Charles A. Stuart

Insulin-Stimulated Translocation of Glucose Transporter (GLUT) 12 Parallels That of GLUT4 in Normal Muscle

CONTEXT GLUT4 is the predominant glucose transporter isoform expressed in fat and muscle. In GLUT4 null mice, insulin-stimulated glucose uptake into muscle was diminished but not eliminated, suggesting that another insulin-sensitive system was present. OBJECTIVE This study was intended to determine whether insulin caused GLUT12 translocation in muscle. DESIGN Six normal volunteers had muscle biopsies before and after euglycemic insulin infusions. SETTING Infusions and biopsies were performed in an outpatient clinic. PARTICIPANTS Subjects were nonobese, young adults with no family history of diabetes. MAIN OUTCOME MEASURES GLUT12, GLUT4, and GLUT1 proteins were quantified in muscle biopsy fractions. Cultured myoblasts were used to determine whether GLUT12 translocation was phosphatidyl inositol-3 kinase (PI3-K)-dependent. INTERVENTION Insulin was infused at 40 mU/m(2) x min for 3 h. RESULTS In human muscle, insulin caused a shift of a portion of GLUT12 from intracellular low-density microsomes to the plasma membrane (PM) fraction (17% in PM at baseline, 38% in PM after insulin). Insulin increased GLUT4 in PM from 13 to 42%. GLUT1 was predominantly in the PM fractions at baseline and did not change significantly after insulin. L6 myoblasts in culture also expressed and translocated GLUT12 in response to insulin, but inhibiting PI3-K prevented the translocation of GLUT12 and GLUT4. CONCLUSIONS Insulin causes GLUT12 to translocate from an intracellular location to the plasma membrane in normal human skeletal muscle. Translocation of GLUT12 in cultured myoblasts was dependent on activation of PI3-K. GLUT12 may have evolutionarily preceded GLUT4 and now provides redundancy to the dominant GLUT4 system in muscle.

Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-κB pathways

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. We have previously shown that morphine induces Fas expression and promotes Fas-mediated apoptosis. Here, we investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells. Morphine-induced apoptosis was inhibited by transfection with a dominant negative Fas-associated death domain (FADD) plasmid, revealing that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression by RNA interference technology considerably attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K), as PI3K inhibition by the PI3K inhibitor LY294002 significantly enhanced morphine-induced apoptosis. Moreover, inhibition of Akt or nuclear factor-kappaB (NF-kappaB) expression by RNA interference technology also dramatically increased morphine-induced apoptosis. Our study thus demonstrates that morphine induces Jurkat cell apoptosis through FADD/p53, anti-apoptotic PI3K/Akt and NF-kappaB pathways.

Slow-Twitch Fiber Proportion in Skeletal Muscle Correlates With Insulin Responsiveness

CONTEXT The metabolic syndrome, characterized by central obesity with dyslipidemia, hypertension, and hyperglycemia, identifies people at high risk for type 2 diabetes. OBJECTIVE Our objective was to determine how the insulin resistance of the metabolic syndrome is related to muscle fiber composition. DESIGN Thirty-nine sedentary men and women (including 22 with the metabolic syndrome) had insulin responsiveness quantified using euglycemic clamps and underwent biopsies of the vastus lateralis muscle. Expression of insulin receptors, insulin receptor substrate-1, glucose transporter 4, and ATP synthase were quantified with immunoblots and immunohistochemistry. PARTICIPANTS AND SETTING Participants were nondiabetic, metabolic syndrome volunteers and sedentary control subjects studied at an outpatient clinic. MAIN OUTCOME MEASURES Insulin responsiveness during an insulin clamp and the fiber composition of a muscle biopsy specimen were evaluated. RESULTS There were fewer type I fibers and more mixed (type IIa) fibers in metabolic syndrome subjects. Insulin responsiveness and maximal oxygen uptake correlated with the proportion of type I fibers. Insulin receptor, insulin receptor substrate-1, and glucose transporter 4 expression were not different in whole muscle but all were significantly less in the type I fibers of metabolic syndrome subjects when adjusted for fiber proportion and fiber size. Fat oxidation and muscle mitochondrial expression were not different in the metabolic syndrome subjects. CONCLUSION Lower proportion of type I fibers in metabolic syndrome muscle correlated with the severity of insulin resistance. Even though whole muscle content was normal, key elements of insulin action were consistently less in type I muscle fibers, suggesting their distribution was important in mediating insulin effects.

Chronic restraint stress promotes immune suppression through toll-like receptor 4-mediated phosphoinositide 3-kinase signaling.

