Browsing by Subject "Skeletal muscle"

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An amino acid mixture enhances insulin-stimulated glucose uptake in isolated epitrochlearis muscle
(2010-08) Kleinert, Maximilian; Ivy, John, 1945-; Farrar, Roger P.
Amino acids are important modulators of skeletal muscle metabolism, but their impact on glucose uptake by skeletal muscle remains unclear. To address the effect of an amino acid (AA) mixture consisting predominately of isoleucine on glucose uptake we first conducted a dose-response experiment, investigating how different concentrations of the AA mixture affect glucose uptake by isolated rat epitrochlearis muscle. In a subsequent experiment we examined how the AA mixture affects insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle. It was found that the AA mixture with as little as 0.5 mM Ile increases [H3]2-deoxy-D-glucose (2-DG) uptake by 76% compared to basal glucose uptake. The AA mixtures with 1, 2 or 4 mM Ile provided no significant additional effect. Next we combined the AA mixture consisting of 2 mM Ile, 0.012 mM Cys, 0.006 mM Val and 0.014 mM Leu with physiological levels (75 μU/ml, sINS) and maximally-stimulating levels (2 mU/ml, mINS) of insulin. The AA mixture only, sINS and mINS significantly increased 2-DG uptake compared to basal by 63, 79 and 298%, respectively. When the AA mixture was combined with sINS and mINS 2-DG uptake was further increased significantly by 26 and 14%, respectively. Western blotting analysis revealed that compared to basal the AA mixture increased AS160 phosphorylation, while phosphorylation of Akt and mTOR did not change. Combining the AA mixture with sINS resulted in no additional phosphorylation compared to sINS alone. Interestingly, addition of the AA mixture to mINS resulted in increased phosphorylation of mTOR, Akt and AS160 compared to mINS alone. Our results suggest that certain AAs (1) increase glucose uptake in the absence of insulin and (2) augment insulin-stimulated glucose uptake in an additive manner. These effects on glucose uptake appear to be mediated via a molecular pathway that is partially independent from the canonical insulin signaling cascade.
Chromium chloride increases insulin-stimulated glucose uptake in the perfused rat hindlimb
(2010-12) Doerner, Phillip Gene; Ivy, John, 1945-
Chromium has been reported to increase glucose clearance in insulin resistant and diabetic populations. Skeletal muscle is the tissue primarily responsible for glucose clearance. We therefore tested the effect of chromium chloride (CrCl3) on skeletal muscle glucose uptake both in the absence and presence of a submaximal level of insulin via the rat hindlimb perfusion technique. 0.096 μM CrCl3 was used with and without 200 μU/ml insulin. Our testing showed that insulin significantly increased [H3]-2 deoxyglucose (2-DG) uptake in both the gastrocnemius and quadriceps muscles. Additionally, the combination of CrCl3 and insulin (Cr-sIns) led to greater amounts of 2-DG uptake than insulin alone (sIns) in both the gastrocnemius (Cr-sIns 6.49±0.75 μmol/g/h, sIns 4.83±0.42 μmol/g/h) and quadriceps (Cr-sIns 6.74±0.62 μmol/g/h, sIns 4.54±0.43 μmol/g/h). However, CrCl3 without insulin (Cr) had no affect on 2-DG uptake above basal (Bas) in both the gastrocnemius (Cr 1.45±0.14 μmol/g/h, Bas 1.61±30 μmol/g/h) and the quadriceps (Cr 1.35±0.15 μmol/g/h, Bas 1.27±0.13 μmol/g/h). It has been speculated that chromium works to increase glucose uptake by increasing insulin signaling. To examine this, we used western blotting analysis to test both Akt and AS160 phosphorylation in the mixed gastrocnemius. We found that insulin increased Akt and AS160 phosphorylation, but chromium had no affect on Akt (Cr-sIns 25%±2%, sIns 22%±4%) or AS160 (Cr-sIns 35%±5%, sIns 36%±4%) phosphorylation in the absence or presence of insulin. Our results suggest that supplementation with CrCl3 can lead to an increase in glucose uptake in skeletal muscle, but only in the presence of insulin. However, this effect of CrCl3 does not appear to be a result of enhanced insulin signaling.
