753 resultados para skeletal muscle gene expression


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RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

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BACKGROUND: Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle. METHODS: Hyperpolarized [1-(13)C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The (13)C magnetic resonance signals of [1-(13)C]acetate and [1-(13)C]acetylcarnitine were recorded in vivo for 1min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios. RESULTS: Although separated by two biochemical transformations, a kinetic analysis of the (13)C label flow from [1-(13)C]acetate to [1-(13)C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were KM=0.35±0.13mM and Vmax=0.199±0.031μmol/g/min. CONCLUSIONS: The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results. GENERAL SIGNIFICANCE: This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.

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BackgroundBipolar disorder is a highly heritable polygenic disorder. Recent enrichment analyses suggest that there may be true risk variants for bipolar disorder in the expression quantitative trait loci (eQTL) in the brain.AimsWe sought to assess the impact of eQTL variants on bipolar disorder risk by combining data from both bipolar disorder genome-wide association studies (GWAS) and brain eQTL.MethodTo detect single nucleotide polymorphisms (SNPs) that influence expression levels of genes associated with bipolar disorder, we jointly analysed data from a bipolar disorder GWAS (7481 cases and 9250 controls) and a genome-wide brain (cortical) eQTL (193 healthy controls) using a Bayesian statistical method, with independent follow-up replications. The identified risk SNP was then further tested for association with hippocampal volume (n = 5775) and cognitive performance (n = 342) among healthy individuals.ResultsIntegrative analysis revealed a significant association between a brain eQTL rs6088662 on chromosome 20q11.22 and bipolar disorder (log Bayes factor = 5.48; bipolar disorder P = 5.85×10(-5)). Follow-up studies across multiple independent samples confirmed the association of the risk SNP (rs6088662) with gene expression and bipolar disorder susceptibility (P = 3.54×10(-8)). Further exploratory analysis revealed that rs6088662 is also associated with hippocampal volume and cognitive performance in healthy individuals.ConclusionsOur findings suggest that 20q11.22 is likely a risk region for bipolar disorder; they also highlight the informative value of integrating functional annotation of genetic variants for gene expression in advancing our understanding of the biological basis underlying complex disorders, such as bipolar disorder.

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This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

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This thesis investigated whole body glucose disposal and the adaptive changes in skeletal muscle carbohydrate metabolism following 28 d of supplementation with 1000 mg R(+)-lipoic acid in young sedentary males (age, 22.1 ± 0.67 yr, body mass, 78.7 ± 10.3 kg, n=9). In certain individuals, lipoic acid decreased the 180-min area under the glucose concentration and insulin concentration curve during an oral glucose tolerance test (OGTT) (n=4). In the same individuals, lipoic acid supplementation decreased pyruvate dehydrogenase kinase activity (PDK) (0.09 ± 0.024 min"^ vs. 0.137 ± 0.023 min'\ n=4). The fasting levels of the activated form of pyruvate dehydrogenase (PDHa) were decreased following lipoic acid (0.42 ± 0.13 mmol-min'kg'^ vs. 0.82 ± 0.32 mmolrnin'^kg"\ n=4), yet increased to a greater extent during the OGTT (1.21 ± 0.34 mmol-min'kg"' vs. 0.81 ±0.13 mmolmin"'kg'\ n=4) following hpoic acid supplementation. No changes were demonstrated in the remaining subjects (n=5). It was concluded that improved glucose clearance during an OGTT following lipoic acid supplementation is assisted by increased muscle glucose oxidation through increased PDHa activation and decreased PDK activity in certain individuals.

