Rats Fed Control Diet Research Articles

The Effects of 1-Kestose on the Abundance of Inflammation-Related Gene mRNA in Adipose Tissue and the Gut Microbiota Composition in Rats Fed a High-Fat Diet.

Chronic inflammation in adipose tissue is thought to contribute to insulin resistance, which involves the gut microbiota. Our previous studies have demonstrated that ingestion of 1-kestose can alter the gut microbiota composition, increase cecal butyrate levels, and improve insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Additionally, we found that 1-kestose supplementation ameliorated insulin resistance in obese rat models fed a high-fat diet (HFD), although the effects of 1-kestose on the abundance of inflammation-related gene in adipose tissue and gut microbiota composition in these rats were not explored. This study aimed to investigate the impact of 1-kestose on these parameters in HFD-fed rats, compared to OLETF rats. Male Sprague-Dawley rats were divided into two dietary groups, control or HFD, for 19 wk. Each group was further subdivided to receive either tap water or tap water supplemented with 2% (w/v) 1-kestose throughout the study. We evaluated gene expression in adipose tissue, as well as short-chain fatty acids (SCFAs) levels and microbial composition in the cecum contents. 1-Kestose intake restored the increased relative abundance of tumor necrosis factor (Tnf) mRNA in adipose tissue and the reduced level of butyrate in the cecum contents of HFD-fed rats to those observed in control diet-fed rats. Additionally, 1-kestose consumption changed the composition of the gut microbiota, increasing Butyricicoccus spp., decreasing UGC-005 and Streptococcus spp., in the cecum contents of HFD-fed rats. Our findings suggest that 1-kestose supplementation reduces adipose tissue inflammation and increases butyrate levels in the gut of HFD-fed rats, associated with changes in the gut microbiota composition, distinct from those seen in OLETF rats.

Feeding soy protein concentrates with low or high isoflavone decreases liver inflammation by reducing lipopolysaccharide translocation

Lipopolysaccharide (LPS) translocation and inflammation contribute to the increased risk of chronic diseases, including non-alcoholic fatty liver disease (NAFLD), associated with obesity. Previously, we reported that feeding soy protein with high or low (negligible) isoflavone reduces liver steatosis in obese Zucker rats, and the reduced steatosis is accompanied by decreased serum C-reactive protein levels. The current study investigated the effect of feeding soy protein concentrate (SPC) with high or low isoflavone (HIF or LIF) on liver inflammation and LPS translocation in obese Zucker rats. Six-week-old male lean (L, n = 21) and obese (O, n = 21) Zucker rats were fed casein control, SPC-LIF, or SPC-HIF diets for 18 weeks. At the end of 18 weeks, the expression levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), arginase 1 (ARG1), lipopolysaccharide binding protein (LBP), myeloperoxidase (MPO), and sterol regulatory element-binding protein 1 (SREBP-1) were significantly higher in obese rats compared to lean rats. Compared to the casein control diet, both the SPC-LIF and SPC-HIF diets significantly decreased TNF-α, MCP-1, iNOS, and LBP expression in obese rats, which is accompanied by significantly less LPS staining in liver slides from SPC-LIF-and SPC-HIF-fed obese rats compared to the casein control diet-fed obese rats. Taken together, the SPC-LIF and SPC-HIF diets attenuated liver inflammation in obese Zucker rats, likely by decreasing LPS translocation.

Quality evaluation of therapeutic noodle from water yam, millet and wheat composite flour and its effect on lipid profile and blood glucose concentration using albino rats

This study investigated the development and sensory qualities of therapeutic noodle from wheat, pearl millet (Pennisetum glaucum) and water yam composite flour and the effect of feeding the product on lipid profile and blood glucose concentration using alloxan induced albino rats. The therapeutic noodle was produced using different ratios of flour blends of wheat, millet and water yam. Sensory analysis was carried out based on color, taste, aroma, slipperiness and overall acceptability on the basis of a 9-point hedonic scale. The therapeutic noodle samples competed well with the commercial noodle (control) sample as they were all rated above average (>5.0) with noodle sample (WMF5) having a very high score of 8.40 in the overall acceptability. Alloxan induced male albino rats were fed the formulated therapeutic noodle for 21 days and assessed for their resultant body weight, blood glucose concentration and lipid profiles. The total cholesterol levels of all diabetic albino rats fed the formulated therapeutic noodle were reduced to within the normal range of below 200mg/dl ideal for adults after the 21days. The therapeutic noodles also helped to significantly (p < 0.05) reduce the high blood glucose concentration of the diabetic rats to below 200mg/dl while diabetic rats fed control diet (100:0) had the highest concentration of blood glucose (485mg/dl). Due to the high anti-diabetic properties of the therapeutic noodles, it can be recommended as a functional food source for the treatment of overweight, obesity, and diabetes.

