Sanghyun Kim

Normal adaptations to exercise despite protection against oxidative stress

It has been reported that supplementation with the antioxidant vitamins C and E prevents the adaptive increases in mitochondrial biogenesis and GLUT4 expression induced by endurance exercise. We reevaluated the effects of these antioxidants on the adaptive responses of rat skeletal muscle to swimming in a short-term study consisting of 9 days of vitamins C and E with exercise during the last 3 days and a longer-term study consisting of 8 wk of antioxidant vitamins with exercise during the last 3 wk. The rats in the antioxidant groups were given 750 mg·kg body wt(-1)·day(-1) vitamin C and 150 mg·kg body wt(-1)·day(-1) vitamin E. In rats euthanized immediately after exercise, plasma TBARs were elevated in the control rats but not in the antioxidant-supplemented rats, providing evidence for an antioxidant effect. In rats euthanized 18 h after exercise there were large increases in insulin responsiveness of glucose transport in epitrochlearis muscles mediated by an approximately twofold increase in GLUT4 expression in both the short- and long-term treatment groups. The protein levels of a number of mitochondrial marker enzymes were also increased about twofold. Superoxide dismutases (SOD) 1 and 2 were increased about twofold in triceps muscle after 3 days of exercise, but only SOD2 was increased after 3 wk of exercise. There were no differences in the magnitudes of any of these adaptive responses between the control and antioxidant groups. These results show that very large doses of antioxidant vitamins do not prevent the exercise-induced adaptive responses of muscle mitochondria, GLUT4, and insulin action to exercise and have no effect on the level of these proteins in sedentary rats.

Effects of resveratrol and SIRT1 on PGC-1α activity and mitochondrial biogenesis: a reevaluation

Feeding resveratrol to rodents has no effect on mitochondrial biogenesis, and deacetylation of PGC-1α results in a decrease, not an increase, in its coactivator activity.

Does calorie restriction induce mitochondrial biogenesis? A reevaluation

It has been reported that 30% calorie restriction (CR) for 3 mo results in large increases in mitochondrial biogenesis in heart, brain, liver, and adipose tissue, with concomitant increases in respiration and ATP synthesis. We found these results surprising, and performed this study to determine whether 30% CR does induce an increase in mitochondria in heart, brain, liver, adipose tissue, and/or skeletal muscle. To this end, we measured the levels of a range of mitochondrial proteins, and mRNAs. With the exception of long‐chain acyl‐CoA dehydrogenase protein level, which was increased ~60% in adipose tissue, none of the mitochondrial proteins or mRNAs that we measured were increased in rats subjected to 30% CR for 14 wk. There was also no increase in citrate synthase activity. Because it is not possible to have an increase in mitochondria without any increase in key mitochondrial proteins, we conclude that 30% CR does not induce an increase in mitochondria in heart, brain, liver, adipose tissue, or skeletal muscle in laboratory rodents.—Hancock, C. R., Han, D.‐H., Higashida, K., Kim, S. H., Holloszy, J. O. Does calorie restriction induce mito‐chondrial biogenesis? A reevaluation. FASEB J. 25, 785–791 (2011). www.fasebj.org

Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

Deficiency of the Mitochondrial Electron Transport Chain in Muscle Does Not Cause Insulin Resistance

Background It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis. Methodology/Principal Findings Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance. Conclusions/Significance The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

PGC‐1α mediates a rapid, exercise‐induced downregulation of glycogenolysis in rat skeletal muscle

Long‐term endurance exercise training results in a reduction in the rates of muscle glycogen depletion and lactic acid accumulation during submaximal exercise; this adaptation is mediated by an increase in muscle mitochondria. There is evidence suggesting that short‐term training induces adaptations that downregulate glycogenolysis before there is an increase in functional mitochondria. We discovered that a single long bout of exercise induces decreases in expression of glycogenolytic and glycolytic enzymes in rat skeletal muscle; this adaptation results in slower rates of glycogenolysis and lactic acid accumulation in muscle during contractile activity. Two additional days of training amplified the adaptive response, which appears to be mediated by PGC‐1α; this adaptation is biologically significant, because glycogen depletion and lactic acid accumulation are major causes of muscle fatigue.

β-Adrenergic stimulation does not activate p38 MAP kinase or induce PGC-1α in skeletal muscle

There are reports that the β-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by β-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of β-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle.

Ginsenoside Re rapidly reverses insulin resistance in muscles of high-fat diet fed rats

OBJECTIVEnIn a previous study, it was found that a ginseng berry extract with a high content of the ginsenoside Re normalized blood glucose in ob/ob mice. The objective of this study was to evaluate the effect of the ginsenoside Re on insulin resistance of glucose transport in muscles of rats made insulin resistant with a high-fat diet.nnnMATERIAL/METHODnRats were fed either rat chow or a high-fat diet for 5 weeks. The rats were then euthanized, and insulin stimulated glucose transport activity was measured in epitrochlearis and soleus muscle strips in vitro.nnnRESULTSnTreatment of muscles with Re alone had no effect on glucose transport. The high-fat diet resulted in ~50% decreases in insulin responsiveness of GLUT4 translocation to the cell surface and glucose transport in epitrochlearis and soleus muscles. Treatment of muscles with Re in vitro for 90 min completely reversed the high-fat diet-induced insulin resistance of glucose transport and GLUT4 translocation. This effect of Re is specific for insulin stimulated glucose transport, as Re treatment did not reverse the high-fat diet-induced resistance of skeletal muscle glucose transport to stimulation by contractions or hypoxia.nnnCONCLUSIONSnOur results show that the ginsenoside Re induces a remarkably rapid reversal of high-fat diet-induced insulin resistance of muscle glucose transport by reversing the impairment of insulin-stimulated GLUT4 translocation to the cell surface.

Effect of Lithium on the Mechanism of Glucose Transport in Skeletal Muscles

While lithium is known to stimulate glucose transport into skeletal muscle, the mechanisms of the increased glucose transport by lithium in skeletal muscle are not well defined yet. We excised epitrochlearis muscles from male Wistar rats and measured the transport rates of a glucose analog into lithium-, insulin-, and muscular contraction-stimulated skeletal muscle tissue and we also analyzed the levels of cell surface glucose transport 4 using a photolabeling and multicolor immunofluorescence method. In addition, we generated a cell line that stably expresses myc-tagged GLUT4 to measure the rates of GLUT4 internalization and externalization. Lithium significantly increased 2-DG glucose transport rate in skeletal muscles; however, it was significantly lower than the stimulation induced by the maximum concentration of insulin or tetanic contraction. But co-treatment of lithium with insulin or tetanic contraction increased glucose transport rate by ∼200% more than lithium alone. When skeletal muscle tissues were treated with lithium, insulin, and muscular contraction, the levels of cell surface GLUT4 protein contents were increased similarly by ∼6-fold compared with the basal levels. When insulin or lithium stimuli were maintained, the rate of GLUT4myc internalization was significantly lower, and lithium was found to suppress the internalization of GLUT4myc more strongly. The lithium-induced increase in glucose uptake of skeletal muscles appears to increase in cell surface GLUT4 levels caused by decreased internalization of GLUT4. It is concluded that co-treatment of lithium with insulin and muscular contraction had a synergistic effect on glucose transport rate in skeletal muscle.