Effect of acute exercise on citrate synthase activity in untrained and trained human skeletal muscle

Maximal citrate synthase activity (CS) is routinely used as a marker of aerobic capacity and mitochondrial density in skeletal muscle. However, reported CS has been notoriously variable, even with similar experimental protocols and sampling from the same muscles. Exercise training has resulted in increases in CS ranging from 0 to 100%. Previously, it has been reported that acute exercise may significantly affect CS. To investigate the hypothesis that the large variation in CS that occurs with training is influenced by alterations during the exercise itself, we studied CS in human vastus lateralis both in the rested and acutely exercised state while trained and untrained (n = 6). Tissues obtained from four biopsies (untrained rested, untrained acutely exercised, trained rested, and trained acutely exercised) were analyzed spectrophotometrically for maximal CS. Exercise training measured in a rested state resulted in an 18.2% increase in CS (12.3 +/- 0.3 to 14.5 +/- 0.3 micromol x min(-1) x g tissue(-1), P < or = 0.05). However, even greater increases were recorded 1 h after acute exercise: 49.4% in the untrained state (12.3 +/- 0.3 to 18.3 +/- 0.5 micromol x min(-1) x g tissue(-1), P < or = 0.05) and 50.8% in the trained state (14.5 +/- 0.3 to 21.8 +/- 0.4 micromol x min(-1) x g tissue(-1), P < or = 0.05). Ultrastructural analysis, by electron microscopy, supported an effect of acute exercise with the finding of numerous swollen mitochondria 1 h after exercise that may result in greater access to the CS itself in the CS assay. In conclusion, although unexplained, the increased CS with acute exercise can clearly confound training responses and artificially elevate CS values. Therefore, the timing of muscle sampling relative to the last exercise session is critical when measuring CS and offers an explanation for the large variation in CS previously reported.     

Detection of 65 kD heat shock protein in cerebrospinal fluid of tuberculous meningitis patients

Background  

Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM.     

Simultaneous Non-Negative Matrix Factorization for Multiple Large Scale Gene Expression Datasets in Toxicology

Non-negative matrix factorization is a useful tool for reducing the dimension of large datasets. This work considers simultaneous non-negative matrix factorization of multiple sources of data. In particular, we perform the first study that involves more than two datasets. We discuss the algorithmic issues required to convert the approach into a practical computational tool and apply the technique to new gene expression data quantifying the molecular changes in four tissue types due to different dosages of an experimental panPPAR agonist in mouse. This study is of interest in toxicology because, whilst PPARs form potential therapeutic targets for diabetes, it is known that they can induce serious side-effects. Our results show that the practical simultaneous non-negative matrix factorization developed here can add value to the data analysis. In particular, we find that factorizing the data as a single object allows us to distinguish between the four tissue types, but does not correctly reproduce the known dosage level groups. Applying our new approach, which treats the four tissue types as providing distinct, but related, datasets, we find that the dosage level groups are respected. The new algorithm then provides separate gene list orderings that can be studied for each tissue type, and compared with the ordering arising from the single factorization. We find that many of our conclusions can be corroborated with known biological behaviour, and others offer new insights into the toxicological effects. Overall, the algorithm shows promise for early detection of toxicity in the drug discovery process.     

Exercise adaptation attenuates VEGF gene expression in human skeletal muscle

Angiogenesis is a component of the multifactoral adaptation to exercise training, and vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation. However, there is limited evidence supporting the role of VEGF in the exercise training response. Thus we studied mRNA levels of VEGF, using quantitative Northern analysis, in untrained and trained human skeletal muscle at rest and after a single bout of exercise. Single leg knee-extension provided the acute exercise stimulus and the training modality. Four biopsies were collected from the vastus lateralis muscle at rest in the untrained and trained conditions before and after exercise. Training resulted in a 35% increase in muscle oxygen consumption and an 18% increase in number of capillaries per muscle fiber. At rest, VEGF/18S mRNA levels were similar before (0.38 +/- 0.04) and after (1.2 +/- 0.4) training. When muscle was untrained, acute exercise greatly elevated VEGF/18S mRNA levels (16.9 +/- 6.7). The VEGF/18S mRNA response to acute exercise in the trained state was markedly attenuated (5.4 +/- 1.3). These data support the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis and appears to be subject to a negative feedback mechanism as exercise adaptations occur.     

