Omega-3 Fatty Acids and Vitamin D Decrease Plasma T-Tau, GFAP, and UCH-L1 in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) results in neuronal, axonal and glial damage. Interventions targeting neuroinflammation to enhance recovery from TBI are needed. Exercise is known to improve cognitive function in TBI patients. Omega-3 fatty acids and vitamin D reportedly reduce inflammation, and in combination, might improve TBI outcomes. This study examined how an anti-inflammatory diet affected plasma TBI biomarkers, voluntary exercise and behaviors following exposure to mild TBI (mTBI). Adult, male rats were individually housed in cages fitted with running wheels and daily running distance was recorded throughout the study. A modified weight drop method induced mTBI, and during 30 days post-injury, rats were fed diets supplemented with omega-3 fatty acids and vitamin D3 (AIDM diet), or non-supplemented AIN-76A diets (CON diet). Behavioral tests were periodically conducted to assess functional deficits. Plasma levels of Total tau (T-tau), glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1) and neurofilament light chain (NF-L) were measured at 48 h, 14 days, and 30 days post-injury. Fatty acid composition of food, plasma, and brain tissues was determined. In rats exposed to mTBI, NF-L levels were significantly elevated at 48 h post-injury (P < 0.005), and decreased to levels seen in uninjured rats by 14 days post-injury. T-tau, GFAP, and UCH-L1 plasma levels did not change at 48 h or 14 days post-injury. However, at 30 days post-injury, T-tau, GFAP and UCH-L1 all significantly increased in rats exposed to mTBI and fed CON diets (P < 0.005), but not in rats fed AIDM diets. Behavioral tests conducted post-injury showed that exercise counteracted cognitive deficits associated with mTBI. The AIDM diets significantly increased docosahexaenoic acid levels in plasma and brain tissue (P < 0.05), and in serum levels of vitamin D (P < 0.05). The temporal response of the four injury biomarkers examined is consistent with studies by others demonstrating acute and chronic neural tissue damage following exposure to TBI. The anti-inflammatory diet significantly altered the temporal profiles of plasma T-tau, GFAP, and UCH-L1 following mTBI. Voluntary exercise protected against mTBI-induced cognitive deficits, but had no impact on plasma levels of neurotrauma biomarkers. Thus, the prophylactic effect of exercise, when combined with an anti-inflammatory diet, may facilitate recovery in patients with mTBI.

Phenotypic characterization of frontal cortex microglia in a rat model of post-traumatic stress disorder.

INTRODUCTION: Post-traumatic stress disorder (PTSD) is an anxiety disorder induced by psychologically traumatic events. Using a rat model, this study aimed to determine whether psychological trauma alters relative expression between pro-inflammatory and anti-inflammatory markers in microglia. To meet this goal, expression of genes encoding i-NOS, arginase, TNF-α, interleukin-10, CD74, and Mannose Receptor C was analyzed on multiple days following trauma exposure. METHODS: Single-prolonged stress (SPS) was used to model PTSD in male Sprague-Dawley rats. Twenty-four rats (12 Controls and 12 SPS-exposed) were sacrificed on Days 1, 3, and 7 post-SPS. Twenty-four (12 Controls and 12 SPS-exposed) additional rats were exposed to classical fear conditioning on Day 7, and fear extinction on Days 8, 9, 10, 15, 16, and 17. Freezing behavior was measured to assess fear resolution. Microglial isolates were collected from the frontal cortex, and RNA was extracted. Changes in relative expression of target genes were quantified via RT-PCR. RESULTS: SPS rats showed significant decreases in IL-10 and TNF-α expression and increases in the i-NOS:Arginase and TNF-α:IL-10 ratios compared to Controls on Day 1, but not on Day 3 or Day 7 for any of the dependent variables. Day 17 SPS rats showed a significant decrease in IL-10 expression and an increase in the TNF-α:IL-10 ratio, further characterized by a significant inverse relationship between IL-10 expression and fear persistence. CONCLUSION: Psychological trauma impacts the immunological phenotype of microglia of the frontal cortex. Consequently, future studies should further evaluate the mechanistic role of microglia in PTSD pathology.