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BACKGROUND: Capillaries function to provide a surface area for nutrient and waste exchange with cells. The capillary supply of skeletal muscle is highly organized, and therefore, represents an excellent choice to study factors regulating diffusion. Muscle is comprised of three specific fibre types, each with specific contractile and metabolic characteristics, which influence the capillary supply of a given muscle; in addition, both environmental and genetic factors influence the capillary supply, including aging, physical training, and various disease processes. OBJECTIVE: The present study was undertaken to develop and assess the functionality of a data base, from which virtual experiments can be conducted on the capillary supply of human muscle, and the adaptations of the capillary bed in muscle to various perturbations. METHODS: To create the database, an extensive search of the literature was conducted using various search engines, and the three key words - "capillary, muscle, and human". This search yielded 169 papers from which the data for the 46 variables on the capillary supply and fibre characteristics of muscle were extracted for inclusion in the database. A series of statistical analyses (ANOVA) were done on the capillary database to examine differences in skeletal muscle capillarization and fibre characteristics between young and old individuals, between healthy and diseased individuals, and between untrained, endurance trained, endurance welltrained, and resistance trained individuals, using SAS. RESULTS: There was a significantly higher capillarization in the young compared to the old individuals, in the healthy compared to the diseased individuals, and in the endurance-trained and endurance well-trained compared to the untrained individuals. CONCLUSIONS: The results of this study support the conclusion that the capillary supply of skeletal muscle is closely regulated by factors aimed at optimizing oxygen and nutrient supply and/or waste removal in response to changes in muscle mass and/or metabolic activity.

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University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

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Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

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The purpose of this study was to examine cell glucose kinetics in rat skeletal muscle during iso-osmotic recovery from hyper- and hypo-osmotic stress. Rat EDL muscles were incubated for sixty minutes in either HYPO (190 mmol/kg), ISO (290 mmol/kg), or HYPER (400 mmol/kg) media (Sigma medium-199, 8 mM glucose) according to an established in vitro whole muscle model. In addition to sixty minute baseline measures in aniso-osmotic conditions, (HYPO-0 n=8; ISO- 0, n=S; HYPER-0, n=8), muscles were subjected to either one minute (HYPO-1 n=8; ISO-1, n=8; HYPER-1, n=8) or five minutes (HYPO-5 n=8; ISO-5, n=8; HYPER-5, n=8) of iso-osmotic recovery media and analyzed for metabolite content and glycogen synthase percent activation. To determine glucose uptake during iso-osmotic recovery, muscles (n=6 per group) were incubated for sixty minutes in either hypo-, iso-, or hyper-osmotic media immediately followed by five minutes of iso-osmotic media containing 3H-glucose and 14 C-mannitol. Increased relative water content/decreased [glucose] (observed in HYPO-0) and decreased water content/increased [glucose] (observed in HYPER-0) returned to ISO levels within 5 minutes of recovery. Glycogen synthase percent activation increased significantly in HYPO-5 over iso-osmotic controls. Glucose uptake measurements revealed no significant differences between groups. It was determined that [glucose] and muscle water content rapidly recovered from osmotic stress demonstrating skeletal muscle's resilience to osmotic stress.

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The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

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Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise and its activity can be down-regulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of PDH in its active form (PDHa) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n=7) underwent 2 fat loading trials spaced at least 2 weeks apart. Subjects consumed saturated (SFA) or polyunsaturated (PUFA) fat over the course of 5 hours. Following this, participants cycled at 65% VO2 max for 15 min. Muscle biopsies were taken prior to and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 ± 0.07 to 0.54 ± 0.19 mM over 5 hours with SFA and from 0.1 1 ± 0.04 to 0.35 ±0.13 mM with PUFA. PDHa activity was unchanged following fat loading, but increased at the onset of exercise in the SFA trial, from 1 .4 ± 0.4 to 2.2 ± 0.4 /xmol/min/kg wet wt. This effect was negated in the PUFA trial (1 .2 ± 0.3 to 1 .3 ± 0.3 pimol/min/kg wet wt.). PDH kinase (PDK) was unchanged in both trials, suggesting that the attenuation of PDHa activity with PUFA was a result of changes in the concentrations of intramitochondrial effectors, more specifically intramitochondrial NADH or Ca^*. Our findings suggest that attenuated PDHa activity participates in the preferential oxidation of PUFA during moderateintensity exercise.

