Standard Rodent Chow Diet Research Articles

Impact of a Standard Rodent Chow Diet on Tissue n-6 Fatty Acids, Δ9-Desaturation Index, and Plasmalogen Mass in Rats Fed for One Year.

Although many studies focus on senescence mechanisms, few habitually consider age as a biological parameter. Considering the effect of interactions between food and age on metabolism, here we depict the lipid framework of 12 tissues isolated from Sprague-Dawley rats fed standard rodent chow over 1year, an age below which animals are commonly studied. The aim is to define relevant markers of lipid metabolism influenced by age in performing a fatty acid (FA) and dimethylacetal profile from total lipids. First, our results confirm impregnation of adipose and muscular tissues with medium-chain FA derived from maternal milk during early infancy. Secondly, when animals were switched to standard croquettes, tissues were remarkably enriched in n-6 FA and especially 18:2n-6. This impregnation over time was coupled with a decrease of the desaturation index and correlated with lower activities of hepatic Δ5- and Δ6-desaturases. In parallel, we emphasize the singular status of testis, where 22:5n-6, 24:4n-6, and 24:5n-6 were exceptionally accumulated with growth. Thirdly, 18:1n-7, usually found as a discrete FA, greatly accrued over the course of time, mostly in liver and coupled with Δ9-desaturase expression. Fourthly, skeletal muscle was characterized by a surprising enrichment of 22:6n-3 in adults, which tended to decline in older rats. Finally, plasmalogen-derived dimethylacetals were specifically abundant in brain, erythrocytes, lung, and heart. Most notably, a shift in the fatty aldehyde moiety was observed, especially in brain and erythrocytes, implying that red blood cell analysis could be a good indicator of brain plasmalogens.

Duodenal jejunal bypass attenuates non-alcoholic fatty liver disease in western diet-obese rats

To evaluate the effects of duodenal-jejunal bypass (DJB) on serum and hepatic profiles of obese rats fed on a western diet (WD). Twenty eight male Wistar rats were fed a standard rodent chow diet (CTL group) or WD ad libitum. After 10 weeks, WD rats were submitted to sham (WD SHAM) or duodenal-jejunal bypass (WD DJB). Body weight, fat pad depots, glycemia, insulinemia, HOMA-IR, TyG, lipids profile and hepatic analyses were evaluated two months after surgery. The WD SHAM group presented greater obesity, hyperglycemia, hyperinsulinemia, insulin resistance, hypertriglyceridemia and hepatic steatosis than the CTL group. WD DJB rats presented decreased serum glucose and insulin resistance, when compared to WD SHAM animals, without changes in insulinemia. In addition, DJB surgery normalized serum TG and attenuated TG accumulation and steatosis in the liver of the WD DJB group. Hepatic ACC and FAS protein expressions were similar in all groups. Duodenal-jejunal bypass attenuates hepatic parameters of non-alcoholic fatty liver disease in obese rats fed on a western diet.

A mouse Col4a4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers

A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on Chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G to A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, for gaining better insights into the mechanisms leading to Alport syndrome and to the variability in the age of onset and associated phenotypes.

Chlorella modulates insulin signaling pathway and prevents high-fat diet-induced insulin resistance in mice