Stress, either psychological or physical, can have a dramatic impact on the immune system. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. We have reported that stress modulates the immune response in a TLR4-dependent manner. However, the mechanisms underlying TLR4-mediated signaling in stress modulation of immune system have not been identified. Here, we demonstrate an essential role for the TLR4-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling. PI3K inhibition by inhibitors wortmannin or LY294002 abrogated protection of stress-induced immune suppression in TLR4-deficient mice compared with TLR4-deficient mice that did not receive the inhibitors. The mechanisms by which PI3K are increased in the TLR4-deficient lymphocytes may involve increased phosphorylation of Akt as well as increased phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta). The stress-mediated suppression of T help 1 (Th1) cytokine and increased production of Th2 cytokine was greatly reduced in TLR4 deficient mice compared with the wild type mice. Moreover, inhibition of PI3K diminished protection of the above Th1 and Th2 changes caused by stress in TLR4-deficient mice compared with non-stressed mice and the wild type mice. Our data demonstrated that TLR4 negatively regulates PI3K activity in wild type mice, leading to the observed the stress-induced immune response. The higher levels of PI3K prevent TLR4 deficient mice from the stress-induced immune response. Therefore, stress modulates the immune system through TLR4-mediated PI3K/Akt signaling.

Oral Branched-Chain Amino Acids Decrease Whole-Body Proteolysis

BACKGROUND This study reports the effects of ingesting branched-chain amino acids (leucine, valine, and isoleucine) on protein metabolism in four men. METHODS To calculate leg protein synthesis and breakdown, we used a new model that utilized the infusion of L-[ring-13C6]phenylalanine and the sampling of the leg arterial-venous difference and muscle biopsies. In addition, protein-bound enrichments provided for the direct calculation of muscle fractional synthetic rate. Four control subjects ingested an equivalent amount of essential amino acids (threonine, methionine, and histidine) to discern the effects of branched-chain amino acid nitrogen vs the effects of essential amino acid nitrogen. Each drink also included 50 g of carbohydrate. RESULTS Consumption of the branched-chain and the essential amino acid solutions produced significant threefold and fourfold elevations in their respective arterial concentrations. Protein synthesis and breakdown were unaffected by branched-chain amino acids, but they increased by 43% (p < .05) and 36% (p < .03), respectively, in the group consuming the essential amino acids. However, net leg balance of phenylalanine was unchanged by either drink. Direct measurement of protein synthesis by tracer incorporation into muscle protein (fractional synthetic rate) revealed no changes within or between drinks. Whole-body phenylalanine flux was significantly suppressed by each solution but to a greater extent by the branched-chain amino acids (15% and 20%, respectively) (p < .001). CONCLUSIONS These results suggest that branched-chain amino acid ingestion suppresses whole-body proteolysis in tissues other than skeletal muscle in normal men.

Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression

Stress, either physical or psychological, can have a dramatic impact on the immune system. Little progress, however, has been made in understanding stress-induced immune suppression. We report here that mice subjected to chronic 12-hour daily physical restraint for two days significantly increased the expression of Toll-like receptor 4 (TLR4). Interestingly, TLR4-deficient mice are resistant to stress-induced lymphocyte reduction. In addition, restraint stress caused dramatic decrease in T help 1 (Th1) cytokine IFN-gamma and IL-2 levels but increase in Th2 cytokine IL-4 in wild type mice. Moreover, the restraint stress significantly inhibits changes of Th1 and Th2 cytokines in TLR4-deficient mice compared with the wild type mice. Therefore, stress modulates the immune system through a TLR4-dependent mechanism.

HIV-1 gp120 primes lymphocytes for opioid-induced, β-arrestin 2-dependent apoptosis

The mechanisms by which opioids affect progression of human immunodeficiency virus type 1 (HIV-1) infection are not well-defined. HIV-1 gp120 is important in the apoptotic death of uninfected, bystander T cells. In this study, we show that co-treatment of human peripheral blood mononuclear cells (PBMC) with HIV-1 gp120/morphine synergistically induces apoptosis in PBMC. Co-treatment of murine splenocytes from mu opiate receptor knockout mice with gp120/morphine resulted in decreased apoptosis when compared to splenocytes from wild type mice. Co-treatment of human PBMC or murine splenocytes with gp120/morphine led to decreased expression of beta-arrestin 2, a protein required for opioid-mediated signaling. The role of beta-arrestin 2 was confirmed in Jurkat lymphocytes, in which 1) over-expression of beta-arrestin 2 inhibited gp120/morphine-induced apoptosis and 2) RNA interference of beta-arrestin 2 expression enhanced gp120/morphine-induced apoptosis. These data suggest a novel mechanism by which HIV-1 gp120 and opioids induce lymphocyte cell death.