Thiazolidinediones upregulate impaired fatty acid uptake in skeletal muscle of type 2 diabetic subjects

We examined the regulation of free fatty acid (FFA, palmitate) uptake into skeletal muscle cells of nondiabetic and type 2 diabetic subjects. Palmitate uptake included a protein-mediated component that was inhibited by phloretin. The protein-mediated component of uptake in muscle cells from type 2 diabetic subjects (78 +/- 13 nmol. mg protein-1. min-1) was reduced compared with that in nondiabetic muscle (150 +/- 17, P < 0.01). Acute insulin exposure caused a modest (16 +/- 5%, P < 0.025) but significant increase in protein-mediated uptake in nondiabetic muscle. There was no significant insulin effect in diabetic muscle (+19 +/- 19%, P = not significant). Chronic (4 day) treatment with a series of thiazolidinediones, troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) increased FFA uptake. Only the phloretin-inhibitable component was increased by treatment, which normalized this activity in diabetic muscle cells. Under the same conditions, FFA oxidation was also increased by thiazolidinedione treatment. Increases in FFA uptake and oxidation were associated with upregulation of fatty acid translocase (FAT/CD36) expression. FAT/CD36 protein was increased by Tgz (90 +/- 22% over control), Rgz (146 +/- 42%), and Pio (111 +/- 37%, P < 0.05 for all 3) treatment. Tgz treatment had no effect on fatty acid transporter protein-1 and membrane-associated plasmalemmal fatty acid-binding protein mRNA expression. We conclude that FFA uptake into cultured muscle cells is, in part, protein mediated and acutely insulin responsive. The basal activity of FFA uptake is impaired in type 2 diabetes. In addition, chronic thiazolidinedione treatment increased FFA uptake and oxidation into cultured human skeletal muscle cells in concert with upregulation of FAT/CD36 expression. Increased FFA uptake and oxidation may contribute to lower circulating FFA levels and reduced insulin resistance in skeletal muscle of individuals with type 2 diabetes following thiazolidinedione treatment.     

Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPARgamma agonists

The impact of type 2 diabetes on the ability of muscle to accumulate and dispose of fatty acids and triglycerides was evaluated in cultured muscle cells from nondiabetic (ND) and type 2 diabetic (T2D) subjects. In the presence of 5 microM palmitate, T2D muscle cells accumulated less lipid than ND cells (11.5 +/- 1.2 vs. 15.1 +/- 1.4 nmol/mg protein, P < 0.05). Chronic treatment (4 days) with the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist troglitazone increased palmitate accumulation, normalizing uptake in T2D cells. There were no significant differences between groups with regard to the relative incorporation of palmitate into neutral lipid species. This distribution was also unaffected by troglitazone treatment. beta-Oxidation of both long-chain (palmitate) and medium-chain (octanoate) fatty acids in T2D muscle cells was reduced by approximately 40% compared with ND cells. Palmitate oxidation occurred primarily in mitochondrial ( approximately 40-50% of total) and peroxisomal (20-30%) compartments. The diabetes-related defect in palmitate oxidation was localized to the mitochondrial component. Both palmitate and octanoate oxidation were stimulated by a series of thiazolidinediones. Oxidation in T2D muscle cells was normalized after treatment. Troglitazone increased the mitochondrial component of palmitate oxidation. Skeletal muscle cells from T2D subjects express defects in free fatty acid metabolism that are retained in vitro, most importantly defects in beta-oxidation. These defects can be corrected by treatment with PPARgamma agonists. Augmentation of fatty acid disposal in skeletal muscle, potentially reducing intramyocellular triglyceride content, may represent one mechanism for the lipid-lowering and insulin-sensitizing effects of thiazolidinediones.     