AimsThe search for natural agents that minimize obesity-associated disorders is receiving special attention. In this regard, the present study aimed to evaluate the prophylactic effect of Chlorella vulgaris (CV) on body weight, lipid profile, blood glucose and insulin signaling in liver, skeletal muscle and adipose tissue of diet-induced obese mice. Main methodsBalb/C mice were fed either with standard rodent chow diet or high-fat diet (HFD) and received concomitant treatment with CV for 12 consecutive weeks. Triglyceride, free fatty acid, total cholesterol and fractions of cholesterol were measured using commercial assay. Insulin and leptin levels were determined by enzyme-linked immunosorbent assay (ELISA). Insulin and glucose tolerance tests were performed. The expression and phosphorylation of IRβ, IRS-1 and Akt were determined by Western blot analyses. Key findingsHerein we demonstrate for the first time in the literature that prevention by CV of high-fat diet-induced insulin resistance in obese mice, as shown by increased glucose and insulin tolerance, is in part due to the improvement in the insulin signaling pathway at its main target tissues, by increasing the phosphorylation levels of proteins such as IR, IRS-1 and Akt. In parallel, the lower phosphorylation levels of IRS-1ser307 were observed in obese mice. We also found that CV administration prevents high-fat diet-induced dyslipidemia by reducing triglyceride, cholesterol and free fatty acid levels. SignificanceWe propose that the modulatory effect of CV treatment preventing the deleterious effects induced by high-fat diet is a good indicator for its use as a prophylactic–therapeutic agent against obesity-related complications.

Preservation of bone structure and function by Lithothamnion sp. derived minerals.

Progressive bone mineral loss and increasing bone fragility are hallmarks of osteoporosis. A combination of minerals isolated from the red marine algae, Lithothamnion sp. was examined for ability to inhibit bone mineral loss in female mice maintained on either a standard rodent chow (control) diet or a high-fat western diet (HFWD) for 5, 12, and 18 months. At each time point, femora were subjected to μ-CT analysis and biomechanical testing. A subset of caudal vertebrae was also analyzed. Following this, individual elements were assessed in bones. Serum levels of the 5b isoform of tartrate-resistant acid phosphatase (TRAP) and procollagen type I propeptide (P1NP) were also measured. Trabecular bone loss occurred in both diets (evident as early as 5 months). Cortical bone increased through month 5 and then declined. Cortical bone loss was primarily in mice on the HFWD. Inclusion of the minerals in the diet reduced bone mineral loss in both diets and improved bone strength. Bone mineral density was also enhanced by these minerals. Of several cationic minerals known to be important to bone health, only strontium was significantly increased in bone tissue from animals fed the mineral diets, but the increase was large (5-10 fold). Serum levels of TRAP were consistently higher in mice receiving the minerals, but levels of P1NP were not. These data suggest that trace minerals derived from marine red algae may be used to prevent progressive bone mineral loss in conjunction with calcium. Mineral supplementation could find use as part of an osteoporosis-prevention strategy.

Transcription factor NRF2 protects mice against dietary iron-induced liver injury by preventing hepatocytic cell death

The liver, being the major site of iron storage, is particularly exposed to the toxic effects of iron. Transcription factor NRF2 is critical for protecting the liver against disease by activating the transcription of genes encoding detoxification/antioxidant enzymes. We aimed to determine if the NRF2 pathway plays a significant role in the protection against hepatic iron overload. Wild-type and Nrf2(-/-) mouse primary hepatocytes were incubated with ferric ammonium citrate. Wild-type and Nrf2(-/-) mice were fed standard rodent chow or iron-rich diet for 2weeks, with or without daily injection of the antioxidant mito-TEMPOL. In mouse hepatocytes, iron induced the nuclear translocation of NRF2 and the expression of cytoprotective genes in an NRF2-dependent manner. Moreover, Nrf2(-/-) hepatocytes were highly susceptible to iron-induced cell death. Wild-type and Nrf2(-/-) mice fed iron-rich diet accumulated similar amounts of iron in the liver and were equally able to increase the expression of hepatic hepcidin and ferritin. Nevertheless, in Nrf2-null mice the iron loading resulted in progressive liver injury, ranging from mild confluent necrosis to severe necroinflammatory lesions. Hepatocytic cell death was associated with gross ultrastructural damage to the mitochondria. Notably, liver injury was prevented in iron-fed animals that received mito-TEMPOL. NRF2 protects the mouse liver against the toxicity of dietary iron overload by preventing hepatocytic cell death. We identify NRF2 as a potential modifier of liver disease in iron overload pathology and show the beneficial effect of the antioxidant mito-TEMPOL in a mouse model of dietary iron-induced liver injury.