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This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

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The time course for the reversal of the adaptive increase in pyruvate dehydrogenase kinase (PDK) activity following a 6d high fat diet (HP: 4.2 ± 0.2 % carbohydrate; 75.6 ± 0.4 % fat; 19.5 ± 0.8 % protein) was investigated in human skeletal muscle (vastus lateralis). HF feeding increased PDK activity by 44% (from 0.081 ± 0.025 min"' to 0.247 ± 0.025 mm\p < 0.05). Following carbohydrate re-feeding, (88% carbohydrate; 5% fat; 7% protein), PDK activity had returned to baseline (0.111 ± 0.014 min"') within 3h of re-feeding. The active fraction of pyruvate dehydrognease (PDHa) was depressed following 6d of the HF diet (from 0.89 ± 0.21 mmol/min/kg WW to 0.32 ± 0.05 mmol/min/kg ww,p <0.05) and increased to pre-HF levels by 45 min of post re-feeding (0.74 ±0.19 mmol/min/kg ww) and remained elevated for 3h. Western blotting analysis of the PDK isoforms, PDK4 and PDK2, revealed a 31% increase in PDK4 protein content following the HF diet, with no change in PDK2 protein. This adaptive increase in PDK4 protein content was reversed with carbohydrate re-feeding. It was concluded that the adaptive up-regulation in PDK activity and PDK4 protein content was fiilly reversed by 3h following carbohydrate re-feeding.

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Resveratrol, a polyphenol found in red wine, has been reported to have antithrombotic, antiatherogenic, and anticancer properties both in vitro and III VIVO. However, possible antidiabetic properties of resveratrol have not been examined. The objective of this study was to investigate the direct effects of resveratrol on basal and insulin-stimulated glucose uptake and to elucidate its mechanism of action in skeletal muscle cells. In addition, the effects of resveratrol on basal and insulin- stimulated amino acid transport and mitogenesis were also examined. Fully differentiated L6 rat skeletal muscle cells were incubated with resveratrol concentrations ranging from 1 to 250 IlM for 15 to 120 min. Maximum stimulation, 201 ± 8.90% of untreated control, (p<0.001), of2eH] deoxy- D- glucose (2DG) uptake was seen with 100 IlM resveratrol after 120 min. Acute, 30 min, exposure of the cells to 100 nM insulin stimulated 2DG uptake to 226 ± 12.52% of untreated control (p<0.001). This appears to be a specific property of resveratrol that is not shared by structurally similar antioxidants such as quercetin and rutin, both of which did not have any stimulatory effect. Resveratrol increased the response of the cells to submaximal insulin concentrations but did not alter the maximum insulin response. Resveratrol action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. L Y294002 and wortmannin, inhibitors of PI3K, abolished both insulin and resveratrolstimulated glucose uptake while phosphorylation of AktlPKB, ERK1I2, JNK1I2, and p38 MAPK were not increased by resveratrol. Resveratrol did not stimulate GLUT4 transporter translocation in GLUT4cmyc overexpressing cells, in contrast to the significant translocation observed with insulin. Furthermore, resveratrol- stimulated glucose transport was not blocked by the presence of the protein kinase C (PKC) inhibitors BIMI and G06983. Despite that, resveratrol- induced glucose transport required an intact actin network, similar to insulin. In contrast to the stimulatory effect seen with resveratrol for glucose transport, e4C]methylaminoisobutyric acid (MeAIB) transport was inhibited. Significant reduction of MeAIB uptake was seen only with 100uM resveratrol (74.2 ± 6.55% of untreated control, p<0.05), which appeared to be maximum. In parallel experiments, insulin (100 nM, 30 min) increased MeAIB transport by 147 ± 5.77% (p<0.00l) compared to untreated control. In addition, resveratrol (100 JlM, 120 min) completely abolished insulin- stimulated amino acid transport (103 ± 7.35% of untreated control,p>0.05). Resveratrol also inhibited cell proliferation in L6 myoblasts with maximal inhibition of eH]thymidine incorporation observed with resveratrol at 50 J.LM after 24 hours (8 ± 1.59% of untreated control, pskeletal muscle cells by a mechanism that is in4ependent of insulin and protein synthesis. Resveratrol- stimulated glucose uptake may be PI3K and actin cytoskeleton- dependent and independent of AktIPKB, PKC, ERK1I2, JNK1I2, p38 MAPK, and GLUT4 translocation. However, unlike glucose transport, resveratrol inhibits both basal and insulin- stimulated amino acid transport and mitogenesis.