TBI weight-drop model with variable impact heights differentially perturbs hippocampus-cerebellum specific transcriptomic profile.

The degree of brain injury is the governing factor for the magnitude of the patient's psycho- and physiological deficits post-injury, and the associated long-term consequences. The present scaling method used to segregate the patients among mild, moderate and severe phases of traumatic brain injury (TBI) has major limitations; however, a more continuous stratification of TBI is still elusive. With the anticipation that differentiating molecular markers could be the backbone of a robust method to triage TBI, we used a modified closed-head injury (CHI) Marmarou model with two impact heights (IH). By definition, IH directly correlates with the impact force causing TBI. In our modified CHI model, the rat skull was fitted with a helmet to permit a diffuse axonal injury. With the frontal cortex as the focal point of injury, the adjacent brain regions (hippocampus, HC and cerebellum, CB) were susceptible to diffuse secondary shock injury. At 8 days post injury (po.i.), rats impacted by 120 cm IH (IH120) took a longer time to find an escape route in the Barnes maze as compared to those impacted by 100 cm IH (IH100). Using a time-resolved interrogation of the transcriptomic landscape of HC and CB tissues, we mined those genes that altered their regulations in correlation with the variable IHs. At 14 days po.i., when all rats demonstrated nearly normal visuomotor performance, the bio-functional analysis suggested an advanced healing mechanism in the HC of IH100 group. In contrast, the HC of IH120 group displayed a delayed healing with evidence of active cell death networks. Combining whole genome rat microarrays with behavioral analysis provided the insight of neuroprotective signals that could be the foundation of the next generation triage for TBI patients.

Dietary Zinc Modulates Matrix Metalloproteinases in Traumatic Brain Injury.

Animal models of mild traumatic brain injury (mTBI) provide opportunity to examine the extent to which dietary interventions can be used to improve recovery after injury. Animal studies also suggest that matrix metalloproteinases (MMPs) play a role in tissue remodeling post-TBI. Because dietary zinc (Zn) improved recovery in nonblast mTBI models, and the MMPs are Zn-requiring enzymes, we evaluated the effects of low- (LoZn) and adequate-Zn (AdZn) diets on MMP expression and behavioral responses, subsequent to exposure to a single blast. MMP messenger RNA expression in soleus muscle and frontal cortex tissues were quantified at 48 h and 14 days post-blast. In muscle, blast resulted in significant upregulation of membrane-type (MT)-MMP, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 at 48 h post-injury in rats consuming AdZn. At 14 days post-blast, there were no blast or dietary effects observed on MMP levels in muscle, supporting the existence of a Zn-responsive, functional repair and remodeling mechanism. In contrast, blast resulted in a significant downregulation of MT-MMP, TIMP-1, and TIMP-2 and a significant upregulation of MMP-3 levels at 48 h post-injury in cortex tissue, whereas at 14 days post-blast, MT-MMP, MMP-2, and TIMP-2 were all downregulated in response to blast, independent of diet, and TIMP-1 were significantly increased in rats fed AdZn diets despite the absence of elevated MMPs. Because the blast injuries occurred while animals were under general anesthesia, the increased immobility observed post-injury in rats consuming LoZn diets suggest that blast mTBI can, in the absence of any psychological stressor, induce post-traumatic stress disorder-related traits that are chronic, but responsive to diet. Taken together, our results support a relationship between marginally Zn-deficient status and a compromised regenerative response post-injury in muscle, likely through the MMP pathway. However, in neuronal tissue, changes in MMP/TIMP levels after blast indicate a variable response to marginally Zn-deficient diets that may help explain compromised repair mechanism(s) previously associated with the systemic hypozincemia that develops in patients with TBI.