P4: Nitrite-mediated, xanthine oxidase-dependent diminution of obesity-related hyperglycemia and cardiopulmonary dysfunction

Background Obesity is associated with elevated tissue and plasma xanthine oxidase (XO) levels and allied enhancement of reactive species formation contributory to systemic inflammation. Despite a long standing association between increased XO activity and negative clinical outcomes, recent reports describe a paradigm shift where XO may mediate beneficial actions by reducing NO 2 - to NO. While provocative, these observations contradict both reports of improved outcomes in similar models when XO is inhibited and reports revealing anoxia as a requisite for XO-mediated NO formation. Herein, we identify a vascular microenvironment where NO 2 - reductase activity of XO is operative in the presence of O2 as well as examine effects of XO inhibition vs. NO 2 - supplementation in a high-fat diet (HFD) model of obesity. Methods Purified XO, XO bound to heparin-Sepharose 6B and XO sequestered by endothelial cell glycosaminoglycans (GAGs) was used to examine NO 2 - reductase activity as assessed by several detection platforms including enhanced chemiluminescence (NO), EPR spin trapping O 2 - and amperometric O2 detection. Male C57Blk/j6 mice were subjected to a HFD (60% calories derived from fat) for 20 weeks and treated (drinking water) with the XO-specific inhibitor febuxostat (0.5 mg/L) or NaNO2 (100 mg/L) or both for the final 6 weeks. Age-matched controls were maintained on a standard rodent chow diet consisting of 15% of adjusted calories from fat. After 20 weeks on diets mice were analyzed for right ventricular (RV) pressure volume (PV) relationship using a PV conductance catheter under steady state conditions. Results Whereas NO 2 - reductase activity of XO free in solution is only operative under anoxia, sequestration of XO on endothelial cell GAGs confers the capacity for substantive NO formation in the presence of O2 (1–2% or ∼13–26 μM) with concomitant diminution of XO-derived ROS production (↓40%). Treatment with febuxostat: reduced fasting blood glucose (223 vs. 181 mg/dL), improved (39%) impaired glucose tolerance and diminished oxidative stress in lung, liver, heart and skeletal muscle. Febuxostat also improved indices related to obesity-mediated right ventricular (RV) dysfunction and the onset of pulmonary arterial hypertension including RV end systolic pressure (RVESP) (41.2 ± 7.3 vs. 27.2 ± 5.2 mmHg), RV contractility index, pulmonary vascular resistance (PVR) (2.51 ± 0.47 vs. 1.96 ± 0.24 mmHg/mL/min), mean pulmonary artery pressure (mPAP) (25.2 ± 3.7 vs. 18.5 ± 3.0 mmHg) and Tau (diastolic function) (6.8 ± 1.3 vs. 4.5 ± 0.9). Febuxostat significantly reduced RV hypertrophy (Fulton Index), pulmonary arteriole smooth muscle (SM) remodeling (α-SM actin staining) and pulmonary tissue macrophage infiltration. Treatment of HFD mice with NO 2 - produced similar yet more pronounced beneficial effects than febuxostat in all the parameters listed above. However, combined treatment with NO 2 - + febuxostat completely abolished these protective effects, suggesting the salutary actions of NO 2 - were mediated by XO. Conclusion These data demonstrate that under hypoxic/inflammatory conditions where vascular XO levels and subsequent XO-GAG interactions are enhanced, XO-catalyzed NO 2 - reduction increases NO generation, decreases ROS production and serves to diminish indices of obesity-mediated pathology. Disclosure Supported by AHA National Scientist Development-10SDG3560005 (E.E.K.).

