Effect of protein restriction on sulfur amino acid catabolism in insulin-dependent diabetes mellitus

Persons with conventionally treated insulin-dependent diabetes mellitus (IDDM) appear to be impaired in their ability to reduce fed-state urea production appropriately in response to dietary protein restriction (Hoffer LJ, Taveroff A, and Schiffrin A. Am J Physiol 272: E59-E67, 1997). To determine whether these conclusions apply to whole body sulfur amino acid (SAA) catabolism, we used samples from this protocol to measure daily urinary sulfate excretion and fed-state sulfate production after a high-protein test meal before and after dietary protein restriction. Eight normal subjects and six IDDM subjects treated with twice-daily intermediate- and short-acting insulin consumed a mixed test meal containing 0.50 g protein/kg after adaptation to 4 days of high protein intake (1.28 g protein/kg body wt) and again after 5 days of dietary protein restriction (0.044 g/kg). Adaptation to protein restriction decreased daily urinary sulfate and urea-N excretion by approximately 80%. Over the first 24 h of protein restriction, urinary sulfate excretion decreased more than urea-N excretion for both the normal and IDDM subjects. Under conditions of a high prior protein intake, fed-state sulfate production was normal for the IDDM subjects; protein restriction reduced fed-state sulfate production by 51% (normal subjects) and 59% (IDDM subjects; not significant). We conclude that whole body SAA metabolism is normal in conventionally treated IDDM before and after dietary protein restriction. SAA catabolism, as measured by fed-state sulfate production, may be a convenient and useful method to determine the extent of whole body protein dysregulation in IDDM.     

Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity

Impaired mitochondrial function and structure and intramyocellular lipid (IMCL) accumulation have been associated with obesity and Type 2 diabetes. We examined whether endurance exercise training and sex influenced IMCL and mitochondrial morphology using electron microscopy, whole-body substrate use, and mitochondrial enzyme activity. Untrained men (n = 5) and women (n = 7) were tested before and after 7 wk of endurance exercise training. Testing included 90 min of cycle ergometry at 60% Vo(2 peak) with preexercise muscle biopsies analyzed for IMCL and mitochondrial size/area using electron microscopy and short-chain beta-hydroxyacyl-CoA dehydrogenase (SCHAD) and citrate synthase (CS) enzyme activity. Training increased the mean lipid area density (P = 0.090), the number of IMCL droplets (P = 0.055), the number of IMCL droplets in contact with mitochondria (P = 0.010), the total mitochondrial area (P < 0.001), and the size of individual mitochondrial fragments (P = 0.006). Women had higher mean lipid area density (P = 0.030) and number of IMCL droplets (P = 0.002) before and after training, but higher individual IMCL area only before training (P = 0.013), compared with men. Women oxidized more fat (P = 0.027) and less carbohydrate (P = 0.032) throughout the study. Training increased Vo(2 peak) (P < 0.001), %fat oxidation (P = 0.018), SCHAD activity (P = 0.003), and CS activity (P = 0.042). In summary, endurance exercise training increased IMCL area density due to an increase in the number of lipid droplets, whereas the increase in total mitochondrial area was due to an increase in the size of individual mitochondrial fragments. In addition, women have higher IMCL content compared with men due mainly to a greater number of individual droplets. Finally, endurance exercise training increased the proportion of IMCL in physical contact with mitochondria.     

One universal common endpoint in mouse models of amyotrophic lateral sclerosis

There is no consensus among research laboratories around the world on the criteria that define endpoint in studies involving rodent models of amyotrophic lateral sclerosis (ALS). Data from 4 nutrition intervention studies using 162 G93A mice, a model of ALS, were analyzed to determine if differences exist between the following endpoint criteria: CS 4 (functional paralysis of both hindlimbs), CS 4+ (CS 4 in addition to the earliest age of body weight loss, body condition deterioration or righting reflex), and CS 5 (CS 4 plus righting reflex >20 s). The age (d; mean ± SD) at which mice reached endpoint was recorded as the unit of measurement. Mice reached CS 4 at 123.9±10.3 d, CS 4+ at 126.6±9.8 d and CS 5 at 127.6±9.8 d, all significantly different from each other (P<0.001). There was a significant positive correlation between CS 4 and CS 5 (r = 0.95, P<0.001), CS 4 and CS 4+ (r = 0.96, P<0.001), and CS 4+ and CS 5 (r = 0.98, P<0.001), with the Bland-Altman plot showing an acceptable bias between all endpoints. Logrank tests showed that mice reached CS 4 24% and 34% faster than CS 4+ (P = 0.046) and CS 5 (P = 0.006), respectively. Adopting CS 4 as endpoint would spare a mouse an average of 4 days (P<0.001) from further neuromuscular disability and poor quality of life compared to CS 5. Alternatively, CS 5 provides information regarding proprioception and severe motor neuron death, both could be important parameters in establishing the efficacy of specific treatments. Converging ethics and discovery, would adopting CS 4 as endpoint compromise the acquisition of insight about the effects of interventions in animal models of ALS?     