Nutritional treatment for traumatic brain injury.

Traumatic brain injury (TBI) is a significant public health concern. On average, 1.7 million persons sustain a TBI annually, and about 5.3 million Americans are living with a TBI-related disability. As the leading cause of death and disability in persons under 45 years old, there is a need for developing evidence-based interventions to reduce morbidity from this injury. So far, despite encouraging preclinical results, almost all neuroprotection trials have failed to show any significant efficacy in the treatment of patients with clinical TBI. The cascade of molecular and cellular changes after TBI involves plasticity in many different neurochemical systems, which represent putative targets for neurotherapeutic interventions. Accordingly, a successful TBI treatment may have to simultaneously attenuate many injury factors. The purpose of this review is to highlight four promising nutritional intervention options that have been identified-omega-3, zinc, vitamin D, and glutamine-and to provide an up-to-date summary regarding their apparent efficacy for affecting TBI.

Conjugated linoleic acid and calcium co-supplementation improves bone health in ovariectomised mice.

Osteoporosis is a significant health concern for the elderly; conjugated linoleic acid (CLA) has been shown to improve overall bone mass when calcium is included as a co-supplement. However, potential effects of CLA and calcium on bone mass during a period of bone loss have not been reported. The purpose of this study was to determine how dietary calcium modulates the effects of conjugated linoleic acid (CLA) in preventing bone loss, using an ovariectomised mouse model. CLA supplementation significantly prevented ovariectomy-associated weight and fat mass gain, compared to non-supplemented controls. CLA significantly increased bone markers without major changes in bone mineral composition in the femur compared to respective controls. CLA treatment increased serum parathyroid hormone (PTH) significantly (p=0.0172), while serum 1,25-dihydroxyvitamin D3 concentration was not changed by CLA. Meanwhile, CLA significantly reduced femur tartrate resistant acid phosphatase (TRAP) activity, suggesting potential reduction of osteoclastogenesis. The data suggest that CLA, along with dietary calcium, has great potential to be used to prevent bone loss and weight gain associated with menopause.

Use of zinc as a treatment for traumatic brain injury in the rat: effects on cognitive and behavioral outcomes.

BACKGROUND: While treatments for the behavioral deficits associated with traumatic brain injury (TBI) are currently limited, animal models suggest that zinc supplementation may increase resilience to TBI. OBJECTIVE: This work tests the hypothesis that zinc supplementation after TBI can be used as treatment to improve behavioral outcomes such as anxiety, depression, and learning and memory. METHODS: TBI was induced by controlled cortical impact to the medial frontal cortex. After TBI, rats were fed either a zinc adequate (ZA, 30 ppm) or zinc supplemented (ZS, 180 ppm) diet. Additional rats in each dietary group (ZA or ZS) were given a single intraperitoneal (ip) injection of zinc (30 mg/kg) 1 hour following injury. RESULTS: Brain injury resulted in significant increases in anxiety-like and depression-like behaviors as well as impairments in learning and memory. None of the zinc treatments (dietary or ip zinc) improved TBI-induced anxiety. The 2-bottle saccharin preference test for anhedonia revealed that dietary ZS also did not improve depression-like behaviors. However, dietary ZS combined with an early ip zinc injection significantly reduced anhedonia (P < .001). Dietary supplementation after injury, but not zinc injection, significantly improved (P < .05) cognitive behavior as measured by the time spent finding the hidden platform in the Morris water maze test compared with injured rats fed a ZA diet. CONCLUSIONS: These data suggest that zinc supplementation may be an effective treatment option for improving behavioral deficits such as cognitive impairment and depression following TBI.

Zinc supplementation provides behavioral resiliency in a rat model of traumatic brain injury.

