The master antioxidant defense is activated during EBV latent infection.

IMPORTANCE: To our knowledge, this is the first report delineating the activation of the master antioxidant defense during EBV latency. We show that EBV-triggered reactive oxygen species production activates the Keap1-NRF2 pathway in EBV-transformed cells, and LMP1 plays a major role in this event, and the stress-related kinase TBK1 is required for NRF2 activation. Moreover, we show that the Keap1-NRF2 pathway is important for cell proliferation and EBV latency maintenance. Our findings disclose how EBV controls the balance between oxidative stress and antioxidant defense, which greatly improve our understanding of EBV latency and pathogenesis and may be leveraged to opportunities toward the improvement of therapeutic outcomes in EBV-associated diseases.

Bioinformatics-Driven Identification of p62 as A Crucial Oncogene in Liver Cancer.

Liver hepatocellular carcinoma (LIHC) is the major form of liver cancer that is the fourth most common cause of cancer death worldwide. It has been reported that the multifunctional protein p62 (also known as SQSTM1) plays a cancer-promoting role in LIHC, but the detailed mechanisms underlying p62 interaction with LIHC remains unclear. To gain a comprehensive understanding of p62 interaction with LIHC in clinical settings, we performed bioinformatic analyses using various online algorithms derived from high throughput profiling. Our results indicate that p62 expression is significantly upregulated, partially due to its promoter demethylation, rather than p62 gene mutation, in LIHC. Mutation of TP53, CTNNB1, or ALB significantly correlates with, and mutation of AXIN1 reversely correlates with, the p62 expression level. Its upregulation occurs as early as liver cirrhosis, and go through all stages of the carcinogenesis. HCV infection makes a significant contribution to p62 upregulation in LIHC. We further identified p62-associated molecular signatures in LIHC, including many genes that are involved in antioxidant stress and metabolism, such as SRX1 and TXNRD1. Regarding to the clinical outcome, p62 expression level reversely correlates with the survival of LIHC patients (p<0.01). Importantly, we experimentally validated that p62 depletion in liver cancer cell lines downregulates the expression of SRX1 and TXNRD1 at both transcriptional and translational levels, and reduces cell proliferation. As the potential mechanisms underlying the tumor-promoting role of p62, we show that p62 upregulation is remarkably associated with reprogramming of pathways mediated by p53, Wnt/ß-catenin, and Keap1-NRF2, which are crucial for oncogenesis in many contexts. Our findings provide a comprehensive insight into the interaction between p62 and LIHC, offering valuable information for understanding of LIHC pathogenesis.

The Ubiquitin Sensor and Adaptor Protein p62 Mediates Signal Transduction of a Viral Oncogenic Pathway.

The Epstein-Barr virus (EBV) protein LMP1 serves as a paradigm that engages complicated ubiquitination-mediated mechanisms to activate multiple transcription factors. p62 is a ubiquitin sensor and a signal-transducing adaptor that has multiple functions in diverse contexts. However, the interaction between p62 and oncogenic viruses is poorly understood. We recently reported a crucial role for p62 in oncovirus-mediated oxidative stress by acting as a selective autophagy receptor. In this following pursuit, we further discovered that p62 is upregulated in EBV type 3 compared to type 1 latency, with a significant contribution from NF-κB and AP1 activities downstream of LMP1 signaling. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination and activation. As expected, short hairpin RNA (shRNA)-mediated knockdown (KD) of p62 transcripts reduces LMP1-TRAF6 interaction and TRAF6 ubiquitination, as well as p65 nuclear translocation, which was assessed by Amnis imaging flow cytometry. Strikingly, LMP1-stimulated NF-κB, AP1, and Akt activities are all markedly reduced in p62-/- mouse embryo fibroblasts (MEFs) and in EBV-negative Burkitt's lymphoma (BL) cell lines with CRISPR-mediated knockout (KO) of the p62-encoding gene. However, EBV-positive BL cell lines (type 3 latency) with CRISPR-mediated KO of the p62-encoding gene failed to survive. In consequence, shRNA-mediated p62 KD impairs the ability of LMP1 to regulate its target gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of lymphoblastic cell lines (LCLs). These important findings have revealed a previously unrecognized novel role for p62 in EBV latency and oncogenesis, which advances our understanding of the mechanism underlying virus-mediated oncogenesis. IMPORTANCE As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-κB activation, which involves the ubiquitin machinery, in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-κB is an open question. In this study, we provide evidence that p62 is upregulated in EBV type 3 latency and, in turn, p62 mediates LMP1 signal transduction to NF-κB, AP1, and Akt by promoting TRAF6 ubiquitination and activation. In consequence, p62 deficiency negatively regulates LMP1-mediated gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of LCLs. These important findings identified p62 as a novel signaling component of the key viral oncogenic signaling pathway.