Chronic Morphine-Induced MicroRNA-124 Promotes Microglial Immunosuppression by Modulating P65 and TRAF6

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects including immunosuppression. However, the mechanisms are unclear. TLRs and acetylcholine are widely expressed in the immune and nervous systems, and play critical roles in immune responses. In this article, we show that morphine suppresses the innate immunity in microglia and bone marrow–derived macrophages through differential regulation of TLRs and acetylcholinesterase. Either morphine or inhibition of acetylcholine significantly promotes upregulation of microRNA-124 (miR-124) in microglia, bone marrow–derived macrophages, and the mouse brain, where miR-124 mediates morphine inhibition of the innate immunity by directly targeting a subunit of NF-κB p65 and TNFR-associated factor 6 (TRAF6). Furthermore, transcription factors AP-1 and CREB inhibited miR-124, whereas p65 bound directly to promoters of miR-124, thereby enhancing miR-124 transcription. Moreover, acute morphine treatment transiently upregulated the expression of p65 and phospho-p65 in both nucleus and cytoplasm priming the expression of miR-124, whereas long exposure of morphine maintained miR-124 expression, which inhibited p65- and TRAF6-dependent TLR signaling. These data suggest that modulation of miRs is capable of preventing opioid-induced damage to microglia.

Impaired Muscle AMPK Activation in the Metabolic Syndrome May Attenuate Improved Insulin Action after Exercise Training

CONTEXT Strength training induces muscle remodeling and may improve insulin responsiveness. OBJECTIVE This study will quantify the impact of resistance training on insulin sensitivity in subjects with the metabolic syndrome and correlate this with activation of intramuscular pathways mediating mitochondrial biogenesis and muscle fiber hypertrophy. DESIGN Ten subjects with the metabolic syndrome (MS) and nine sedentary controls underwent 8 wk of supervised resistance exercise training with pre- and posttraining anthropometric and muscle biochemical assessments. SETTING Resistance exercise training took place in a sports laboratory on a college campus. MAIN OUTCOME MEASURES Pre- and posttraining insulin responsiveness was quantified using a euglycemic clamp. Changes in expression of muscle 5-AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathways were quantified using immunoblots. RESULTS Strength and stamina increased in both groups. Insulin sensitivity increased in controls (steady-state glucose infusion rate = 7.0 ± 2.0 mg/kg · min pretraining training vs. 8.7 ± 3.1 mg/kg · min posttraining; P < 0.01) but did not improve in MS subjects (3.3 ± 1.3 pre vs. 3.1 ± 1.0 post). Muscle glucose transporter 4 increased 67% in controls and 36% in the MS subjects. Control subjects increased muscle phospho-AMPK (43%), peroxisome proliferator-activated receptor γ coactivator 1α (57%), and ATP synthase (60%), more than MS subjects (8, 28, and 21%, respectively). In contrast, muscle phospho-mTOR increased most in the MS group (57 vs. 32%). CONCLUSION Failure of resistance training to improve insulin responsiveness in MS subjects was coincident with diminished phosphorylation of muscle AMPK, but increased phosphorylation of mTOR, suggesting activation of the mTOR pathway could be involved in inhibition of exercise training-related increases in AMPK and its activation and downstream events.

Brain glucose transporter (Glut3) haploinsufficiency does not impair mouse brain glucose uptake

Mouse brain expresses three principal glucose transporters. Glut1 is an endothelial marker and is the principal glucose transporter of the blood-brain barrier. Glut3 and Glut6 are expressed in glial cells and neural cells. A mouse line with a null allele for Glut3 has been developed. The Glut3(-/-) genotype is intrauterine lethal by 7days post-coitis, but the heterozygous (Glut3(+/-)) littermate survives, exhibiting rapid post-natal weight gain, but no seizures or other behavioral aberrations. At 12weeks of age, brain uptake of tail vein-injected ((3))H-2-deoxy glucose in Glut3(+/-) mice was not different from Glut3(+/+) littermates, despite 50% less Glut3 protein expression in the brain. The brain uptake of injected ((18))F-2-fluoro-2-deoxy glucose was similarly not different from Glut3(+/-) littermates in the total amount, time course, or brain imaging in the Glut3(+/-) mice. Glut1 and Glut6 protein expressions evaluated by immunoblots were not affected by the diminished Glut3 expression in the Glut3(+/-) mice. We conclude that a 50% decrease in Glut3 is not limiting for the uptake of glucose into the mouse brain, since Glut3 haploinsufficiency does not impair brain glucose uptake or utilization.