Depression, anxiety, and impairments in learning and memory are all associated with traumatic brain injury (TBI). Because of the strong link between zinc deficiency, depression, and anxiety, in both humans and rodent models, we hypothesized that dietary zinc supplementation prior to injury could provide behavioral resiliency to lessen the severity of these outcomes after TBI. Rats were fed a marginal zinc deficient (5 ppm), zinc adequate (30 ppm), or zinc supplemented (180 ppm) diet for 4 weeks followed by a moderately-severe TBI using the well-established model of controlled cortical impact (CCI). Following CCI, rats displayed depression-like behaviors as measured by the 2-bottle saccharin preference test for anhedonia. Injury also resulted in evidence of stress and impairments in Morris water maze (MWM) performance compared to sham-injured controls. While moderate zinc deficiency did not worsen outcomes following TBI, rats that were fed the zinc supplemented diet for 4 weeks showed significantly attenuated increases in adrenal weight (p<0.05) as well as reduced depression-like behaviors (p<0.001). Supplementation prior to injury improved resilience such that there was not only significant improvements in cognitive behavior compared to injured rats fed an adequate diet (p<0.01), there were no significant differences between supplemented and sham-operated rats in MWM performance at any point in the 10-day trial. These data suggest a role for supplemental zinc in preventing cognitive and behavioral deficits associated with TBI.

Zinc transporter expression profiles in the rat prostate following alterations in dietary zinc.

Zinc plays important roles in numerous cellular activities and physiological functions. Intracellular zinc levels are strictly maintained by zinc homeostatic mechanisms. Zinc concentrations in the prostate are the highest of all soft tissues and could be important for prostate health. However, the mechanisms by which the prostate maintains high zinc levels are still unclear. In addition, the response of the prostate to alterations in dietary zinc is unknown. The current study explored cellular zinc levels and zinc transporter expression profiles in the lobes of the prostate during dietary marginal zinc depletion. Rats were given either zinc-adequate (ZA, 30 mg Zn/kg) or marginal zinc-deficient (MZD, 5 mg Zn/kg) diet for 9 weeks. In addition, a subgroup of the MZD rats was supplemented with phytase (1,500 unit/kg diet) to improve zinc bioavailability. We found that both zinc concentrations and ZnT2 expression in the prostate dorsolateral lobes were substantially higher than in the ventral lobes (P < 0.05). Marginal zinc depletion significantly decreased ZnT2 expression in the dorsolateral lobes (P < 0.05), and phytase supplementation had a trend to increase ZnT2 expression. In addition, of all measured zinc transporters, only ZnT2 mRNA abundance was significantly correlated to the zinc concentrations in the dorsolateral lobe. No correlations were found between zinc transporter expression and zinc concentrations in the ventral lobes. These results indicate that ZnT2 may play a significant role in the maintenance of zinc homeostasis in the prostate.

Marginal zinc deficiency increases oxidative DNA damage in the prostate after chronic exercise.

Approximately 12% of Americans do not consume the recommended level of zinc and could be at risk for marginal zinc deficiency. Zinc functions in antioxidant defense and DNA repair and could be important for prostate health. We hypothesized that marginal zinc deficiency sensitizes the prostate to oxidative stress and DNA damage. Rats were fed a zinc-adequate (ZA; 30 mg Zn/kg) or marginally zinc-deficient (MZD; 5-6 mg Zn/kg) diet for 6 weeks. MZD increased p53 and PARP expression but no change in 8-hydroxy-2'-deoxyguanosine levels was detected. To examine the susceptibility to exogenous oxidative stress, rats fed a ZA or MZD diet were assigned to exercising (EXE) or sedentary (SED) groups for 9 weeks. MZD or EXE alone did not affect oxidative DNA damage in the prostate; however, combined MZD + EXE increased DNA damage in the dorsolateral lobe. PARP and p53 expression was not further induced with MZD + EXE, suggesting that MZD interferes with DNA repair responses to stress. Finally, the addition of phytase to the MZD diet successfully restored zinc levels in the prostate and decreased DNA damage back to ZA levels. Overall, this study suggests that marginal zinc deficiency sensitizes the prostate to oxidative stress and demonstrates the importance of maintaining optimal zinc nutrition in physically active populations.