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Diabetes mellitus is a disorder of inadequate insulin action and consequent high blood glucose levels. Type 2 diabetes accounts for the majority of cases of the disease and is characterized by insulin resistance and relative insulin deficiency resulting in metabolic deregulation. It is a complex disorder to treat as its pathogenesis is not fully understood and involves a variety of defects including ~-cell failure, insulin resistance in the classic target tissues (adipose, muscle, liver), as well as defects in a-cells and kidney, brain, and gastrointestinal tissue. Present oral treatments, which aim at mimicking the effects of insulin, remain limited in their efficacy and therefore the study of the effects of novel compounds on insulin target tissues is an important area of research both for potentially finding more treatment options as well as for increasing our knowledge of metabolic regulation in health and disease. In recent years the extensively studied polyphenol, resveratrol, has been reported to have antidiabetic effects showing that it increases glucose uptake by skeletal muscle cells and prevents fatty acid-induced insulin resistance in vitro and in vivo. Naringenin, a citrus flavonoid with structural similarities to resveratrol, is reported to have antioxidan.t, antiproliferative, anticancer, and anti-inflammatory properties. Effects on glucose and lipid metabolism have also been reported including blood glucose and lipid lowering effects. However, whether naringenin has insulinlike effects is not clear. In the present study the effects of naringenin on glucose uptake in skeletal muscle cells are examined and compared with those of insulin. Naringenin treatment of L6 myotubes increased glucose uptake in a dose- and time dependent manner and independent of insulin. The effects of naringenin on glucose uptake achieved similar levels as seen with maximum insulin stimulation and its effect was additive with sub-maximal insulin treatment. Like insulin naringenin treatment did not increase glucose uptake in myoblasts. To elucidate the mechanism involved in naringenin action we looked at its effect on phosphatidylinositol 3-kinase (PI3K) and Akt, two signalling molecules that are involved in the insulin signalling cascade leading to glucose uptake. Naringenin did not stimulate basal or insulinstimulated Akt phosphorylation but inhibition of PI3K by wortmannin partially repressed the naringenin-induced glucose uptake. We also examined naringenin's effect on AMP-activated protein kinase (AMPK), a molecule that is involved in mediating glucose uptake by a variety of stimuli. Naringenin stimulated AMPK phosphorylation and this effect was not inhibited by wortmannin. To deduce the nature of the naringenin-stimulated AMPK phosphorylation and its impact on glucose uptake we examined the role of several molecules implicated in mod.ulating AMPK activity including SIRTl, LKB 1, and ca2+ Icalmodulin-dependent protein kinase kinase (CaMKK). Our results indicate that inhibition of SIRTI did not prevent the naringeninstimulated glucose uptake Of. AMPK phosphorylation; naringenin did not stimulate LKB 1 phosphorylation; and inhibition of CaMKK did not prevent naringeninstimulated glucose uptake. Inhibition of AMPK by compound C also did not prevent naringenin-stimulated glucose uptake but effectively inhibited the phosphorylation of AMPK suggesting that AMPK may not be required for the naringenin-stimulated glucose uptake.