Hypercholesterolemia Induced by Cholesterol- or Cystine-Enriched Diets is Characterized by Different Plasma Lipoprotein and Apolipoprotein Concentrations in Rats

This study examined the effects of diet-induced hypercholesterolemia on plasma apolipoprotein (apo) concentrations and hepatic apolipoprotein mRNA levels in rats. Hypercholesterolemia was induced by feeding rats diets containing an excess of either cholesterol or cystine. After cholesterol feeding, plasma apo E and apo B concentrations were lower (-65%, P < 0.001) and greater (+39%, P < 0.01), respectively, compared with control diet-fed rats. After cystine feeding, plasma apo B and apo E concentrations were greater (+46%, P < 0.01 and +75%, P < 0.001, respectively) and plasma apo A-IV concentration was lower (-29%, P < 0.001) than in rats fed control diet. After cholesterol or cystine feeding, a tendency (one-way ANOVA, P = 0.08) for greater apo B mRNA level (+42% and +47%, respectively) was observed compared with control diet-fed rats. No difference emerged between groups for apo E and apo A-I mRNA levels. An opposite effect of cholesterol and cystine feeding was shown for apo A-IV mRNA level, i.e., higher after cholesterol feeding (+47%, P < 0.05) and lower after cystine feeding (-65%, P < 0.01). From this work, it seems that hypercholesterolemia induced by dietary cholesterol or by increased cholesterogenesis in cystine-fed rats is characterized by different plasma lipoprotein and apolipoprotein concentrations and is associated with different apolipoprotein gene expression in the liver.

Alcohol-induced increase in BMP levels promotes fatty liver disease in male prediabetic stage Otsuka Long-Evans Tokushima Fatty rats.

Alcohol consumption exacerbates liver abnormalities in animal models, but whether it increases the severity of liver disease in early diabetic or prediabetic rats is unclear. To investigate the molecular mechanisms underlying alcohol-induced liver steatosis or hepatitis, we used a prediabetic animal model. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Fatty (LETO) rats were pair-fed with an ethanol-containing liquid diet for 6 weeks. Compared with controls, OLETF and LETO rats displayed more pronounced liver steatosis and higher plasma levels of serum glutamic oxaloacetic transaminase(SGOT)andserumglutamatepyruvatetransaminase(SPGT), indicating liver injury. Ethanol-fed LETO (Pd-L-E) rats showed mild liver steatosis and no inflammation compared with ethanol-fed OLETF (Pd-O-E) rats. Although precursor and active SREBP-1 levels in the liver of ethanol-fed OLETF rats significantly increased compared with control diet-fed OLETF rats (Pd-O-C), those of Pd-L-E rats did not. Bone morphogenetic protein (BMP) and TGF-β1 balance in Pd-O-E rats was significantly altered because BMP signaling was upregulated by inducing BMP2, BMP4, BMP7, BMP9, Smad1, and Smad4, whereas TGF-β1, Smad3, and Erk were downregulated. Activation of TGF-β/Smad signaling inhibited BMP2 and BMP9 expression and increased epithelial-mesenchymal transition (EMT) marker levels (Hepcidin, Snail, and Twist) in the liver of LETO rats. Livers of ethanol-fed OLETF rats showed increased levels of vimentin, FSP-1, α-SMA, MMP1, MMP13, and collagen III compared with rats of other groups, whereas EMT marker levels did not change. Thus, BMP exerted anti- and/or pro-fibrotic effects in ethanol-fed rats. Therefore, BMP and TGF-β, two key members of the TGF-β superfamily, play important but diverse roles in liver steatosis in young LETO and OLETF rats.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1was also investigated using a chromatin immunoprecipitation assay. Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300mRNA transcripts. These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

The Influence of Diet and Sex on the Gut Microbiota of Lean and Obese JCR:LA-cp Rats