Phytase supplementation increases bone mineral density, lean body mass and voluntary physical activity in rats fed a low-zinc diet.

Phytic acid forms insoluble complexes with nutritionally essential minerals, including zinc (Zn). Animal studies show that addition of microbial phytase (P) to low-Zn diets improves Zn status and bone strength. The present study determined the effects of phytase supplementation on bone mineral density (BMD), body composition and voluntary running activity of male rats fed a high phytic acid, low-Zn diet. In a factorial design, rats were assigned to ZnLO (5 mg/kg diet), ZnLO+P (ZnLO diet with 1500 U phytase/kg) or ZnAD (30 mg/kg diet) groups and were divided into voluntary exercise (EX) or sedentary (SED) groups, for 9 weeks. SED rats were significantly heavier from the second week, and no catch-up growth occurred in EX rats. Feed intakes were not different between groups throughout the study. ZnLO animals had decreased food efficiency ratios compared to both phytase-supplemented (ZnLO+P) and Zn-adequate (ZnAD) animals (P<.01 compared to ZnLO). The ZnLO+P and ZnAD rats ran 56-75 km more total distance than ZnLO rats (P<.05), with the ZnLO+P rats running more kilometers per week than the ZnLO rats by Week 6. In vivo DEXA analyses indicate that rats fed phytase-supplemented diets had higher lean body mass (LBM) than those fed ZnLO diets; and that rats fed the Zn-adequate diets had the highest LBM. Body fat (%) was significantly lower in EX rats and was both Zn- and phytase insensitive. Rats fed phytase-supplemented diets had higher bone mineral content (BMC), bone area (BA) and BMD than rats fed ZnLO diets; and in rats fed ZnAD diets these indices were the highest. The dietary effects on BMC, BA and BMD were independent of activity level. We conclude that consuming supplemental dietary phytase or dietary Zn additively enhances Zn status to increase BMD, LBM and voluntary physical activity in rats fed a low-Zn diet. While the findings confirm that bone health is vulnerable to disruption by moderate Zn deficiency in rats, this new data suggests that if dietary Zn is limiting, supplemental phytase may have beneficial effects on LBM and performance activity.

Zinc and micronutrient combinations to combat gastrointestinal inflammation.

PURPOSE OF REVIEW: To examine current evidence for dietary supplementation with zinc and other micronutrients for primary prevention of multiple micronutrient deficiencies that are known to result from therapies used in the treatment of gastrointestinal inflammatory disorders. RECENT FINDINGS: Epidemiological observations and clinical findings have strengthened the concept that both nutritional deficiencies and nutritional excesses impair the gastrointestinal response(s) and alter susceptibility to inflammation and other diseases. The interaction of micronutrient intake, biochemical indicators of nutritional status, and four specific gastrointestinal inflammation states are reviewed. These conditions include celiac disease and concomitant micronutrient deficiencies resulting from the sustained adherence to a gluten-free diet; micronutrient nutrition as an important determinant of immunity for two major types of inflammatory bowel disease: ulcerative colitis and Crohn's disease; and HIV/AIDS-related diarrhea and concomitant micronutrient deficiencies which may be exacerbated by the initiation of highly active antiretroviral therapy. SUMMARY: For each inflammation 'state', enhancement of micronutrient status can improve immunocompetance and minimize therapeutic side-effects. The impact of single-micronutrient deficiencies on immune responses, and the possible impact of uncorrected micronutrient status are discussed.

Zinc and diarrheal disease: current status and future perspectives.