Algorithm-Based Meta-Analysis Reveals the Mechanistic Interaction of the Tumor Suppressor LIMD1 With Non-Small-Cell Lung Carcinoma.

Non-small-cell lung carcinoma (NSCLC) is the major type of lung cancer, which is among the leading causes of cancer-related deaths worldwide. LIMD1 was previously identified as a tumor suppressor in lung cancer, but their detailed interaction in this setting remains unclear. In this study, we have carried out multiple genome-wide bioinformatic analyses for a comprehensive understanding of LIMD1 in NSCLC, using various online algorithm platforms that have been built for mega databases derived from both clinical and cell line samples. Our results indicate that LIMD1 expression level is significantly downregulated at both mRNA and protein levels in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with a considerable contribution from its promoter methylation rather than its gene mutations. The Limd1 gene undergoes mutation only at a low rate in NSCLC (0.712%). We have further identified LIMD1-associated molecular signatures in NSCLC, including its natural antisense long non-coding RNA LIMD1-AS1 and a pool of membrane trafficking regulators. We have also identified a subgroup of tumor-infiltrating lymphocytes, especially neutrophils, whose tumor infiltration levels significantly correlate with LIMD1 level in both LUAD and LUSC. However, a significant correlation of LIMD1 with a subset of immune regulatory molecules, such as IL6R and TAP1, was only found in LUAD. Regarding the clinical outcomes, LIMD1 expression level only significantly correlates with the survival of LUAD (p<0.01) but not with that of LUSC (p>0.1) patients. These findings indicate that LIMD1 plays a survival role in LUAD patients at least by acting as an immune regulatory protein. To further understand the mechanisms underlying the tumor-suppressing function of LIMD1 in NSCLC, we show that LIMD1 downregulation remarkably correlates with the deregulation of multiple pathways that play decisive roles in the oncogenesis of NSCLC, especially those mediated by EGFR, KRAS, PIK3CA, Keap1, and p63, in both LUAD and LUSC, and those mediated by p53 and CDKN2A only in LUAD. This study has disclosed that LIMD1 can serve as a survival prognostic marker for LUAD patients and provides mechanistic insights into the interaction of LIMD1 with NSCLC, which provide valuable information for clinical applications.

p62-mediated Selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress.

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.

LIMD1 is induced by and required for LMP1 signaling, and protects EBV-transformed cells from DNA damage-induced cell death.

LIMD1 (LIM domain-containing protein 1) is considered as a tumor suppressor, being deregulated in many cancers to include hematological malignancies; however, very little is known about the underlying mechanisms of its deregulation and its roles in carcinogenesis. Epstein-Barr Virus (EBV) is associated with a panel of malignancies of lymphocytic and epithelial origin. Using high throughput expression profiling, we have previously identified LIMD1 as a common marker associated with the oncogenic transcription factor IRF4 in EBV-related lymphomas and other hematological malignancies. In this study, we have identified potential conserved IRF4- and NFκB-binding motifs in the LIMD1 gene promoter, and both are demonstrated functional by promoter-reporter assays. We further show that LIMD1 is partially upregulated by EBV latent membrane protein 1 (LMP1) via IRF4 and NFκB in EBV latency. As to its role in the setting of EBV latent infection, we show that LIMD1 interacts with TRAF6, a crucial mediator of LMP1 signal transduction. Importantly, LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apoptosis, and inhibits p62-mediated selective autophagy. Taken together, these results show that LIMD1 is upregulated in EBV latency and plays an oncogenic role rather than that of a tumor suppressor. Our findings have identified LIMD1 as a novel player in EBV latency and oncogenesis, and open a novel research avenue, in which LIMD1 and p62 play crucial roles in linking DNA damage response (DDR), apoptosis, and autophagy and their potential interplay during viral oncogenesis.