There is an increased interest in the gut microbiota as it relates to health and obesity. The impact of diet and sex on the gut microbiota in conjunction with obesity also demands extensive systemic investigation. Thus, the influence of sex, diet, and flaxseed supplementation on the gut microbiota was examined in the JCR:LA-cp rat model of genetic obesity. Male and female obese rats were randomized into four groups (n = 8) to receive, for 12 weeks, either (a) control diet (Con), (b) control diet supplemented with 10% ground flaxseed (CFlax), (c) a high-fat, high sucrose (HFHS) diet, or (d) HFHS supplemented with 10% ground flaxseed (HFlax). Male and female JCR:LA-cp lean rats served as genetic controls and received similar dietary interventions. Illumine MiSeq sequencing revealed a richer microbiota in rats fed control diets rather than HFHS diets. Obese female rats had lower alpha-diversity than lean female; however, both sexes of obese and lean JCR rats differed significantly in β-diversity, as their gut microbiota was composed of different abundances of bacterial types. The feeding of an HFHS diet affected the diversity by increasing the phylum Bacteroidetes and reducing bacterial species from phylum Firmicutes. Fecal short-chain fatty acids such as acetate, propionate, and butyrate-producing bacterial species were correspondingly impacted by the HFHS diet. Flax supplementation improved the gut microbiota by decreasing the abundance of Blautia and Eubacterium dolichum. Collectively, our data show that an HFHS diet results in gut microbiota dysbiosis in a sex-dependent manner. Flaxseed supplementation to the diet had a significant impact on gut microbiota diversity under both flax control and HFHS dietary conditions.

Contrasting Effects of Fasting on Liver-Adipose Axis in Alcohol-Associated and Non-alcoholic Fatty Liver.

Background: Fatty liver, a major health problem worldwide, is the earliest pathological change in the progression of alcohol-associated (AFL) and non-alcoholic fatty liver disease (NAFL). Though the causes of AFL and NAFL differ, both share similar histological and some common pathophysiological characteristics. In this study, we sought to examine mechanisms responsible for lipid dynamics in liver and adipose tissue in the setting of AFL and NAFL in response to 48 h of fasting.Methods: Male rats were fed Lieber-DeCarli liquid control or alcohol-containing diet (AFL model), chow or high-fat pellet diet (NAFL model). After 6–8 weeks of feeding, half of the rats from each group were fasted for 48 h while the other half remained on their respective diets. Following sacrifice, blood, adipose, and the liver were collected for analysis.Results: Though rats fed AFL and NAFL diets both showed fatty liver, the physiological mechanisms involved in the development of each was different. Here, we show that increased hepatic de novo fatty acid synthesis, increased uptake of adipose-derived free fatty acids, and impaired triglyceride breakdown contribute to the development of AFL. In the case of NAFL, however, increased dietary fatty acid uptake is the major contributor to hepatic steatosis. Likewise, the response to starvation in the two fatty liver disease models also varied. While there was a decrease in hepatic steatosis after fasting in ethanol-fed rats, the control, chow and high-fat diet-fed rats showed higher levels of hepatic steatosis than pair-fed counterparts. This diverse response was a result of increased adipose lipolysis in all experimental groups except fasted ethanol-fed rats.Conclusion: Even though AFL and NAFL are nearly histologically indistinguishable, the physiological mechanisms that cause hepatic fat accumulation are different as are their responses to starvation.

Content of essential and toxic trace elements in organs of obese Wistar and Zucker leprfa rats receiving quercetin