High intensity resistance training induced angiogenesis and muscle hypertrophy enhance skeletal muscle regeneration in volumetric muscle loss rats
(2015-12) Lee, Kwangjun; Farrar, Roger P.; Brothers, Matthew R
Skeletal muscle has an outstanding regenerative capacity when damaged muscle mass is less than 20%. The volumetric muscle loss (VML) injury, muscle loss beyond self-repair capacity, results in functional and morphological disability. This study investigated the effect of myofibers injection into a decellurized extracellular matrix (ECM) with resistance training on skeletal muscle growth following a VML injury. Male fisher 344 and 2 month old F344-Tg (UBC-EGFP) rats, as myofiber donors, were used in this study. Approximately 20% of the mass of the lateral gastrocnemius (LGAS) was excised, which was replaced by ECM in same dimensions. 30 myofibers were injected into the injury site in 7 days of post injury, Ladder climbing began at 10 days post defect surgery, and rats were subjected to climb a ladder every third day with weight for 6 weeks. Following 56 days of recovery, EXE group (5122±92μm2) increased cross sectional area of intact muscle area significantly compared to ECM (4668±79μm2), FIB (4795±82μm2), and FIB+EXE (47642±97μm2) groups. Quantification of the number of blood vessel larger than 20μm in the entire area showed a significant difference only in EXE group (46±4.2) compared to ECM group (35±3.89). EXE group (34±3.2) significantly increased the number of blood vessel compared to ECM group (22±3.3). A significant difference in connective tissue area in middle region of the ECM was quantified between EXE (45±1.6%) and ECM (69±1.9%) groups (Figure 10). Moreover, small muscle fiber area within ECM was significantly higher in EXE group (1.39±30.15 μm2) than ECM and FIB groups (0.67±0.15μm2). The data suggest that ECM transplantation with resistance training can repair a volumetric muscle loss injury by hypertrophy, angiogenesis, and myofiber infiltration through entire ECM regions.
IGF-1 conjugated to a PEGylated-Fibrin hydrogel as a therapeutic modality for eccentric muscle damage in rats
(2011-12) Treff, Jessica Caitlin; Farrar, Roger P.
We evaluated the efficacy of treating eccentric muscle damage with IGF-1 PEGylated to a fibrin biomatrix. With one injection, delivered one hour after the induction of eccentric muscle damage we saw an attenuation of force loss early in recovery, maintenance of muscle weight, and progression to the repair/regeneration of the damaged fibers at a greater speed and magnitude in the first week of recovery. As opposed to introducing an unbound bolus of IGF-1, we believe the ability of the PEGylated-fibrin to stabilize and sustain delivery of the molecule results in significantly better recovery. Coupling IGF-1, which has multiple beneficial effects in tissue repair, with this system of delivery provides a simple and easy to administer treatment for eccentric muscle damage. With this form of damage being the most prevalent of all skeletal muscle damage types, since it is underlies all muscle strain, a simple and effective treatment is important for increasing functional recovery after injury.
Investigation of the physiological and biochemical function of mitochondrial uncoupling protein 3
(2010-12) Kenaston, Monte Alexander; Mills, Edward M.; Bratton, Shawn B.; Gore, Andrea C.; Hursting, Stephen D.; Sprague, Jon E.