Cholesterol enhances amyloid β deposition in mouse retina by modulating the activities of Aβ-regulating enzymes in retinal pigment epithelial cells

Subretinally-deposited amyloid β (Aβ) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing Aβ deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on Aβ production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10μg/ml cholesterol for 48h. Aβ amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate Aβ production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased Aβ production in cultured RPE cells. Activities of Aβ degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; α-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of β- or γ-secretase. mRNA levels of NEP and α-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing Aβ, whereas age-matched mice fed standard rodent chow diet did not. Activities and mRNA levels of NEP and α-secretase were significantly lower in native RPE cells freshly isolated from cholesterol-enriched chow fed mice compared to standard rodent chow fed mice. These findings suggest that cholesterol enhances subretinal Aβ accumulation by modulating the activities of enzymes degrading and processing Aβ in RPE cells in senescent subjects.

Abstract 292: ETC-1002 Reduces Body Weight Gain and Hepatic Triglyceride Content and Improves Glycemic Control in a Mouse Model of Diet-Induced Obesity

ETC-1002 is an investigational drug currently in Phase 2 clinical development to treat dyslipidemia and other cardiometabolic risk factors. Previously, ETC-1002 prevented hyperlipidemia and atherosclerosis in rodent models; and improved hepatic triglycerides (TG) as well as fasting blood glucose and insulin in the KKA y insulin resistant mouse model via putative mechanisms including activation of AMP-activated protein kinase. In the present study we investigated the effect of ETC-1002 on body weight, hepatic TG and insulin sensitivity in a diet-induced obese (DIO) mouse model. C57BL/6 mice were fed a 60% high-fat diet beginning at 11 weeks of age. At 12 weeks of age mice were assigned to treatment groups and administered vehicle or ETC-1002 at 3, 10, or 30 mg/kg/day for 9 weeks. A separate cohort of mice was maintained on standard rodent chow diet throughout the study as a comparator. Food consumption, body weight, hepatic TG content, fasting blood glucose, fasting plasma insulin, insulin tolerance tests, and glucose tolerance tests were measured. Mice developed obesity, hyperinsulinemia, mild hyperglycemia and elevated hepatic triglycerides in response to the high-fat diet. ETC-1002 results were dose-dependent and statistically significant at doses of 10 and 30 mg/kg/day. ETC-1002 attenuated body weight gain 8% and 15% with no effect on food consumption. Body weight changes were associated with 12% and 32% decreases in epididymal fat pad mass. Hepatic TG content was also reduced with ETC-1002 by 34% and 46%; respectively. ETC-1002 treatment reduced fasting blood glucose 11% and 16%; plasma insulin 80% and 95%; and resulted in significant improvements in insulin tolerance tests (19% and 22% reduction in AUC) with modestly improved glucose tolerance (not significant). In an intervention study with a 12 week lead-in on high-fat diet, comparable effects on body weight, hepatic TG, and insulin sensitivity were observed. In summary, ETC-1002 reduced obesity and hepatic TG and improved glycemic parameters in a high-fat fed diet-induced mouse model of disease. The present data in the DIO mouse, combined with previously reported efficacy in rodent models supports ETC-1002 as a regulator of imbalances in lipid and carbohydrate metabolism.

Abstract 296: Decreased Hepatic Avpr1a Gene Expression and Elevated Serum Bile Acid in Mouse Model of Metabolic Syndrome