BackgroundThe levels of a number of essential and toxic trace elements in organs and tissues are affected by the disruptions in body homeostasis caused by obesity. Some of these elements may also be influenced by the consumption of biologically active substances of polyphenolic origin, which possess potent abilities to complex with transition metal ions. AimsThe aim of this study was to determine the content of essential and toxic trace elements in Wistar outbred and hereditary obese Zucker Leprfa (Z) rats consuming a standard balanced diet or hypercaloric diet with excess fat and fructose, supplemented with quercetin or not supplemented. Materials and methodsMale Wistar and Z rats were fed a control AIN-93M-based semi-synthetic diet or a high-fat-high-carbohydrate diet (HFCD, with 30% fat by weight and 20% fructose provided in the drinking water). A portion of the animals in each line and diet group was administered quercetin at 50 mg/kg body weight. Essential trace elements were included in the diets as a high-purity salt mixture. After the termination of feeding on day 63, the livers, kidneys, and brains of the rats were excised and the content of 16 elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, Ni, Al, Cd, As, Pb, V, Cs, and Ag) was measured by inductively coupled plasma mass spectrometry (ICP-MS). ResultsIn the livers of the Z rats, the contents of Co, Zn, Mg, Fe, Se, and V were reduced and the content of Cr was increased compared to that of the Wistar rats. Supplementation with quercetin significantly decreased liver Fe, V, and Se content, which was more noticeable in the Wistar rats than in the Z rats. In kidneys of Z rats consuming control diet, the contents of Co, Cu, and Cs were decreased whereas those of Ni, Al, and Se were increased compared with the contents in the Wistar rats. The same trend was observed with HFCD feeding except for Cs content. Quercetin reduced kidney V content in both rat lines fed both diets, whereas it reduced Se and Cs only in the Z rats fed control diet. In the brains of the Z rats, a large increase was observed in some trace elements including Pb, Cd, Al, Cr, Ni, Fe, and V compared with the levels in the Wistar rat brains. Supplementation of the control diet with quercetin decreased Al and Ni in the brains of the Z rats. ConclusionThere were significant differences in the mineral content of organs between the Wistar and Z rats, with different propensities for obesity. Moreover some of these effects had no straightforward association with decreased feed consumption or hepatic fat accumulation. When introduced into the diets, quercetin affected the content of essential and toxic elements, but with ambiguous physiological significance. Thus, indicators of essential and toxic trace elements deserve to be used in the protocols of preclinical as well as clinical trials of biologically active substances and food supplements.

Relationship between hyperlipidemia and the gut microbiome of rats, characterized using high-throughput sequencing

To determine the effects of a high-fat diet (HFD) on the gut microbiome in rats, to explore the relationship between the intestinal flora and blood lipid profile. Sprague–Dawley rats were fed an HFD for four weeks to induce hyperlipidemia, then 16S rRNA sequencing was used to compare the intestinal flora between hyperlipidemic and control diet-fed rats. The microbiome of rats fed an HFD for four weeks differed from that of control diet-fed rats. Bacterial species that were less abundant were most affected by HFD feeding, among which were many pathogenic species, which became significantly more abundant. Eighteen genera were present in significantly different numbers in hyperlipidemic and control rats, more than half of which have been linked to infection and inflammation, or energy intake and obesity. The results indicated a type of stress response of the flora to a high-fat environment. In addition, the age of the rats tended to influence the gut microbial composition. These findings suggest that HFD may induce hyperlipidemia by affecting the gut microbial composition. Changes in the abundance of pro-inflammatory and pathogenic bacteria, and those that influence energy intake and obesity, may be important mediators of this.

High fat diet in female Dahl salt sensitive rats causes non‐neurogenic hypertension and minimal renal injury

There is evidence in human subjects and animal models that obesity‐associated hypertension has a smaller neurogenic component in females than males. In addition, females typically show less severe hypertension, immune cell infiltration in the kidney and renal injury than males. The mechanistic links between these phenomena, however, are not clear. We previously found that feeding Dahl salt‐sensitive (SS) rats a high fat diet (HFD) from weaning through 6 months caused equivalent hypertension in male and female animals when compared to SS rats fed control diet (CD). Yet female rats exhibited significantly less renal injury than males. Therefore, in the current study, we quantified the contribution of neurogenic and other blood pressure control mechanisms to HFD associated hypertension in female Dahl SS rats. We also reexamined the impact of long‐term hypertension on histological indices of renal injury in these rats. After 24 weeks on diet feeding starting at weaning, all rats were hypertensive (via telemetric measurements), but HFD females (60% kcal from fat, 0.3% NaCl n=10) had significantly higher mean arterial pressure (MAP) than females fed the CD (10% kcal from fat, 0.3% NaCl, n=7) (MAP 165±4mmHg vs 141±3mmHg, p&lt;0.05). Thereafter, all rats were treated sequentially with prazosin (150mg/L, 5days), atenolol (500mg/L, 7days), prazosin+atenolol (7days), trichlormethiazide (250mg/L, 5 days), and enalapril (250mg/L, 7 days) via the drinking water. Each treatment was followed by washout for 7 days. We found that MAP was moderately and similarly reduced by treatment with prazosin (−14±1mmHg vs −17±1mmHg), atenolol (−31±2mmHg vs −27±2mmHg), prazosin+atenolol (−26±1mmHg vs −29±2mmHg) and trichlormethiazide (−16±2mmHg vs −17±2mmHg) in CD versus HFD fed rats. Enalapril treatment caused more reduction of blood pressure than any other treatment, but the decreases of MAP were similar in CD and HFD fed rats (−36±5 mmHg vs −49±9 mmHg) (P&gt;0.05). Renal histological alterations and immune cell infiltration were determined at 34–36 weeks on diet feeding. Overall CD and HFD fed rats showed similar, and low grade, renal injury scores and renal T‐cell (CD3+) and macrophage (CD68+) infiltration. However, a few HFD rats did show severe renal injury and inflammation, and we noted that in each of these animals MAP levels were consistently &gt; 170 mmHg. Splenic immune cell analysis by flow‐cytometry did not support HFD associated alternations in immune cell population/distributions (CD4+, CD8+, NK cells, B cells et al). Taken together the data indicate that in the Dahl SS rats, HFD causes hypertension via mechanisms that do not include increased neurogenic support of blood pressure or volume expansion. Increased activity of the renin‐angiotensin system may play a part but is not wholly responsible. Female Dahl SS rats on HFD are strongly protected against hypertension‐associated renal inflammation and injury, but the mechanisms responsible remain unknown.Support or Funding InformationNHLBI 2P01HL070687 for G.D.F, J.J.G. and H.X