Effects of SGLT2 Inhibition in Human Kidney Proximal Tubular Cells—Renoprotection in Diabetic Nephropathy?

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.     

The Role of TLR2 and 4-Mediated Inflammatory Pathways in Endothelial Cells Exposed to High Glucose

Postprandial hyperglycemia induces inflammation and endothelial dysfunction resulting in vascular complications in patients with diabetes. Toll-like receptors (TLRs) are central to the regulation of inflammatory responses through activation of nuclear factor-kappa B (NF-ĸB). This study examined the role of TLR2 and 4 in regulating inflammation and endothelial dysfunction when exposed to fluctuating glucose concentrations. HMEC-1 cells (a human microvascular endothelial cell line) were exposed to control (5 mM), 30 mM (high), fluctuating (5/30 mM) and 11.2 mM glucose (approximate glycaemic criteria for the diagnosis of diabetes mellitus) for 72 h. Cells were assessed for TLR2, 4, high mobility group box -1 (HMGB1), NF-ĸB, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Fluctuating glucose concentrations maximally upregulated TLR4 but not TLR2 expression with increased NF-ĸB activation, IL-8 and ICAM-1 expression. HMGB1 was increased in the supernatants of cells exposed to 30 mM and 11.2 mM glucose compared to control. The addition of recombinant HMGB1 induced NF-ĸB activation and synthesis of proinflammatory cytokines and chemokines, which were prevented by TLR2 or 4 signalling inhibition. An additive effect when both TLR2 and 4 signalling pathways were inhibited was observed. However, only inhibition of TLR4 signalling suppressed the synthesis of MCP-1, IL-8 and ICAM-1. In vivo, streptozotocin-induced diabetic mice exhibited an increase in glomerular ICAM-1 which was not evident in TLR2^-/- or TLR4^-/- diabetic mice. Collectively, our results suggest that targeting the signalling pathway of TLR2 and 4 may be of therapeutic benefit in attenuating vascular inflammation in diabetic microangiopathy.     

Adipocyte Differentiation‐related Protein in Human Skeletal Muscle: Relationship to Insulin Sensitivity

Objective: To determine whether adipocyte differentiation‐related protein (ADRP), a lipid droplet—associated protein that binds to and sequesters intracellular fatty acids, is 1) expressed in human skeletal muscle and 2) differentially regulated in human skeletal muscle obtained from obese non‐diabetic (OND) and obese diabetic (OD) subjects. Research Methods and Procedures: Ten OND subjects and 15 OD subjects underwent a weight loss or pharmacological intervention program to improve insulin sensitivity. Anthropometric data, hemoglobin A_1C, fasting glucose, lipids, and glucose disposal rate were determined at baseline and at completion of studies. Biopsies of the vastus lateralis muscle (SkM) were obtained in the fasting state from OND and OD subjects. Protein expression was determined by Western blotting. Results: ADRP was highly expressed in SkM from OND (4.4 ± 1.54 AU/10 μg, protein, n = 10) and OD (5.02 ± 1.33 AU/10 μg, n = 12) subjects. OND subjects undergoing weight loss had decreased triglyceride levels and improved insulin action. SkM ADRP content increased with weight loss from 5.14 ± 2.15 AU/10 μg to 9.92 ± 1.57 AU/10 μg (p < 0.025). OD subjects were treated with either troglitazone or metformin, together with glyburide, for 3 to 4 months. Both treatments attained similar levels of glycemic control. OD subjects with lower baseline ADRP content (2.85 ± 1.07 AU/10 μg, n = 6) displayed up‐regulation of ADRP expression (to 9.27 ± 2.76 AU/10 μg, p < 0.025). Discussion: ADRP is the predominant lipid droplet—associated protein in SkM, and low ADRP expression is up‐regulated in circumstances of improved glucose tolerance. Up‐regulation of ADRP may act to sequester fatty acids as triglycerides in discrete lipid droplets that could protect muscle from the detrimental effects of fatty acids on insulin action and glucose tolerance.