The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

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.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

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.

Human skeletal muscle pyruvate dehydrogenase phosphatase activity and expression : the effect of aerobic capacity

Activation of pyruvate dehydrogenase (PDH), which converts pyruvate into acetyl-CoA, is accomplished by a pair of specific phosphatases (PDP 1 & 2). A cross-sectional study investigating the effect of aerobic capacity on PDP activity and expression found that: 1) PDP activity and PDP! protein expression were positively correlated with most aerobic capacity measures in males (n=lS), but not females (n=12); 2) only males showed a positive correlation between PDP activity and PDPl protein expression (r=0.47; p=O.05), indicating that the increase in PDP activity in males is largely explained by increased PDPl protein expression, but that females rely on another level for PDP activity regulation; and 3) PDP} and Ela protein expression increase in unison when expressed relative to the E2 core. These data suggest that with increased aerobic capacity there is an increased capacity for carbohydrate oxidation through PDH, via El a, and an increased ability to activate PDH, via PDP, when exercising maximally.

Protein Turnover in Rat Skeletal Muscle During In Vitro Hypo-Osmotic Stress

Hypo-osmolality influences tissue metabolism, but research on protein turnover in skeletal muscle is limited. The purpose of this investigation was to examine the effects of hypo-osmotic stress on protein turnover in rat skeletal muscle. We hypothesized increased protein synthesis and reduced degradation following hypo-osmotic exposure. EDL muscles (n=8/group) were incubated in iso-osmotic (290 Osm/kg) or hypo-osmotic (190 Osm/kg) modified medium 199 (95% O2, 5% CO2, pH 7.4, 30±2 °C) for 60 min, followed by 75 min incubations with L-U[14C]phenylalanine or cycloheximide to determine protein synthesis and degradation. Immunoblotting was performed to assess signalling pathways involved. Phenylalanine uptake and incorporation were increased by 199% and 169% respectively in HYPO from ISO (p < 0.05). This was supported by elevated phosphorylation of mTOR Ser2448 (+12.5%) and increased Thr389 phosphorylation on p70s6 kinase (+23.6%) (p < 0.05). Hypo-osmotic stress increased protein synthesis and potentially amino acid uptake. Future studies should examine the upstream mechanisms involved.

Phosphorylation of Skeletal Muscle Pyruvate Dehydrogenase Phosphatase in Response to Insulin Stimulation

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the pyruvate dehydrogenase (PDH) complex. PDP activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In culture myoblasts, both PDP1 and PDP2 undergo covalent activation in response to insulin–stimulation by protein kinase C delta (PKCδ). Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation in skeletal muscle. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, 1g of tension) for 30min in basal or insulin–stimulated (10 mU/mL) media. PDH activity increased 58% following stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.047) and PDP2 (p=0.006) increased by 29% and 48%, respectively (n=8), and mitochondrial PKCδ protein content was enriched by 45% in response to stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.

Skeletal muscle protein content of lipolytic inhibitor G(0)/G(1) switch gene-2 protein: the effect of endurance training

The first and rate-limiting step of lipolysis is the removal of the first fatty acid from a triglyceride molecule; it is catalyzed by adipose triglyceride lipase (ATGL). ATGL is co-activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 protein (G0S2). G0S2 has also recently been identified as a positive regulator of oxidative phosphorylation within the mitochondria. Previous research has demonstrated in cell culture, a dose dependent mechanism for inhibition by G0S2 on ATGL. However our data is not consistent with this hypothesis. There was no change in G0S2 protein content during an acute lipolytic inducing set of contractions in both whole muscle, and isolated mitochondria yet both ATGL and G0S2 increase following endurance training, in spite of the fact that there should be increased reliance on intramuscular lipolysis. Therefore, inhibition of ATGL by G0S2 appears to be regulated through more complicated intracellular or post-translation regulation.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

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.

The effects of R(+)-lipoic acid supplementation on regulation of human skeletal muscle pyruvate dehydrogenase

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.

Effects of naringenin on glucose uptake in L6 skeletal muscle cells

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.