High-fat diet-induced obesity causes an inflammatory microenvironment in the kidneys of aging Long-Evans rats

BackgroundObesity is a risk factor for chronic kidney disease (CKD). While the exact mechanisms remain unclear, inflammation may be a consequence of obesity that directly impacts the kidneys. The aim of this study was to examine the inflammatory status of the kidneys and potential ongoing renal damage, i.e., tubular damage and fibrosis after long-term obesity maintained through persistent consumption of a high-fat diet (HFD).ResultsTwenty-four-week-old male Long-Evans (LEV) rats were continuously fed a control diet (CD) or HFD for 51 weeks. The mean body weight was higher in HFD-fed rats than in control diet-fed rats and markedly elevated during the last 24 weeks. Blood analyses revealed no substantial alterations in renal functional parameters by HFD consumption but a substantial increase in creatine kinase, a muscle loss marker. Magnetic resonance imaging (MRI) was utilized to quantify rat quadriceps muscle mass. The data showed that HFD-induced obesity in LEV rats was accompanied by minor decreases in muscle mass and strength at 75 weeks of age. Rat kidney inflammatory status was evaluated using histological and immunohistological techniques. The number of foci with immune cell infiltrates and infiltrating monocytes/macrophages was significantly increased in HFD-fed rat kidneys at week 75. Renal fibrosis parameters, including glomerulosclerosis and tubular damage, were also markedly increased in renal tissues from HFD-fed rats compared to the controls. The significant increase in tubular protein casts in HFD-fed rat tissues indicated that renal function was already disturbed. Rat kidney inflammatory status was further evaluated using the simultaneous profiling of twenty-two inflammatory markers in kidney tissue extracts. Consistently, MCP-1 and eotaxin (CCL11) levels were elevated in obese LEV rat kidneys.ConclusionsCompared to CD-fed rats, HFD-fed obese LEV rats show significant damage of renal structures with aging. These subtle changes may sensitize the kidneys to the development of progressive CKD.

Vitamin D Replacement Mitigates Menopause-Associated Dyslipidaemia and Atherogenic Indices in Ovariectomized Rats; A Biochemical Study.