PURPOSE OF REVIEW: To evaluate clinical data indicating the benefits of oral zinc supplementation to prevent and/or treat diarrhea in children and extend these findings to adults. RECENT FINDINGS: Zinc plays an important role in modulating host resistance to infectious agents and reducing the risk, severity, and duration of diarrheal diseases. Zinc is important in the developing world and in low-income and middle-income countries where mild-to-moderate zinc deficiency is highly prevalent.The WHO/UNICEF recommendations for zinc supplementation are based on meta-analyses of randomized, controlled intervention trials on children: 20 mg zinc/day for 10-14 days for children with acute diarrhea and 10 mg/day for infants under 6 months of age. Effective forms include sulfate, gluconate, or acetate. No similar studies have been conducted on adults. Thus, carefully conducted clinical trials are necessary to ascertain the efficacy of zinc in prevention of acute and persistent diarrhea in adults. SUMMARY: Faced with rising antibiotic resistance and the lack of effective antidiarrheal vaccines, oral zinc provides substantial benefit in the reduction of stool output and disease duration combined with safety, selectivity of action, and low cost. Thus, oral zinc supplementation is a practical therapeutic intervention for the treatment of diarrhea in children, and by extension, should be provided to adults.

Moderate zinc restriction affects intestinal health and immune function in lipopolysaccharide-challenged mice.

Zinc (Zn) is an essential nutrient that affects immune function, especially within the digestive system, although the underlying mechanisms are not well understood. This study examined the effects of short-term moderate Zn restriction on intestinal health and immune function in lipopolysaccharide (LPS)-challenged mice through plasma cytokine profiling and histological evaluation of intestinal tissue sections. Adult male mice were fed with a Zn-adequate (40 ppm) or a Zn-marginal (4 ppm) diet for 4 weeks, and then a bacterial challenge was simulated by intraperitoneal injection of LPS (10 microg/g body weight [BW]) or saline (control). BW was recorded weekly, and feed intake was recorded daily over the last week. Voluntary locomotor activity was assessed 6 and 24 h after the challenge. Plasma and tissues were collected 0, 6 or 24 h after the challenge for analysis. Histological analysis of intestinal samples included evaluation of villi length and width, lamina propria (LP) width, crypt depth and intraepithelial as well as LP leukocyte numbers. Plasma was analyzed for IL-1beta, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, interferon gamma and tumor necrosis factor alpha. Diet did not affect BW and feed intake. The LPS challenge led to decreased voluntary locomotor activity (P<.05). Moderate Zn restriction led to greater leukocyte infiltration in the LP after the LPS challenge (P<.05) and higher plasma IL-6 and IL-10 levels 24 h after the LPS challenge (P<.01). Results indicate that Zn status impacts intestinal responses to LPS through modulation of the cytokine response and leukocyte recruitment, and this impact is evident even with short-term (4 weeks) moderate Zn restriction.

Effect of supplemental dietary zinc on the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle and liver from post-absorptive mice.

Zinc (Zn) is an essential trace element that functions in cellular signaling. The mammalian target of rapamycin (mTOR) regulates the initiation of protein synthesis. The objective of this study was to determine whether Zn could stimulate protein phosphorylation in the mTOR pathway in vivo. Mice (C57BL/6J, n = 30) were fed Zn marginal diets (ZM, 5 mg/kg) for 4 weeks, followed by fasting (F) and/or refeeding with ZM or Zn supplemental (300 mg/kg, ZS) diets for 3 or 6 h. Plasma insulin was greater (P < 0.05) in refed animals as compared to F animals. Protein phosphorylation was detected using multiplex analysis and Western blotting. Multiplex analysis indicated greater (P < 0.05) p70 S6 kinase (p70S6K) and glycogen synthase kinase 3 (GSK-3 alpha/beta) phosphorylation in livers from 6-h refed ZS animals as compared to F animals. Western blots indicated increased (P < 0.05) Akt (Ser 473) phosphorylation in skeletal muscle from animals refed ZS diets for 3 and 6 h as compared to F animals. The ZS diet affected phosphorylation of GSK-3 (alpha/beta) in liver, as 3-h ZS refed animals had greater (P < 0.01) phosphorylation than F animals. These findings indicate that Zn may contribute to the initiation of protein synthesis as a signaling molecule in vivo.