The KK/HIJ mouse has been demonstrated to have polygenic obesity and insulin resistance and serve as a model of metabolic syndrome. From microarray studies in this mouse strain, we observed hepatic gene expression of arginine vasopressin receptor 1A (Avpr1a) as the most differentially elevated gene in the liver following 3-week of voluntary wheel running activity. Subsequent studies validated that hepatic Avpr1a gene expression is significantly upregulated following voluntary activity and also highly suppressed (4 to 8-fold) in 2 independent models of insulin resistance (including obesity and lipoatrophic models). Although Avpr1a is highly abundant in the liver, its physiologic role is not well described. One proposed role for hepatic Avpr1a is mediation of vasopressin-induced bile acid secretion. To further evaluate the relationship between hepatic Avpr1a and bile acid homeostasis, we determined the age-related change in these variables in female KK mice. To investigate, adipose tissue, liver and serum were collected from female KK mice from before sexual maturity (PRE: 4.5 weeks old, n=9) and after sexual maturation (POST: 6 to 30 weeks old, n=12). Consistent with previous studies using this obesity-prone strain, we observed a robust increase in adiposity with age despite a standard rodent chow diet. RT-PCR studies of hepatic gene expression revealed a 53% lower Avpr1a in POST compared to PRE mice (p<0.00001). In parallel with the drop in hepatic Avpr1a gene expression was an increase in serum bile acids (PRE: 26.56± 9.98μmol/L; POST: 39.40± 9.63μmol/L; p<0.01). A negative correlation was evident between hepatic Avpr1a gene expression and serum bile acid level (R= -0.51). The change in Avpr1a and bile acids was pronounced at the age of onset of estrous cycling. In conclusion, female KK mice have a significant increase in fat mass with age in parallel with an elevation of serum bile acids and downregulation of hepatic Avpr1a gene expression. We propose that suppression of hepatic Avpr1a increases hydrophobic bile acids in the liver and serum and promotes hepatic inflammation, contributing to symptoms of the metabolic syndrome.

Duodenal–Jejunal Bypass Surgery Enhances Glucose Tolerance and Beta-Cell Function in Western Diet Obese Rats

The aim of this study was to investigate the effect of the duodenal-jejunal bypass (DJB) on glucose homeostasis and islet insulin secretion in Western diet (WD) obese rats. Male Wistar rats received a standard rodent chow diet (CTL group) or WD ad libitum. After 32 weeks of diet, WD rats were submitted to duodenal-jejunal bypass (WD DJB) or sham (WD S-DJB) operation. Intraperitoneal (ip) glucose tolerance test was performed 1 week after surgery. Body weight, fat pad depots, glycemia, insulinemia, HOMA-IR, and glucose-induced insulin secretion were evaluated 1 month after surgery. Body weight and fat pads of the WD group were higher than those of the CTL group. Sham and DJB surgeries did not alter these parameters. WD and WD S-DJB rats were glucose intolerant, insulin resistant, and hyperinsulinemic. WD DJB rats showed similar glucose tolerance, insulin sensitivity, and plasma insulin levels to those of CTL rats. WD rats presented higher fat and glycogen contents in the liver. DJB surgery normalized fat and glycogen stores in the liver of the WD DJB group. Insulin release at 11.1-mM glucose, in isolated islets from WD and WD S-DJB rats, was higher than from islets of CTL rats. In contrast, DJB surgery improves the beta-cell secretory capacity with increased glucose-induced secretion at 5.6-, 11.1-, and 22.2-mM glucose in WD DJB islets, compared with CTL islets. DJB surgery improves glucose homeostasis and enhances beta-cell glucose responsiveness in rats submitted to the WD diet without any modification in adiposity.

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Calcium insufficiency accelerates type 1 diabetes in vitamin D receptor-deficient nonobese diabetic (NOD) mice.

Vitamin D exerts important regulatory effects on the endocrine and immune systems. Autoimmune type 1 diabetes (T1D) development in the inbred NOD mouse strain can be accelerated by vitamin D insufficiency or suppressed by chronic treatment with high levels of 1α,25-dihydroxyvitamin D(3). Consequently, a report that T1D development was unaffected in NOD mice genetically lacking the vitamin D receptor (VDR) was unexpected. To further assess this result, the mutant stock was imported to The Jackson Laboratory, backcrossed once to NOD/ShiLtJ, and progeny rederived through embryo transfer. VDR-deficient NOD mice of both sexes showed significant acceleration of T1D. This acceleration was not associated with alterations in immune cells targeting pancreatic β-cells. Rather, the capacity of β-cells to produce and/or secrete insulin was severely impaired by the hypocalcaemia developing in VDR-deficient NOD mice fed a standard rodent chow diet. Feeding a high-lactose calcium rescue diet that circumvents a VDR requirement for calcium absorption from the intestine normalized serum calcium levels, restored β-cell insulin secretion, corrected glucose intolerance, and eliminated accelerated T1D in VDR-deficient NOD mice. These findings suggest that calcium and/or vitamin D supplementation may improve disease outcomes in some T1D-prone individuals that are calcium deficient.

Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

Intermittent Fasting in Mice Does Not Improve Hindlimb Motor Performance after Spinal Cord Injury

Previously, we reported that every-other-day-fasting (EODF) in Sprague-Dawley rats initiated after cervical spinal cord injury (SCI) effectively promoted functional recovery, reduced lesion size, and enhanced sprouting of the corticospinal tract. More recently, we also showed improved behavioral recovery with EODF after a moderate thoracic contusion injury in rats. In order to make use of transgenic mouse models to study molecular mechanisms of EODF, we tested here whether this intermittent fasting regimen was also beneficial in mice after SCI. Starting after SCI, C57BL/6 mice were fed a standard rodent chow diet either with unrestricted access or feeding every other day. Over a 14-week post-injury period, we assessed hindlimb locomotor function with the Basso Mouse Scale (BMS) open-field test and horizontal ladder, and the spinal cords were evaluated histologically to measure white and grey matter sparing. EODF resulted in an overall caloric restriction of 20% compared to animals fed ad libitum (AL). The EODF-treated animals exhibited a ∼ 14% reduction in body weight compared to AL mice, and never recovered to their pre-operative body weight. In contrast to rats on an intermittent fasting regimen, mice exhibited no increase in blood ketone bodies by the end of the second, third, and fourth day of fasting. EODF had no beneficial effect on tissue sparing and failed to improve behavioral recovery of hindlimb function. Hence this observation stands in stark contrast to our earlier observations in Sprague-Dawley rats. This is likely due to the difference in the metabolic response to intermittent fasting as evidenced by different ketone levels during the first week of the EODF regimen.

Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP−/−) have a systemic alpha-tocopherol (AT) deficiency, with their lung AT levels being < 10% of those in AT-replete ATTP+/+ mice when fed a standard rodent chow diet. ATTP+/+ and ATTP−/− mice (4 wk old male mice, n = 16 per group) were fed a standard diet (35 IU AT/kg diet) for 8–12 wk, exposed 6 h/day for 3 days to either to O3 (0.5 ppm) or filtered air, then sacrificed. No significant differences in plasma or lung AT concentrations were observed in response to this level of O3 exposure. Lung genomic responses of the lungs to O3 were determined using Affymetrix 430A 2.0 arrays containing over 22,600 probe sets representing 14,000 well-characterized mouse genes. As compared with filtered air exposure, O3 exposure resulted in 99 genes being differentially expressed in ATTP−/− mice, as compared to 52 differentially expressed genes in ATTP+/+ mice. The data revealed an O3-induced upregulation of genes related to cell proliferation/DNA repair and inflammatory-immune responses in both ATTP+/+ and ATTP−/− mice, with the expression of 22 genes being common to both, whereas 30 and 77 genes were unique to ATTP+/+ and ATTP−/− mice, respectively. The expressions of O3 sensitive genes—Timp1, Areg, Birc5 and Tnc—were seen to be further modulated by AT status. The present study reveals AT modulation of adaptive response of lung genome to O3 exposure.