Dyslipidaemia is highly prevalent among postmenopausal women and its management represents a keystone in the prevention of the worldwide increase in cardiovascular morbidity and mortality. Therapy choices for menopause-associated dyslipidaemia are limited and a matter of debate. So, it becomes prudent to search for natural safe alternatives. Vitamin D (VD) has been acknowledged as an essential factor in cardiovascular health. Thus, we aimed to illustrate the impact of different VD status on dyslipidaemia and atherogenic indices. 5 groups of rats were conducted; SHAM group fed control diet, ovariectomized rats fed control diet (OVX), ovariectomized rats fed VD-sufficient-high fat diet (HFD) (1 000 IU/ kg diet), ovariectomized rats fed VD-deficient-HFD (25 IU/ kg diet), and ovariectomized rats fed VD-replete-HFD (10 000 IU/ kg diet) for 16 weeks. Dyslipidaemia with an increased atherogenic index of plasma, atherosclerosis coefficient, cardiac risk ratio, and aortic total cholesterol accumulation in addition to reduced serum 25-hydroxy-VD levels was observed in the OVX and VD-sufficient HFD versus SHAM. These findings were aggravated by VD-deficient-HFD while reversed by VD-replete-HFD. The VD-mediated abundance of aortic ATP-binding cassette transporter A1 (ABCA1) expression, reduced activity of the inflammatory Jun N-terminal kinases (JNK), and downregulation of aortic cluster of differentiation-36 (CD36) receptors expression together with increased serum total antioxidant capacity and reduced serum malondialdehyde were among the supposed mechanisms. Our study sheds light on alarming levels of VD deficiency among ovariectomized rats. VD repletion improved the menopause-associated dyslipidaemia and atherogenic indices through hypolipidemic, antioxidant, and anti-inflammatory effects.

The anomeric nature of glucose and its implications on its analyses and the influence of diet: are routine glycaemia measurements reliable enough?

Background: Glucose is the main inter-organ energy supplying metabolite in humans and other vertebrates. In clinical analyses, its measurement is probably the most performed and used for diagnostic, monitoring and control of the physiological status. However, glucose chemical structure, specially its anomeric forms (alpha/beta), may deeply interfere in their own analyses, often resulting in misleading results. Methods: These effects on glucose estimation were studied by using a common glucose oxidase/peroxidase based method, in the presence or absence of added mutarotase, which speeds up the alpha/beta conversion rate. Glucose concentrations were measured in pure standards and plasma samples from control and cafeteria diet-fed rats. Results: The addition of mutarotase resulted in higher glucose readings, independently of glucose concentration and its initial anomeric proportions in the sample. In the absence of mutarotase, cafeteria-fed rats had higher glucose levels than controls, but the differences disappeared in its presence, because under experimental conditions, a proportion of the alpha-anomer was not isomerized and thus was not measured. Conclusions: Diet altered the proportion of anomers, suggesting that glucose usage by physiological processes affects the anomers’ ratio and may have an important metabolic meaning, which deserves a detailed study in addition to the need to correct the methods in use to obtain real “total glucose” readings. J Endocrinol Metab. 2019;9(3):63-70 doi: https://doi.org/10.14740/jem555

Association of high-fat diet with neuroinflammation, anxiety-like defensive behavioral responses, and altered thermoregulatory responses in male rats

Overweight and obesity are a worldwide pandemic affecting billions of people. These conditions have been associated with a chronic low-grade inflammatory state that is recognized as a risk factor for a range of somatic diseases as well as neurodevelopmental disorders, anxiety disorders, trauma- and stressor-related disorders, and affective disorders. We previously reported that the ingestion of a high-fat diet (HFD; 45% fat kcal/g) for nine weeks was capable of inducing obesity in rats in association with increased reactivity to stress and increased anxiety-related defensive behavior. In this study, we conducted a nine-week diet protocol to induce obesity in rats, followed by investigation of anxiety-related defensive behavioral responses using the elevated T-maze (ETM), numbers of FOS-immunoreactive cells after exposure of rats to the avoidance or escape task of the ETM, and neuroinflammatory cytokine expression in hypothalamic and amygdaloid nuclei. In addition, we investigated stress-induced cutaneous thermoregulatory responses during exposure to an open-field (OF). Here we demonstrated that nine weeks of HFD intake induced obesity, in association with increased abdominal fat pad weight, increased anxiety-related defensive behavioral responses, and increased proinflammatory cytokines in hypothalamic and amygdaloid nuclei. In addition, HFD exposure altered avoidance- or escape task-induced FOS-immunoreactivity within brain structures involved in control of neuroendocrine, autonomic, and behavioral responses to aversive stimuli, including the basolateral amygdala (BLA) and dorsomedial (DMH), paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei. Furthermore, rats exposed to HFD, relative to control diet-fed rats, responded with increased tail skin temperature at baseline and throughout exposure to an open-field apparatus. These data are consistent with the hypothesis that HFD induces neuroinflammation, alters excitability of brain nuclei controlling neuroendocrine, autonomic, and behavioral responses to stressful stimuli, and enhances stress reactivity and anxiety-like defensive behavioral responses.