Uncoupling proteins (UCPs) are highly conserved inner mitochondrial membrane proteins that have been found in plants, nematodes, flies, and vertebrates. UCPs dissipate the proton gradient formed by the electron transport chain in an energy-expending process that generates heat. In mammals, the brown fat-specific UCP1 is thought to be the dominant, if not the only significant mediator of thermogenic responses. However, adult humans express only negligible amounts of brown fat and UCP1, yet still show significant non-shivering thermogenic responses (e.g. amphetamine-induced hyperthermia, diet induced thermogenesis, fever). Thus, the fact that human thermogenic mechanisms haven't been identified is a huge gap in our understanding of human thermoregulation. UCP3 is primarily expressed in skeletal muscle, an established thermogenic organ which is a major target of amphetamine-induced pathology. UCP3 knockout mice have a near complete loss (~80%) of amphetamine-induced thermogenesis and are completely protected from amphetamine-induced death over a range of lethal doses. With regard to mechanisms of UCP3 activation, we observed that norepinephrine and free fatty acids are elevated in the bloodstream prior to peak amphetamine-induced hyperthermia. However, little is known about the anatomic location of UCP3-dependent thermogenesis or the mechanisms by which fatty acids regulate UCP function. Thus, we sought to investigate the physiology and biochemical activation of UCP3 to establish the thermogenic potential of skeletal muscle uncoupling and elucidate the mechanisms of UCP3 function. The overall goal of this research was to identify the tissue target(s) and mechanisms involved in amphetamine-induced UCP3-dependent thermogenesis. Herein, we show that in addition to a deficit in induced thermogenesis, UCP3-null mice also lack responses to other physiologically-relevant stimuli (i.e. catecholamines and bacterial pathogens). Conversely, UCP3 knockout mice, engineered to express UCP3 only in skeletal muscle have an augmented thermogenic response to amphetamines. In order to explore UCP3's mechanism of activation, we performed a modified yeast two-hybrid analysis and identified [Delta][superscript 3,5][Delta][superscript 2,4]dienoyl-CoA isomerase (DCI) as a UCP3 binding partner. DCI, an auxiliary fatty acid oxidation enzyme, protects cells from the accumulation of toxic lipid metabolites. Using immunoprecipitation and fatty acid oxidation (FAO) assays, we determined that UCP3 and DCI directly bind in the mitochondrial matrix in order to augment lipid metabolism. These findings support a novel model in which skeletal muscle UCP3 is responsible for inducible thermogenesis through cooperation with binding partners such as DCI which enhance oxidation of fatty acids. Together, these studies shed light on thermogenic pathways in rodents that are likely to be relevant to humans.
The underlying mechanisms of UCP3-dependent thermogenesis in skeletal muscle
(2015-12) Dao, Christine Ky Linh; Mills, Edward Michael; Wright, Casey; Bratton, Shawn; Mukhopadhyay, Somshuvra; Ivy, John
Mitochondrial uncoupling proteins (UCPs) are anion / solute transporters that dissipate the proton gradient used to drive ATP generation. By allowing protons to flow down their electrochemical gradient, UCP activation releases the energy generated from mitochondrial substrate oxidation as heat. This thermogenic process is important in normal thermoregulation (i.e. non-shivering thermogenesis), and also serves as an attractive target in the treatment of obesity by lowering metabolic efficiency. The skeletal muscle (SKM) enriched UCP homologue, UCP3, is associated with increased energy expenditure, fatty acid metabolism, and insulin sensitivity. Unlike the cold-induced prototypical pathway of UCP1-mediated non-shivering thermogenesis in brown adipose tissue (BAT), the mechanisms underlying the thermogenic actions of UCP3 in SKM are not well characterized. Although global UCP3 knockout mice exhibit normal thermoregulatory responses to cold under fed conditions, they exhibit an attenuated hyperthermic response when administered amphetamine-type drugs. In our initial investigation, we show that selective overexpression of UCP3 in SKM by the human α-skeletal actin promoter restored methamphetamine (Meth)-induced hyperthermia in the UCP3⁻/⁻ background (TgSKM UCP3⁻/⁻), but not in the UCP1/UCP3 double knockout background (TgSKM UCP1⁻/⁻+UCP3⁻/⁻). Taken together, these findings further bolster the role of UCP3 as a thermogenic mediator in SKM, and suggest a novel mechanism of crosstalk between BAT UCP1 and SKM UCP3 in Meth-induced hyperthermia. In the second aspect of my project, we characterized the underlying mechanisms of UCP3-dependent thermogenesis within SKM by utilizing an immunoprecipitation- based mass spectrometry approach to identify interacting partners of UCP3. These analyses corroborated previous work performed by our lab, and demonstrated that UCP3 interacts with a subset of fatty acid metabolizing enzymes. Interestingly, one such enzyme, enoyl-CoA hydratase-1 (ECH1), is involved in the metabolism of oleic acid, a known ligand activator of UCP3. This work reveals that ECH1:UCP3 complex formation enhances uncoupled-respiration and fatty acid metabolism, and that genetic mouse models in vivo show that UCP3 and ECH1 participate in a common pathway of thermogenesis. These findings support a new model by which UCP3-dependent thermogenesis in SKM is mediated in part through its cooperation with ECH1, and suggest new approaches for treatment of obesity and related metabolic diseases.