Muscle hypertrophy in prediabetic men after 16 wk of resistance training.

Resistance training of healthy young men typically results in muscle hypertrophy and a shift in vastus lateralis composition away from type IIx fibers to an increase in IIa fiber content. Our previous studies of 8 wk of resistance training found that many metabolic syndrome men and women paradoxically increased IIx fibers with a decrease in IIa fibers. To confirm the hypothesis that obese subjects might have muscle remodeling after resistance training very different from healthy lean subjects, we subjected a group of nine obese male volunteers to progressive resistance training for a total of 16 wk. In these studies, weight loss was discouraged so that muscle changes would be attributed to the training alone. Detailed assessments included comparisons of histological examinations of needle biopsies of vastus lateralis muscle pretraining and at 8 and 16 wk. Prolonging the training from 8 to 16 wk resulted in increased strength, improved body composition, and more muscle fiber hypertrophy, but euglycemic clamp-quantified insulin responsiveness did not improve. Similar to prior studies, muscle fiber composition shifted toward more fast-twitch type IIx fibers (23 to 42%). Eight weeks of resistance training increased the muscle expression of phosphorylated Akt2 and mTOR. Muscle GLUT4 expression increased, although insulin receptor and IRS-1 expression did not change. We conclude that resistance training of prediabetic obese subjects is effective at changing muscle, resulting in fiber hypertrophy and increased type IIx fiber content, and these changes continue up to 16 wk of training.NEW & NOTEWORTHY Obese, insulin-resistant men responded to 16 wk of progressive resistance training with muscle hypertrophy and increased strength and a shift in muscle fiber composition toward fast-twitch, type IIx fibers. Activation of muscle mTOR was increased by 8 wk but did not increase further at 16 wk despite continued augmentation of peak power and rate of force generation.

Pre-Training Muscle Characteristics of Subjects Who Are Obese Determine How Well Exercise Training Will Improve Their Insulin Responsiveness.

Stuart, CA, Lee, ML, South, MA, Howell, MEA, Cartwright, BM, Ramsey, MW, and Stone, MH. Pre-training muscle characteristics of subjects who are obese determine how well exercise training will improve their insulin responsiveness. J Strength Cond Res 31(3): 798-808, 2017-Only half of prediabetic subjects who are obese who underwent exercise training without weight loss increased their insulin responsiveness. We hypothesized that those who improved their insulin responsiveness might have pretraining characteristics favoring a positive response to exercise training. Thirty nondiabetic subjects who were obese volunteered for 8 weeks of either strength training or endurance training. During training, subjects increased their caloric intake to prevent weight loss. Insulin responsiveness by euglycemic clamps and muscle fiber composition, and expression of muscle key biochemical pathways were quantified. Positive responders initially had 52% higher intermediate muscle fibers (fiber type IIa) with 27% lower slow-twitch fibers (type I) and 23% lower expression of muscle insulin receptors. Whether after weight training or stationary bike training, positive responders' fiber type shifted away from type I and type IIa fibers to an increased proportion of type IIx fibers (fast twitch). Muscle insulin receptor expression and glucose transporter type 4 (GLUT4) expression increased in all trained subjects, but these moderate changes did not consistently translate to improvement in whole-body insulin responsiveness. Exercise training of previously sedentary subjects who are obese can result in muscle remodeling and increased expression of key elements of the insulin pathway, but in the absence of weight loss, insulin sensitivity improvement was modest and limited to about half of the participants. Our data suggest rather than responders being more fit, they may have been less fit, only catching up to the other half of subjects who are obese whose insulin responsiveness did not increase beyond their pretraining baseline.

Effects of Short-Term Free-Weight and Semiblock Periodization Resistance Training on Metabolic Syndrome.