Identification of Suitable Reference Genes for Quantitative Gene Expression Analysis in Innervated and Denervated Adipose Tissue from Cafeteria Diet-Fed Rats.

Our previous studies show that cafeteria diet increases body adiposity, plasma insulin levels, and sympathetic activity to brown adipose tissue (BAT) and white adipose tissue (WAT) of Wistar rats, leading to rapid and progressive changes in the metabolic profile. The identification of suitable reference genes that are not affected by the experimental conditions is a critical step in accurate normalization of the reverse transcription quantitative real-time PCR (qRT-PCR), a commonly used assay to elucidate changes in the gene expression profile. In the present study, the effects of the cafeteria diet and sympathetic innervation on the gene expression of adrenoceptor beta 3 (Adrb3) from BAT and WAT were assessed using one of the most stable and one of the least stable genes as normalizers. Rats were fed the cafeteria diet and on the 17th day, interscapular BAT or retroperitoneal WAT was denervated and, 7 days after surgery, the contralateral innervated tissue was used as control. Ten reference genes were evaluated (18S, B2m, Actb, CypA, Gapdh, Hprt1, Rpl32, Tbp, Ubc, and Ywhaz) and ranked according to their stability using the following algorithms: geNorm, NormFinder, BestKeeper, and comparative delta threshold cycle (ΔC t ) method. According to the algorithms employed, the normalization of Adrb3 expression by the least stable genes produced opposite results compared with the most stable genes and literature data. In cafeteria and control diet-fed rats, the three most stable genes were Hprt1, Tbp, and Rpl32 for interscapular BAT and Tbp, B2m, and Hprt1 for retroperitoneal WAT, while the least stable genes were 18S, Actb, and Gapdh for both tissues.

The Anomeric Nature of Glucose and Its Implications on Its Analyses and the Influence of Diet: Are Routine Glycaemia Measurements Reliable Enough?

Background: Glucose is the main inter-organ energy supplying metabolite in humans and other vertebrates. In clinical analyses, its measurement is probably the most performed and used for diagnostic, monitoring and control of the physiological status. However, glucose chemical structure, specially its anomeric forms (alpha/beta), may deeply interfere in their own analyses, often resulting in misleading results. Methods: These effects on glucose estimation were studied by using a common glucose oxidase/peroxidase based method, in the presence or absence of added mutarotase, which speeds up the alpha/beta conversion rate. Glucose concentrations were measured in pure standards and plasma samples from control and cafeteria diet-fed rats. Results: The addition of mutarotase resulted in higher glucose readings, independently of glucose concentration and its initial anomeric proportions in the sample. In the absence of mutarotase, cafeteria-fed rats had higher glucose levels than controls, but the differences disappeared in its presence, because under experimental conditions, a proportion of the alpha-anomer was not isomerized and thus was not measured. Conclusions: Diet altered the proportion of anomers, suggesting that glucose usage by physiological processes affects the anomers’ ratio and may have an important metabolic meaning, which deserves a detailed study in addition to the need to correct the methods in use to obtain real “total glucose” readings. J Endocrinol Metab. 2019;9(3):63-70 doi: https://doi.org/10.14740/jem555

Soy-Induced Fecal Metabolome Changes in Ovariectomized and Intact Female Rats: Relationship with Cardiometabolic Health

Phytoestrogens are plant-derived compounds found in a variety of foods, most notably, soy. These compounds have been shown to improve immuno-metabolic health, yet mechanisms remain uncertain. We demonstrated previously that dietary phytoestrogen-rich soy (SOY) rescued metabolic dysfunction/inflammation following ovariectomy (OVX) in female rats; we also noted remarkable shifts in gut microbiota in SOY vs control diet-fed rats. Importantly, specific bacteria that significantly increased in those fed the SOY correlated positively with several favorable host metabolic parameters. One mechanism by which gut microbes might lead to such host effects is through production of bacterial metabolites. To test this possibility, we utilized non-targeted gas chromatography–mass spectrometry (GCMS) to assess the fecal metabolome in those previously studied animals. Partial least square discriminant analysis (PLSDA) revealed clear separation of fecal metabolomes based on diet and ovarian state. In particular, SOY-fed animals had greater fecal concentrations of the beneficial bacterial metabolite, S-equol, which was positively associated with several of the bacteria upregulated in the SOY group. S-equol was inversely correlated with important indicators of metabolic dysfunction and inflammation, suggesting that this metabolite might be a key mediator between SOY and gut microbiome-positive host health outcomes.

