaptive trait and mitochondrial dysfunction in type 2 diabetes in vivo is based both on primarily impaired ATP synthesis and an adaptive loss of mitochondrial mass.

Impaired TCA cycle flux in mitochondria in skeletal muscle from type 2 diabetic subjects: Marker or maker of the diabetic phenotype?

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Recent research has shown that diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux. A reduced TCA cycle flux has also been shown both in insulin resistant offspring of T2D patients and exercising T2D patients in vivo. This review will discuss the latest advances in the understanding of the molecular mechanisms regulating the TCA cycle with focus on possible underlying mechanism which could explain the impaired TCA flux in insulin resistant human skeletal muscle in type 2 diabetes. A reduced TCA is both a marker and a maker of the diabetic phenotype.

Intact Primary Mitochondrial Function in Myotubes Established from Women with PCOS

CONTEXT Polycystic ovary syndrome (PCOS) affects 5-8% of fertile women and is often accompanied by insulin resistance, leading to increased risk of developing type 2 diabetes. Skeletal muscle from insulin-resistant PCOS subjects display reduced expression of nuclear encoded genes involved in mitochondrial oxidative metabolism. OBJECTIVE We aimed to investigate whether there was a primary mitochondrial dysfunction or difference in mitochondria content that might contribute to the in vivo detected insulin resistance. DESIGN The ATP synthesis with and without ATP use and the mitochondrial mass was determined in mitochondria isolated from myotubes established from PCOS subjects and control subjects. PATIENTS Myotubes were established from eight insulin-resistant PCOS subjects (verified by euglycemic hyperinsulinemic clamp) and eight healthy weight- and age-matched controls. RESULTS Mitochondrial mass and measurable mitochondrial ATP synthesis, with and without ATP use, were not different between PCOS subjects and control subjects. CONCLUSION We found no evidence for a primary impaired mitochondrial function or content in myotubes established from PCOS subjects, and our results suggest that reduced expression of oxidative genes in PCOS subjects is an adaptive trait.

Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging.

Hydrogen Peroxide Production Is Not Primarily Increased in Human Myotubes Established from Type 2 Diabetic Subjects

CONTEXT Increased oxidative stress and mitochondrial dysfunction have been implicated in the development of insulin resistance in type 2 diabetes. To date, it is unknown whether increased mitochondrial reactive oxygen species (ROS) production in skeletal muscle from patients with type 2 diabetes is primarily increased or a secondary adaptation to environmental, lifestyle, and hormonal factors. OBJECTIVE This study investigates whether ROS production is primarily increased in isolated diabetic myotubes. SETTING Mitochondrial membrane potential, hydrogen peroxide (H(2)O(2)), superoxide, and mitochondrial mass were determined in human myotubes precultured under normophysiological conditions. Furthermore, the corresponding ATP synthesis was measured in isolated mitochondria. PATIENTS Muscle biopsies were taken from 10 lean subjects, 10 obese subjects, and 10 subjects with type 2 diabetes; satellite cells were isolated, cultured, and differentiated to myotubes. RESULTS Mitochondrial mass, membrane potential/mitochondrial mass, and superoxide-production/mitochondrial mass were not different between groups. In contrast, H(2)O(2) production/mitochondrial mass and ATP production were significantly reduced in diabetic myotubes compared to lean controls (P < 0.05). The ATP/H(2)O(2) ratios were not significantly different between groups. CONCLUSIONS Our result indicates that the ROS production is not primarily increased in diabetic myotubes but rather is reduced. Moreover, the comparable ATP/H(2)O(2) ratios indicate that the reduced ROS production in diabetic myotubes parallels the reduced ATP production because ROS production in diabetic myotubes must be considered to be in a proportion comparable to lean. Thus, the increased ROS production seen in skeletal muscle of type 2 diabetic patients is an adaptation to the in vivo conditions.

The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

Although, most studies of human skeletal muscle in vivo have reported the co-existence of impaired insulin sensitivity and reduced expression of oxidative phosphorylation genes, there is so far no clear evidence for whether the intrinsic ATP synthesis is primarily decreased or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological conditions in order to verify intrinsic impairments. To resemble dynamic equilibrium present in whole cells between ATP synthesis and utilization, ATP was measured in the presence of an ATP consuming enzyme, hexokinase, under steady state. Mitochondria were isolated using an affinity based method which selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis.

Pyruvate carboxylase is expressed in human skeletal muscle.

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable by streptavidin blot and describe the presence of considerable amounts of PC in cultured human myotubes and in human muscle tissue.