South, MA, Layne, AS, Stuart, CA, Triplett, NT, Ramsey, MW, Howell, ME, Sands, WA, Mizuguchi, S, Hornsby, WG, Kavanaugh, AA, and Stone, MH. Effects of short-term free-weight and semiblock periodization resistance training on metabolic syndrome. J Strength Cond Res 30(10): 2682-2696, 2016-The effects of short-term resistance training on performance and health variables associated with prolonged sedentary lifestyle and metabolic syndrome (MS) were investigated. Resistance training may alter a number of health-related, physiological, and performance variables. As a result, resistance training can be used as a valuable tool in ameliorating the effects of a sedentary lifestyle including those associated with MS. Nineteen previously sedentary subjects (10 with MS and 9 with nonmetabolic syndrome [NMS]) underwent 8 weeks of supervised resistance training. Maximum strength was measured using an isometric midthigh pull and resulting force-time curve. Vertical jump height (JH) and power were measured using a force plate. The muscle cross-sectional area (CSA) and type were examined using muscle biopsy and standard analysis techniques. Aerobic power was measured on a cycle ergometer using a ParvoMedics 2400 Metabolic system. Endurance was measured as time to exhaustion on a cycle ergometer. After training, maximum isometric strength, JH, jump power, and V[Combining Dot Above]O2peak increased by approximately 10% (or more) in both the metabolic and NMS groups (both male and female subjects). Over 8 weeks of training, body mass did not change statistically, but percent body fat decreased in subjects with the MS and in women, and lean body mass increased in all groups (p ≤ 0.05). Few alterations were noted in the fiber type. Men had larger CSAs compared those of with women, and there was a fiber-specific trend toward hypertrophy over time. In summary, 8 weeks of semiblock free-weight resistance training improved several performance variables and some cardiovascular factors associated with MS.

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle.

Insulin resistance and muscle insulin receptor substrate-1 serine hyperphosphorylation.

Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin-responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate-1 (IRS-1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS-1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS-1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS-1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS-1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c-Jun N-terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS-1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS-1 diminishes the transmission of the insulin signal and thereby decreases the insulin-stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS-1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss.

Insulin responsiveness in metabolic syndrome after eight weeks of cycle training.

INTRODUCTION: Insulin resistance in obesity is decreased after successful diet and exercise. Aerobic exercise training alone was evaluated as an intervention in subjects with the metabolic syndrome. METHODS: Eighteen nondiabetic, sedentary subjects, 11 with the metabolic syndrome, participated in 8 wk of increasing intensity stationary cycle training. RESULTS: Cycle training without weight loss did not change insulin resistance in metabolic syndrome subjects or sedentary control subjects. Maximal oxygen consumption (V·O 2max), activated muscle AMP-dependent kinase, and muscle mitochondrial marker ATP synthase all increased. Strength, lean body mass, and fat mass did not change. The activated mammalian target of rapamycin was not different after training. Training induced a shift in muscle fiber composition in both groups but in opposite directions. The proportion of type 2× fibers decreased with a concomitant increase in type 2a mixed fibers in the control subjects, but in metabolic syndrome, type 2× fiber proportion increased and type 1 fibers decreased. Muscle fiber diameters increased in all three fiber types in metabolic syndrome subjects. Muscle insulin receptor expression increased in both groups, and GLUT4 expression increased in the metabolic syndrome subjects. The excess phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser337 in metabolic syndrome muscle tended to increase further after training in spite of a decrease in total IRS-1. CONCLUSIONS: In the absence of weight loss, the cycle training of metabolic syndrome subjects resulted in enhanced mitochondrial biogenesis and increased the expression of insulin receptors and GLUT4 in muscle but did not decrease the insulin resistance. The failure for the insulin signal to proceed past IRS-1 tyrosine phosphorylation may be related to excess serine phosphorylation at IRS-1 Ser337, and this is not ameliorated by 8 wk of endurance exercise training.

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.

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.

Cycle training increased GLUT4 and activation of mammalian target of rapamycin in fast twitch muscle fibers.