Moderate zinc deficiency negatively affects biomechanical properties of rat tibiae independently of body composition.

To guide development of novel nutritional strategies aimed at reducing the incidence of stress fractures, we observed the effects of manipulating dietary zinc (Zn) content on bone integrity in Sprague-Dawley rats fed either a severely Zn-deficient (ZnD; 1 ppm), a moderately Zn-deficient (MZnD; 5 ppm) or a Zn-adequate (ZnAD; 30 ppm) diet for 6 weeks. At the completion of the diet period, body composition, bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were determined in vivo by using dual-energy X-ray absorptiometry. Following euthanasia, long bones were collected for determination of Zn content and biomechanical strength testing. Despite significant positive correlations between dietary Zn and both body weight (BW) and bone Zn content for the entire cohort (r = .77 and r = .83, respectively), rats fed MZnD or ZnAD diets did not differ in feed intakes, body composition, BMC, BA, BMD or BW. Tibial bones, but not femur bones, appear to be more responsive to dietary Zn manipulation, as all bone biomechanical strength indices in the ZnAD-fed rats were significantly greater than in rats fed the ZnD diets. Rats fed either MZnD or ZnAD diets had stronger tibiae (129% increase in maximum load and stress at maximum load, P<.01) compared with those fed ZnD diets. The load at breakage for the tibial bones of rats fed MZnD diets was not different from the ZnD rats, but lower (P<.05) than that of the ZnAD rats. These results suggest that since feed intakes, body composition, BMC, BA, BMD and BW were not significantly different between the MZnD- and ZnAD-fed animals, the reduced bone integrity observed in the MZnD-fed rats resulted from dietary Zn inadequacy, and not as a result of the reduced growth that is typically associated with Zn deficiency.

Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle.

Zinc deficiency negatively affects muscle function, but there are limited biochemical data identifying the cause of this reduction in function. The objective of the present study was to identify soluble proteins in rat soleus muscle that were responsive to different levels of dietary zinc. Rats (n=21) were fed diets containing three concentrations of zinc: 5, 30 and 200 ppm for 42 days. There was no difference in body weights of the rats consuming the 5-ppm zinc diet compared to the rats consuming the 30- or 200-ppm zinc diets; however, bone zinc levels were significantly decreased in the 5-ppm dietary zinc group. Individual soluble protein fractions were isolated from these muscles and the samples were prepared for two-dimensional polyacrylamide gel electrophoresis. The expression levels of four proteins were significantly depressed by dietary Zn depletion and supplementation, S-glutathiolated carbonic anhydrase, myosin light polypeptide 3, heat shock protein 20 and heart fatty acid binding protein. This is the first report that indicates that both Zn depletion and supplementation result in protein expression profiles that may negatively affect skeletal muscle function. These results indicate that there are specific signaling pathways that require proper Zn nutriture for maintaining optimal muscle function and suggest that the consumption of pharmacologic doses of Zn may be detrimental to muscle function.

Alterations in mRNA expression and protein products following spinal cord injury in humans.

