No abstract

To define the effects of acute hyperglycemia per se (i.e., without the confounding effect of hyperinsulinemia) in human tissues in vivo, we performed global gene expression analysis using microarrays in vastus lateralis muscle and subcutaneous abdominal adipose tissue of seven healthy men during a hyperglycemic-euinsulinemic clamp with infusion of somatostatin to inhibit endogenous insulin release. We found that doubling fasting blood glucose values while maintaining plasma insulin in the fasting range modifies the expression of 316 genes in skeletal muscle and 336 genes in adipose tissue. More than 80% of them were downregulated during the clamp, indicating a drastic effect of acute high glucose, in the absence of insulin, on mRNA levels in human fat and muscle tissues. Almost all the biological pathways were affected, suggesting a generalized effect of hyperglycemia. The induction of genes from the metallothionein family, related to detoxification and free radical scavenging, indicated that hyperglycemia-induced oxidative stress could be involved in the observed modifications. Because the duration and the concentration of the experimental hyperglycemia were close to what is observed during a postprandial glucose excursion in diabetic patients, these data suggest that modifications of gene expression could be an additional effect of glucose toxicity in vivo. Diabetes 56:992-999, 2007 A ltered glycemic control in individuals with type 1 and type 2 diabetes is associated with increased risk of micro-and macrovascular complications (1). The mechanisms of the deleterious effects of hyperglycemia, which is referred to as glucotoxicity, have been largely investigated. It is accepted that oxidative stress induced by hyperglycemia could be the main cause of the different pathways leading to diabetes complications (2,3). Importantly, acute glucose fluctuations exhibit a more specific triggering effect on oxidative stress than chronic sustained hyperglycemia (4). Furthermore, acute hyperglycemia induces deleterious effects in various tissues and, from epidemiological studies, the harmful effect of hyperglycemia for cardiovascular complications appears to be mainly related to postprandial glucose excursion (5).On the other hand, excess carbohydrate leads to the activation of several genes that promote storage of glycogen and triglycerides in liver, skeletal muscle, and adipose tissue (6). Although these effects are generally produced through a combined action with insulin, the identification of a glucose-responsive transcription factor named ChREBP (carbohydrate responsive element binding protein) (7,8) has recently shed new light on the mechanisms whereby glucose could directly affect gene transcription.Until now, the effects of high glucose concentrations have mostly been studied in cell culture experiments and using animal models, and little is known about the in vivo molecular mechanisms of hyperglycemia in human tissues. The development of microarray technology offers powerful tools for characterizing the consequence...

Objective: Adiponutrin is a new transmembrane protein specifically expressed in adipose tissue. In obese subjects, short-or long-term calorie restriction diets were associated with a reduction in adiponutrin gene expression. Adiponut.rin mRNA level was previously shown to be negatively correlated with fasting glucose plasma levels and associated with insulin sensitivity of non-diabetic obese and nonobese subjects. The purpose of the present work was to get more insight into the regulation of adiponutrin gene expression by insulin and/or glucose using clamp studies and to examine its potential dysregulation in subjects with a deterioration of glucose homeostasis. Methods: Adiponutrin gene expression was quantified by reverse transcriptase-quantitative PCR in s.c. adipose tissue of healthy lean subjects after an euglycemic hyperinsulinemic clamp (EGHI), a hyperglycemic euinsulinemic clamp, and a hyperglycemic hyperinsulinemic (HGHI) clamp. Adiponutrin gene expression was also analyzed in patients with different levels of insulin resistance. Results: During EGHI, insulin infusion induced adiponutrin gene expression 8.4-fold (PZ0.008). Its expression was also induced by glucose infusion, although to a lesser extend (2.2-fold, PZ0.03). Infusion of both insulin and glucose (HGHI) had an additive effect on the adiponutrin expression (tenfold, PZ0.008). In a pathological context, adiponutrin gene was highly expressed in the adipose tissue of type-1 diabetic patients with chronic hyperglycemia compared with healthy subjects. Conversely, adiponutrin gene expression was significantly reduced in type-2 diabetics (PZ0.01), but remained moderately regulated in these patients after the EGHI clamp (2.5-fold increased). Conclusion: These results suggest a strong relationship between adiponutrin expression, insulin sensitivity, and glucose metabolism in human adipose tissue. European Journal of Endocrinology 155 461-468