PURPOSE: To determine whether cycle training of sedentary subjects would increase the expression of the principle muscle glucose transporters, six volunteers completed 6 wk of progressively increasing intensity stationary cycle cycling. METHODS: In vastus lateralis muscle biopsies, changes in expression of GLUT1, GLUT4, GLUT5, and GLUT12 were compared using quantitative immunoblots with specific protein standards. Regulatory pathway components were evaluated by immunoblots of muscle homogenates and immunohistochemistry of microscopic sections. RESULTS: GLUT1 was unchanged, GLUT4 increased 66%, GLUT12 increased 104%, and GLUT5 decreased 72%. A mitochondrial marker (cytochrome c) and regulators of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha and phospho-5'-adenosine monophosphate-activated protein kinase) were unchanged, but the muscle hypertrophy pathway component, phospho-mammalian target of rapamycin (mTOR), increased 83% after the exercise program. In baseline biopsies, GLUT4 by immunohistochemical techniques was 37% greater in Type I (slow twitch, red) muscle fibers, but the exercise training increased GLUT4 expression in Type II (fast twitch, white) fibers by 50%, achieving parity with the Type I fibers. Baseline phospho-mTOR expression was 50% higher in Type II fibers and increased more in Type II fibers (62%) with training but also increased in Type I fibers (34%). CONCLUSION: Progressive intensity stationary cycle training of previously sedentary subjects increased muscle insulin-responsive glucose transporters (GLUT4 and GLUT12) and decreased the fructose transporter (GLUT5). The increase in GLUT4 occurred primarily in Type II muscle fibers, and this coincided with activation of the mTOR muscle hypertrophy pathway. There was little impact on Type I fiber GLUT4 expression and no evidence of change in mitochondrial biogenesis.

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.

Overexpression of GLUT5 in diabetic muscle is reversed by pioglitazone.

OBJECTIVE: This study was undertaken to quantify the expression of muscle GLUT in type 2 diabetes and to determine if treatment with an insulin-enhancing thiazolidenedione drug, pioglitazone, would alter its expression. RESEARCH DESIGN AND METHODS: Twelve patients with type 2 diabetes were randomly assigned to treatment with either pioglitazone or placebo in a double-blinded 8-week protocol. Protein and mRNA for GLUT4 and GLUT5 were quantified in muscle homogenates from biopsies of vastus lateralis before and after treatment. The five additional GLUT family isoforms expressed in muscle had mRNA quantified in these samples. RESULTS: Baseline and posttreatment repeat measurements of GLUT4 protein were not different from control measurements. Compared with normal subjects, GLUT5 protein increased 2.5-fold, and GLUT5 mRNA was 82% higher in the pretreatment samples from the diabetic subjects. Concentrations of mRNA for the six other GLUTs (GLUT1, GLUT3, GLUT4, GLUT8, GLUT11, and GLUT12) were not different from control subjects before or after treatment. The proportion of type I (red) fibers (46%) in diabetic muscle was not affected by pioglitazone treatment. Pioglitazone treatment decreased muscle GLUT5 mRNA and protein by 52 and 40%, respectively, whereas placebo did not alter GLUT5 expression. Both red and white fibers had higher GLUT5 expression in the baseline diabetic muscle samples, and a pioglitazone-related decrease in GLUT5 protein also occurred in both. CONCLUSIONS: GLUT5 was dramatically increased in diabetic muscle, and pioglitazone treatment reversed this overexpression. The role of this fructose transporter expression in the insulin-enhancing effect of pioglitazone in muscle is unclear.

Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle.

In the past few years, 8 additional members of the facilitative hexose transporter family have been identified, giving a total of 14 members of the SLC2A family of membrane-bound hexose transporters. To determine which of the new hexose transporters were expressed in muscle, mRNA concentrations of 11 glucose transporters (GLUTs) were quantified and compared. RNA from muscle from 10 normal volunteers was subjected to RT-PCR. Primers were designed that amplified 78- to 241-base fragments, and cDNA standards were cloned for GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, and GAPDH. Seven of these eleven hexose transporters were detectable in normal human muscle. The rank order was GLUT4, GLUT5, GLUT12, GLUT8, GLUT11, GLUT3, and GLUT1, with corresponding concentrations of 404 +/- 49, 131 +/- 14, 33 +/- 4, 5.5 +/- 0.5, 4.1 +/- 0.4, 1.2 +/- .0.1, and 0.9 +/- 0.2 copies/ng RNA (means +/- SE), respectively, for the 10 subjects. Concentrations of mRNA for GLUT4, GLUT5, and GLUT12 were much higher than those for the remainder of the GLUTs and together accounted for 98% of the total GLUT isoform mRNA. Immunoblots of muscle homogenates verified that the respective proteins for GLUT4, GLUT5, and GLUT12 were present in normal human muscle. Immunofluorescent studies demonstrated that GLUT4 and GLUT12 were predominantly expressed in type I oxidative fibers; however, GLUT5 was expressed predominantly in type II (white) fibers.