We examined the effects of spinal cord injury (SCI) on alterations in gene expression and respective protein products in human skeletal muscle 2 days and 5 days post-SCI. Biopsies were taken from skeletal muscle of 9 men and 1 woman (n = 10) (43.9 +/- 6.7 years) 2 days and 5 days post-SCI and from 5 healthy young men who served as controls (20.4 +/- 0.5 years). Global changes in gene expression were analysed using Affymetrix GeneChips on a subsample of subjects (n = 3). Candidate genes were then pursued via qRT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes. Immunohistochemistry (IHC) was used to localize proteins. Groups of transcripts showing the greatest percentage of altered expression, the most robust fold-changes, and indicative of involvement of an entire pathway using the GeneChip included genes involved in the ubiquitin proteasome pathway (UPP), metallothionein function, and protease inhibition. qRT-PCR analysis confirmed increases in gene expression for UPP components (UBE3C, Atrogin-1, MURF1, and PSMD11), the metallothioneins (MT1A, MT1F, MT1H), and the protease inhibitor, SLPI (P < 0.05) at 2 days and 5 days post-SCI. Protein levels of the proteasome subunit (PSMD11) and the metallothioneins were increased 5 days post-SCI. Protein levels of UBE3C, Atrogin-1, MURF1 and SLPI were unchanged (P > 0.05). IHC showed increased staining for PSMD11 and the metallothioneins 5 days post-SCI, along the peripheral region of the cells. IHC also showed altered staining for Atrogin-1 at 5 days post-SCI along the membrane region. Thus, there was a profound increase in gene expression of UPP components, the metallothioneins, and the protease inhibitor, SLPI, within 5 days of SCI. Increased protein levels for PSMD11 and the metallothioneins 5 days post-SCI, specifically along the cell periphery, indicate that proteins in this region may be early targets for degradation post-SCI.

Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components.

We examined the effects of 48 h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48 h of immobilization would increase gene expression and respective protein products for ubiquitin-proteasome pathway (UPP) components. Also, we used microarray analysis to identify novel pathways. Biopsies were taken from the vastus muscle of five men (20.4 +/- 0.5 yr) before and after 48-h immobilization. Global changes in gene expression were analyzed by use of Affymetrix GeneChips. Candidate genes were confirmed via quantitative RT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry was used to localize proteins found to be altered when assessed via WB. The greatest percentage of genes showing altered expression with the GeneChip included genes involved in the UPP, metallothionein function, and extracellular matrix (ECM) integrity. Quantitative RT-PCR analysis confirmed increases in mRNA for UPP components [USP-6, small ubiquitin-related modifier (SUMO-1)] and the metallothioneins (MT2A, MT1F, MT1H, MT1X) and decreases in mRNA content for matrix metalloproteinases (MMP-28, TIMP-1) and ECM structural components [collagen III (COLIII) and IV (COLIV)]. Only phosphorylated Akt (Ser473, Thr308), COLIII and COLIV protein levels were significantly different postimmobilization (25, 10, 88, and 28% decrease, respectively). Immunohistochemistry confirmed WB showing decreased staining for collagens postimmobilization. Our results suggest that 48 h of immobilization increases mRNA content for components of the UPP and metallothionein function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.

Effects of dietary phytase on body weight gain, body composition and bone strength in growing rats fed a low-zinc diet.

Phytic acid, a major phosphorous storage compound found in foodstuffs, is known to form insoluble complexes with nutritionally essential minerals, including zinc (Zn). Phytases are enzymes that catalyze the removal of these minerals from phytic acid, improving their bioavailability. The objective of the present study was to determine the ability of dietary phytase to affect body weight, body composition, and bone strength in growing rats fed a high phytic acid, low Zn diet. Rats (n = 20) were fed either a control (AIN-93) or phytase supplemented (Natuphos, BASF, 1,500 phytase units (FTU)/kg) diet for a period of 8 weeks. Phytase supplementation resulted in increased (P<.05) bone and plasma Zn, but no change in plasma inorganic phosphorous or bone levels of Ca, Fe, or Mg. The addition of phytase to the diets resulted in a 22.4% increase (P<.05) in body weight at the end of the study as compared with rats fed a control diet. Dual x-ray absorptiometry (DXA) revealed that phytase supplementation resulted in increase lean body mass (LBM, P<.001) and increased bone mineral content (BMC, P<.001) as compared with feeding the control diet. Bone studies indicated that femurs and tibias from phytase supplemented rats had greater mass (P<.05) and were stronger (P<.05) than rats fed the control diet. This data suggest that the addition of phytase to low Zn diets results in improved Zn status, which may be responsible for beneficial effects on growth, body composition, and bone strength.