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy in children and young adults. Currently, there is no cure for the disease. The transplantation of healthy myoblasts is an experimental therapeutic strategy, since it could restore the expression of dystrophin in DMD muscles. Nevertheless, this cellular therapy is limited by immune reaction, low migration of the implanted cells, and high early cell death that could be at least partially due to anoikis. To avoid the lack of attachment of the cells to an extracellular matrix after the transplantation, which is the cause of anoikis, we tested the use of a fibrin gel for myoblast transplantation. In vitro, three concentrations of fibrinogen were compared (3, 20, and 50 mg/ ml) to form a fibrin gel. A stiffer fibrin gel leads to less degradability and less proliferation of the cells. A concentration of 3 mg/ml fibrin gel enhanced the differentiation of the myoblasts earlier as a culture in monolayer. Human myoblasts were also transplanted in muscles of Rag/mdx mice in a fibrin gel or in a saline solution (control). The use of 3 mg/ml fibrin gel for cell transplantation increased not only the survival of the cells as measured after 5 days but also the number of fibers expressing dystrophin after 21 days, compared to the control. Moreover, the fibrin gel was also compared to a prosurvival cocktail. The survival of the myoblasts at 5 days was increased in both conditions compared to the control but the efficacy of the prosurvival cocktail was not significantly higher than the fibrin gel.Key words: Duchenne muscular dystrophy (DMD); Fibrin gel; Cell transplantation; Cell survival; Graft success INTRODUCTIONproliferate and differentiate as myoblasts, which subsequently activate myogenic differentiation to fuse among themselves to form new myofibers (17,33). These Duchenne muscular dystrophy (DMD) is the most common genetic muscle disorder in children. The incimyoblasts can be proliferated in culture and transplanted in muscles to fuse together and form new fibers or to dence is about 1/3,500 live male births and currently there is no efficient treatment (15). The cause of DMD fuse with host myofibers, introducing in these hybrid fibers nuclei able to produce dystrophin in the case of is a severe deficiency of dystrophin, a major component of the dystrophin-glycoprotein complex in muscle fibers, DMD. Unfortunately, transplanted proliferating myogenic cells undergo rapid and massive cell death within responsible for the maintenance of sarcolemma integrity (10). Following the loss of a functional dystrophin proa few days following implantation into skeletal muscles, as a result of cell dissociation, trophic factor withdrawal, tein, the muscles of DMD patients progressively degenerate as a result of continuous myofiber necrosis, leading oxidative stress, excitotoxicity, hypoxia, and possibly anoikis. Anoikis is a type of apoptosis that is triggered to death into the second/third decade of the patient's life (17,31).by detachment from the extracellular ma...

The MAPKKs MEK1 and MEK2 are activated by phosphorylation, but little is known about how these enzymes are inactivated. Here Mitogen-activated protein kinase (MAPK) 1 pathways are evolutionarily conserved signaling modules by which cells transduce extracellular chemical and physical signals into intracellular responses (reviewed in Refs. 1-3). These modules are organized into an architecture of three sequentially acting protein kinases comprising a MAPK kinase kinase (MAPKKK or MEK kinase), a MAPK kinase (MAPKK or MEK), and the MAPK itself. The propagation of the signal through MAPK pathways is facilitated by specific protein-protein interactions between individual components of the pathway and scaffolding proteins (3, 4).The prototypical and most studied MAPK pathway is the ERK1/2 pathway, which controls cell proliferation, differentiation, and development (1). Stimulation of cells with growth and differentiation factors leads to the activation of the MAPKKK Raf by a complicated mechanism involving cellular relocalization and multiple phosphorylation events (5, 6). Activated Raf isoforms bind to and activate the MAPKKs MEK1 and MEK2 by phosphorylation of two serine residues (corresponding to Ser 218 and Ser 222 in MEK1) in their activation loop (7,8). Substitution of the two regulatory serines with acidic residues is sufficient to enhance the basal activity of MEK1/2 (7-12). The dual-specificity kinases MEK1 and MEK2 then catalyze the phosphorylation of the MAPKs ERK1 and ERK2 at threonine and tyrosine residues within the activation loop motif Thr-Glu-Tyr (13), causing a reorientation of the loop and activation of the enzyme (14). Both MEK1 and MEK2 stably associate with ERK1/2, and this association is required for efficient activation of the latter in cells (15, 16). The binding site for ERK1/2 is located at the N terminus of MEK1/2 and consists of a short basic region known as the D domain (16). MEK1 and MEK2 also contain a unique proline-rich insert between subdomains IX and X, which is required for full activation of ERK1/2 in intact cells (17,18).The magnitude and duration of MAPK activation are important determinants of the cellular response to extracellular signals (19,20). Therefore, a tightly regulated balance between activation and inactivation mechanisms must exist to control the cellular activity of ERK1/2. Inactivation of the ERK1/2 enzymes is mainly achieved by dephosphorylation of the activating threonine and tyrosine residues. Biochemical and genetic studies have implicated both tyrosine-specific phosphatases and dual-specificity MAPK phosphatases in the negative regulation of ERK1/2 and other MAPKs (21,22). Much less is known about the mechanisms that negatively regulate the pathway at the MAPKK level. The serine/threonine phosphatase protein phosphatase 2A was identified as the major phosphatase inactivating MEK1 in lysates of PC12 cells (23). Furthermore, overexpression of SV40 small t antigen, which binds to the A subunit of protein phosphatase 2A and inactivates the enzyme, was found to s...