Pyruvate dehydrogenase activity in response to skeletal muscle contraction at two stimulation frequencies in pyruvate dehydrogenase kinase 2 knockout mice

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PD H is deactivated by a set of PD H kinases (PD K 1-4) with PDK2 and 4 being the predominant isoforms in skeletal muscle. PDK2 is highly sensitive to pyruvate inhibition, and is the most abundant isoform, while PDKI and 4 protein content are normally lower. This study examined the PDK isoform content and PDHa activation in muscle at rest and 10 and 40 Hz stimulation from PDK2 knockout (PDK2KO) mice to delineate the role of PDK2 in activating the PDH complex during low and moderate intensity muscle contraction. PDHa activity was lower in PDK2KO mice during contraction while total PDK actitvity was -4 fold lower. PDK4 protein was not different, however PDKI partially compensated for the lack of PDK2 and was -56% higher than WT. PDKI is a very potent inhibitor of the PDH complex due to its phosphorylation site specificity and allosteric regulation. These results suggest that the site specificity and allosteric regulatory properties of the individual PDK isoforms are more important than total PDK activity in determining transformation of the complex and PDHa activity during acute muscle contraction.

The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.

Changes in mitochondrial PLIN3 and PLIN5 protein content in rat skeletal muscle following acute contraction and endurance training

Surrounding lipid droplets in skeletal muscle are the perilipin (PLIN2-5) family of proteins, regulating lipid droplet metabolism. During exercise lipid droplets provide fatty acids to the mitochondria for oxidation while increasing their proximity to each other. Whether PLIN3 and PLIN5 associate with mitochondria following contraction has not been examined. To determine whether contraction altered mitochondrial PLIN3 and PLIN5 content, sedentary and endurance trained rats underwent acute contraction. The main outcomes are; 1) mitochondrial PLIN3 content is unaltered while mitochondrial PLIN5 content is increased following an acute contraction 2) mitochondrial PLIN3 content is higher in endurance trained rats when compared to sedentary and mitochondrial PLIN5 content is similar in both conditions 3) only PLIN5 mitochondrial content is increased similarly in both groups following acute contraction. This work highlights the dynamics of these two PLIN proteins, which may have roles not only on the lipid droplet but also on the mitochondria.

Investigation of the Time Dependent Influence of Extracellular Osmotic Stress on Protein Turnover in Skeletal Muscle Cells

Acute alterations in cell volume can substantively modulate subsequent metabolism of substrates. However, how such alterations in skeletal muscle modulate protein metabolism is limited. The purpose of this study was to determine the time dependent influence of extracellular osmotic stress on protein turnover in skeletal muscle cells. L6 cells were incubated in hyperosmotic (HYPER; 425.3 ± 1.8mmol/kg), hypo-osmotic (HYPO; 235.4 ± 1.0mmol/kg) or control (CON; 333.5 ± 1.4mmol/kg) media for 4, 8, 12, or 24hrs. During the final 4hrs, incorporation of L-[ring-3,5-3H]-tyrosine was measured to estimate protein synthesis. Western blotting measured markers of protein synthesis and degradation. No differences were observed in any outcomes except p70S6K phosphorylation whereby HYPO was lower (p<0.05) than CON and HYPER; which remained similar except for a large increase at 8hrs for HYPER. These findings suggest that regardless of duration, extracellular osmotic stress does not significantly affect protein metabolism in L6 cells.

INFLUENCE OF INCREASED EXTRACELLULAR LEUCINE ON THE PROTEIN METABOLIC RESPONSES DURING OSMOTIC STRESS IN SKELETAL MUSCLE

The purpose of this study was to examine the effects of increased extracellular leucine concentration on protein metabolism in skeletal muscle cells when exposed to 3 different osmotic stresses. L6 skeletal muscle cells were incubated in either a normal or supplemental leucine (1.5mM) medium set to hypo-osmotic (230 ± 10 Osm), iso-osmotic (330 ± 10 Osm) or hyper-osmotic (440 ± 10 Osm) conditions. 3H-tyrosine was used to quantify protein synthesis. Western blotting analysis was performed to determine the activation of mTOR, p70S6k, ubiquitin, actin, and μ-calpain. Hypo-osmotic stress resulted in the greatest increase in protein synthesis rate under the normal-leucine condition while iso-osmotic stress has the greatest increase under the elevated-leucine condition. Elevated-leucine condition had a decreased rate in protein degradation over the normal condition within the ubiquitin proteasome pathway (p<0.05). Leucine and hypo-osmotic stress therefore creates a favourable environment for anabolic events to occur.