NF-kappaB-induced oxidative stress contributes to mitochondrial and cardiac dysfunction in type II diabetes.

AIMS: Inflammatory molecules and their transcription factor, nuclear factor kappa-B (NF-kappaB), are thought to play important roles in diabetes-induced cardiac dysfunction. Here, we investigated the effects of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, in diabetic mice. METHODS AND RESULTS: Obese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-kappaB activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-kappaB, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups. CONCLUSION: This study demonstrates that NF-kappaB blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.

Effect of dietary fat on serum and intramyocellular lipids and running performance.

PURPOSE: This study evaluated whether lowering IMCL stores via 3-d consumption of very-low-fat (LFAT) diet impairs endurance performance relative to a moderate-fat diet (MFAT), and whether such a diet unfavorably alters lipid profiles. METHODS: Twenty-one male and female endurance-trained runners followed a controlled diet and training regimen for 3 d prior to consuming either a LFAT (10% fat) or MFAT (35% fat) isoenergetic diet for another 3 d in random crossover fashion. On day 7, runners followed a glycogen normalization protocol (to equalize glycogen stores) and then underwent performance testing (90-min preload run at 62 +/- 1% VO2max followed by a 10-km time trial) on the morning of day 8. Muscle biopsies obtained from vastus lateralis before and after performance testing were analyzed for IMCL (via electron microscopy) and glycogen content (via enzymatic methodology). RESULTS: Despite approximately 30% lower IMCL (0.220 +/- 0.032% LFAT, 0.316 +/- 0.049% MFAT; P = 0.045) and approximately 22% higher muscle glycogen stores at the start of performance testing (P = 0.10), 10-km performance time was not significantly different following the two diet treatments (43.5 +/- 1.4 min LFAT vs 43.7 +/- 1.2 min MFAT). However, LFAT produced less favorable lipid profiles (P < 0.01) by increasing fasting triglycerides (baseline = 84.9 +/- 8.6; LFAT = 118.7 +/- 10.0 mg.dL(-1)) and the total cholesterol:HDL cholesterol ratio (baseline = 3.42 +/- 0.13:1; LFAT = 3.75 +/- 0.20:1), whereas MFAT lowered triglycerides (baseline = 97.5 +/- 12.2; MFAT = 70.9 +/- 7.1 mg.dL(-1)) and the total cholesterol:HDL cholesterol ratio (baseline = 3.47 +/- 0.18:1; MFAT = 3.33 +/- 0.14:1). CONCLUSION: The results suggest that reducing IMCL via 3-d consumption of a LFAT diet does not impair running performance lasting a little over 2 h (compared with 3-d consumption of a MFAT diet plus 1-d glycogen normalization), but that even short-term consumption of a LFAT diet may unfavorably alter serum lipids, even in healthy, endurance-trained runners.

Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow.

OBJECTIVE: To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). STUDY DESIGN: In vitro experimental study. ANIMALS: Adult Thoroughbred horses (n=11). METHODS: BM (5 horses; mean [+/-SD] age, 4+/-1.4 years) or adipose tissue (6 horses; mean age, 3.5+/-1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis+/-transforming growth factor-3 (TGFbeta(3)) and bone morphogenic factor-6 (BMP-6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross-sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. RESULTS: Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline-like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. CONCLUSION: Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. CLINICAL RELEVANCE: Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.

Nuclear and cytoplasmic localization of interferon-tau in in vitro-produced bovine blastocysts.

Experiments were conducted to detect interferon-tau in bovine in vitro-derived blastocysts by transmission electron (TEM) and confocal microscopy. TEM showed the presence of IFN-tau in the cytoplasm and the nuclei of expanded blastocysts. Confocal microscopy similarly confirmed the presence of IFN-tau in the trophectoderm of blastocysts. The distribution of IFN-tau appeared variable with some cells showing strong labeling while others appeared to be devoid of the protein.

Overexpression of gK in gK-transformed cells collapses the Golgi apparatus into the endoplasmic reticulum inhibiting virion egress, glycoprotein transport, and virus-induced cell fusion.

Intracellular transport and egress of alphaherpesviruses require the coordinate function of multiple proteins and glycoproteins. Recently, we showed that gK is expressed on infected cell surfaces and that gK cell-surface expression required the presence of the UL20 protein [J. Virol. 77 (2003), 499]. Overexpression of gK by gK-transformed cells blocked transport of enveloped virions from perinuclear spaces and inhibited virus-induced cell fusion caused by gK syncytial mutants [J. Virol. 69 (1995), 5401]. Therefore, we investigated whether altered synthesis and transport of gK was responsible for the observed gK-mediated interference phenomena. HSV-1 infection of the gK-transformed cell line Vero (gK9) caused a profound entrapment of gK in the endoplasmic reticulum and total inhibition of gK cell surface expression. In addition, gK drastically inhibited intracellular transport and maturation of gD and caused substantial defects in Golgi-dependent glycosylation of gB. Visualization of intracellular organelles via confocal microscopy revealed a profound collapse of the Golgi apparatus into the endoplasmic reticulum. These results were analogous to those observed in the presence of brefeldin A, a known Golgi disruptor. Therefore, virion entrapment within perinuclear spaces and inhibition of glycoprotein transport are due to gK-mediated collapse of the Golgi apparatus.