Abstract 1125: Developmental pluripotency associated 4 oncogene interacts with Wnt/β-catenin signaling and stem cells regulatory genes to control pituitary tumor cells invasiveness

Abstract The developmental pluripotency associated 4 (DPPA4) gene has an important role in self-renewal and pluripotency in embryonic stem cells. It is re-expressed in several malignant tumors and is identified as a new pluripotency-related oncogene. We have recently identified elevated expression of DPPA4 in aggressive pituitary tumors induced by estrogen in fetal alcohol exposed rats. In this study, we determined the role of DPPA4 in pituitary tumor growth and invasiveness by employing CRISPR/Cas9 knock-down of DPPA4 and conducting cell proliferation, in vitro soft agar anchorage-independent growth assays, and in vivo tumor formation assay using cell spheres from pituitary tumors of alcohol-fed and control-fed animals. Pituitary tumor cells of fetal alcohol-fed rats and control diet-fed rats were grown in ultra-low attachment plate to develop pituispheres. In vitro and in vivo studies showed pituispheres of alcohol-fed rats had higher cell proliferation rate, increased colony formation in soft agar, and higher tumor development in mice xenografts compared to those in pituispheres of alcohol-fed rats. Pituispheres of alcohol-fed rats also had increased mRNA levels of cell growth, cell stemness and EMT regulatory genes. CRISPR-Cas9 knockdown of DPPA4 in pituispheres reduced the tumor cell ability to proliferate, migrate and form colony in soft agar and grow in mice xenografts. Pituispheres of alcohol-fed rats also showed increased protein levels of Wnt/beta-catenin signaling molecules (Wnt 3, beta-catenin, LPR5, GSK3), stemness regulatory factors (KLF4, Sox2, Nanog) and EMT factors as compared to those in control-fed pituispheres. DPPA4 knockdown reduced the levels of Wnt signaling and stem cells regulatory proteins and EMT factors in alcohol-fed rat pituispheres. These data suggest that DPPA4 is expressed in aggressive pituitary tumors and regulates the growth and invasion of these tumors via activation of Wnt/beta-catenin signaling and stem cells regulatory genes. (This work is supported by a National Institute of Health grant R01 AA11591). Citation Format: Dipak K. Sarkar, Shaima Jabbar, Omkaram Gangisetty. Developmental pluripotency associated 4 oncogene interacts with Wnt/β-catenin signaling and stem cells regulatory genes to control pituitary tumor cells invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1125.

Increase in liver cytosolic lipases activities and VLDL-TAG secretion rate do not prevent the non-alcoholic fatty liver disease in cafeteria diet-fed rats

We have previously shown that the cafeteria diet increases body fat mass, plasma triacylglycerol (TAG) and insulin levels, glucose uptake by white and brown adipose tissues, as well as the sympathetic activity to both adipose tissues in Wistar rats. The metabolic pathways responsible for the development of non-alcoholic fatty liver disease (NAFLD) were examined in cafeteria diet-fed rats. After 3 weeks offering cafeteria diet, we evaluated: (i) activity of the sympathetic nervous system by norepinephrine turnover rates; (ii) de novo fatty acid synthesis in vivo using 3H2O; (iii) secretion of very low density lipoprotein (VLDL)-TAG secretion measuring serum TAG levels after administration of lipase lipoprotein inhibitor, (iv) liver cytosolic lipases activities and (v) liver mRNA expression of enzymes involved in lipids secretion and oxidation by RT-PCR. The cafeteria diet induced an increase in TAG (120%) and cholesterol (30%) liver contents. Cafeteria diet did not change the sympathetic nervous system activity to liver, but induced a marked increase in the lipogenesis (approximately four-fold) and significant increase in cytosolic lipases activities (46%) and VLDL-TAG secretion (22%) compared to control diet-fed rats. The cafeteria diet also increased the microsomal triglyceride transfer protein (30%) and carnitine palmitoyltransferase I (130%) mRNA expression but decreased the apolipoprotein B100 (26%) mRNA expression. Our findings demonstrate that the increase in the cytosolic lipases activities and VLDL-TAG secretion rates were not able to compensate for the increased lipogenesis rates induced by the cafeteria diet, resulting in NAFLD.