Integration of clinical and gene expression endpoints to explore furan-mediated hepatotoxicity

Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days. Furan induced an initial degenerative and necrotic phenotype that was followed by inflammation and fibrosis, consistent with previous observations for this compound. RNA was harvested from each lobe of the liver at several time points to observe whether lobe-specific gene expression effects occurred. Similar gene expression changes were observed in all lobes, however the magnitude of gene expression change was more pronounced in the right lobe. Finally, to help determine the correlation between gene expression changes and liver pathology, we applied traditional microarray visualization tools to the assessment of clinical chemistry and pathology parameters.     

Impact of treadmill running and sex on hippocampal neurogenesis in the mouse model of amyotrophic lateral sclerosis

Hippocampal neurogenesis in the subgranular zone (SGZ) of dentate gyrus (DG) occurs throughout life and is regulated by pathological and physiological processes. The role of oxidative stress in hippocampal neurogenesis and its response to exercise or neurodegenerative diseases remains controversial. The present study was designed to investigate the impact of oxidative stress, treadmill exercise and sex on hippocampal neurogenesis in a murine model of heightened oxidative stress (G93A mice). G93A and wild type (WT) mice were randomized to a treadmill running (EX) or a sedentary (SED) group for 1 or 4 wk. Immunohistochemistry was used to detect bromodeoxyuridine (BrdU) labeled proliferating cells, surviving cells, and their phenotype, as well as for determination of oxidative stress (3-NT; 8-OHdG). BDNF and IGF1 mRNA expression was assessed by in situ hybridization. Results showed that: (1) G93A-SED mice had greater hippocampal neurogenesis, BDNF mRNA, and 3-NT, as compared to WT-SED mice. (2) Treadmill running promoted hippocampal neurogenesis and BDNF mRNA content and lowered DNA oxidative damage (8-OHdG) in WT mice. (3) Male G93A mice showed significantly higher cell proliferation but a lower level of survival vs. female G93A mice. We conclude that G93A mice show higher hippocampal neurogenesis, in association with higher BDNF expression, yet running did not further enhance these phenomena in G93A mice, probably due to a 'ceiling effect' of an already heightened basal levels of hippocampal neurogenesis and BDNF expression.     

Dietary Vitamin D3 Restriction Exacerbates Disease Pathophysiology in the Spinal Cord of the G93A Mouse Model of Amyotrophic Lateral Sclerosis

BackgroundDietary vitamin D₃ (D₃) restriction reduces paw grip endurance and motor performance in G93A mice, and increases inflammation and apoptosis in the quadríceps of females. ALS, a neuromuscular disease, causes progressive degeneration of motor neurons in the brain and spinal cord.ObjectiveWe analyzed the spinal cords of G93A mice following dietary D₃ restriction at 2.5% the adequate intake (AI) for oxidative damage (4-HNE, 3-NY), antioxidant enzymes (SOD2, catalase, GPx1), inflammation (TNF-α, IL-6, IL-10), apoptosis (bax/bcl-2 ratio, cleaved/pro-caspase 3 ratio), neurotrophic factor (GDNF) and neuron count (ChAT, SMI-36/SMI-32 ratio).MethodsBeginning at age 25 d, 42 G93A mice were provided food ad libitum with either adequate (AI;1 IU D₃/g feed; 12 M, 11 F) or deficient (DEF; 0.025 IU D₃/g feed; 10 M, 9 F) D₃. At age 113 d, the spinal cords were analyzed for protein content. Differences were considered significant at P ≤ 0.10, since this was a pilot study.ResultsDEF mice had 16% higher 4-HNE (P = 0.056), 12% higher GPx1 (P = 0.057) and 23% higher Bax/Bcl2 ratio (P = 0.076) vs. AI. DEF females had 29% higher GPx1 (P = 0.001) and 22% higher IL-6 (P = 0.077) vs. AI females. DEF males had 23% higher 4-HNE (P = 0.066) and 18% lower SOD2 (P = 0.034) vs. AI males. DEF males had 27% lower SOD2 (P = 0.004), 17% lower GPx1 (P = 0.070), 29% lower IL-6 (P = 0.023) and 22% lower ChAT (P = 0.082) vs. DEF females.ConclusionD₃ deficiency exacerbates disease pathophysiology in the spinal cord of G93A mice, the exact mechanisms are sex-specific. This is in accord with our previous results in the quadriceps, as well as functional and disease outcomes.     

Arsenic disrupts cellular levels of p53 and mdm2: a potential mechanism of carcinogenesis.

The antitumor protein p53 plays a critical role in DNA repair. Inorganic arsenic exposure is associated with a wide variety of human tumors, particularly of the skin. To investigate how inorganic arsenic might interfere with DNA repair and lead to greater incidence of hyperkeratosis and skin tumors, we exposed human keratinocytes (HaCaT) to environmentally relevant concentrations of arsenite for 14 days. Arsenite reduced p53 levels while concomitantly increasing the p53 regulatory protein mdm2 levels in a dose- and time-dependent manner. We propose the disruption of the p53-mdm2 loop regulating cell cycle arrest as a model for arsenic-related skin carcinogenesis and it may be important in tumors with elevated mdm2 levels.