Lifestyle of Lactobacillus plantarum in the mouse caecum

Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts. Strains of L. plantarum are also marketed as probiotics intended to confer beneficial health effects upon delivery to the human gut. To understand how L. plantarum adapts to its gut habitat, we used whole genome transcriptional profiling to characterize the transcriptome of strain WCFS1 during colonization of the caeca of adult germ-free C57Bl/6 J mice fed a standard low-fat rodent chow diet rich in complex plant polysaccharides or a prototypic Western diet high in simple sugars and fat. Lactobacillus plantarum colonized the digestive tracts of these animals to high levels, although L. plantarum was found in 10-fold higher amounts in the caeca of mice fed the standard chow. Metabolic reconstructions based on the transcriptional data sets revealed that genes involved in carbohydrate transport and metabolism form the principal functional group that is upregulated in vivo compared with exponential phase cells grown in three different culture media, and that a Western diet provides a more nutritionally restricted, growth limiting milieu for the microbe in the distal gut. A set of bacterial genes encoding cell surface-related functions were differentially regulated in both groups of mice. This set included downregulated genes required for the d-alanylation of lipoteichoic acids, extracellular structures of L. plantarum that mediate interactions with the host immune system. These results, obtained in a reductionist gnotobiotic mouse model of the gut ecosystem, provide insights about the niches (professions) of this lactic acid bacterium, and a context for systematically testing features that affect epithelial and immune cell responses to this organism in the digestive tract.

Improvement in motor and exploratory behavior in Rett syndrome mice with restricted ketogenic and standard diets

Rett syndrome (RTT) is a rare X-linked autistic-spectrum neurological disorder associated with impaired energy metabolism, seizure susceptibility, progressive social behavioral regression, and motor impairment primarily in young girls. The objective of this study was to examine the influence of restricted diets, including a ketogenic diet (KD) and a standard rodent chow diet (SD), on behavior in male Mecp2 308/y mice, a model of RTT. The KD is a high-fat, low-carbohydrate diet that has anticonvulsant efficacy in children with intractable epilepsy and may be therapeutic in children with RTT. Following an 11-day pretrial period, adult wild-type and mutant Rett mice were separated into groups that were fed either an SD in unrestricted or restricted amounts or a ketogenic diet (KetoCal) in restricted amounts for a total of 30 days. The restricted diets were administered to reduce mouse body weight by 20–23% compared to the body weight of each mouse before the initiation of the diet. All mice were subjected to a battery of behavioral tests to determine the influence of the diet on the RTT phenotype. We found that performance in tests of motor behavior and anxiety was significantly worse in male RTT mice compared to wild-type mice and that restriction of either the KD or the SD improved motor behavior and reduced anxiety. We conclude that although both restricted diets increased the tendency of Rett mice to explore a novel environment, the beneficial effects of the KD were due more to calorie restriction than to the composition of the diet. Our findings suggest that calorically restricted diets could be effective in reducing the anxiety and in improving motor behavior in girls with RTT.

Dietary n-3 Polyunsaturated Fatty Acids Enhance Hormone Ablation Therapy in Androgen-Dependent Prostate Cancer

Hormone ablation therapy typically causes regression of prostate cancer and represents an important means of treating this disease, particularly after metastasis. However, hormone therapy inevitably loses its effectiveness as tumors become androgen-independent, and this conversion often leads to death because of subsequent poor responses to other forms of treatment. Because environmental factors, such as diet, have been strongly linked to prostate cancer, we examined the affects of dietary polyunsaturated fatty acids (PUFAs; at 1.5 wt%) on growth of androgen-dependent (CWR22) and androgen-independent (CWR22R) human prostatic cancer xenografts, the acute response of CWR22 tumors to ablation therapy, and their progression to androgen independence. Significant diet-induced changes in tumor n-3 or n-6 PUFA content had no affect on CWR22 or CWR22R tumors growing with or without androgen support, respectively. However, dietary changes that increased tumor eicosapentaenoic acid and linoleic acid content enhanced responses to ablation therapy, measured by cancer cell apoptosis and mitosis. In addition, relapse to androgen-independent growth (measured by renewed increases in tumor volume and serum prostate-specific antigen after ablation) positively correlated with tumor arachidonic acid content. There was no correlation between expression of 15-lipoxygenase isozymes or their products and tumor growth or responses to ablation. In conclusion, dietary n-3 PUFA may enhance the response of prostate cancer to ablation therapy and retard progression to androgen-independent growth by altering tumor PUFA content.

Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.