Effects of a High-Fat Diet and Strain on Hypothalamic Gene Expression in Rats

Objective: This study was designed to investigate whether dietary fat and genetic background might differentially alter the expression of hypothalamic genes involved in food intake. Research Methods and Procedures: Three-month-old Osborne-Mendel (OM) and S5B/Pl rats were fed either a high-fat or a low-fat diet for 14 days. mRNA for neuropeptide Y (NPY), corticotrophin-releasing hormone, NPY Y-1 receptor and Y-5 receptor, and serotonin 2c (5-HT2c) receptors were measured using Northern blotting or ribonuclease protection assays. Results: OM rats showed an increased expression of NPY and corticotrophin-releasing hormone compared with S5B/Pl rats. The expression of NPY-Y1 and -Y5 receptor mRNA was significantly higher in the hypothalamus of OM rats compared with S5B/Pl rats. The expression of 5HT-2c receptor mRNA was significantly reduced in both strains of rats eating a high-fat diet when compared with the animals eating the low-fat diet. Discussion: These data suggest that over activity of the NPY system may contribute to the development of obesity in OM rats and that expression of the 5HT-2c receptor gene may be modulated by dietary fat.     

姜黄素对抗高脂饮食诱导SD大鼠血脂水平升高及改善血管内皮依赖性舒张功能的研究

目的：观察高脂饮食对SD大鼠血脂水平及胸主动脉环舒张功能的影响及姜黄素对以上改变的影响。方法：健康SD大鼠30只,分高脂饮食组（10只）、正常饮食对照组、高脂饮食＋姜黄素组（10只）,大鼠行适应性饲养1周后分别给予高脂饮食及正常饮食;于实验开始时、10周及实验结束前测各组大鼠体重,20周后取血测定血清血脂浓度,取胸主动脉测定血管环舒张功能。结果：①高脂饮食喂养的大鼠体重明显高于其他各组大鼠,姜黄素可明显对抗高脂饮食导致的体重升高。②与对照组比较,高脂饮食组TC、TG、LDL-C明显升高（P〈0.01和P〈0.05）;③与正常对照组及姜黄素对照组比较,高脂饮食组胸主动脉环的内皮依赖性舒张功能显著减弱（P〈0.05）。结论：①SD大鼠给予高脂饮食后使大鼠血脂水平明显升高,胸主动脉环内皮依赖性舒张功能显著减弱。②姜黄素具有防治高脂饮食导致的血脂升高及改善高脂饮食导致的血管内皮依赖性的舒张功能减退。     

高脂饮食对小鼠脂质代谢和leptin基因表达水平的影响

目的建立高脂饮食诱导小鼠肥胖模型,分析高脂饲料对小鼠脂质代谢和leptin基因表达水平的影响。方法用高脂饲料饲喂小鼠,每周定时称重和断尾采血一次,分别测定血清中血糖、胆固醇、甘油三酯、胰岛素和leptin的浓度;5周后,分离、称重小鼠体脂并提取腹部脂肪组织RNA,半定量RT-PCR分析leptin基因表达水平。结果从第2周开始,实验组小鼠体重明显高于对照组小鼠,4周后,体重差异显著（P〈0．05）;血清中血糖、胆固醇、甘油三酯、胰岛素和leptin的含量随体重增加明显增高,4周后,差异显著（P〈0．05）;实验组体脂含量明显高于对照组（P〈0．05）,半定量RT-PCR分析表明,肥胖小鼠脂肪组织leptin基因表达水平高于对照组（P〈0．05）。结论高脂饮食诱导可建立小鼠肥胖模型,并能够引起高胰岛素和高leptin血症,为进一步研究肥胖的发病机制奠定基础。     

Differential Effects of Undernourishment and Nutritional Rehabilitation on Serum Leptin Levels in Male and Female Rats

Leptin, a peptide hormone, is secreted by adipose tissue and is crucial to the regulation of feeding behaviour. The present study has shown that both male and female rats which have been undernourished since day six of gestation, show significantly decreased serum leptin levels on postnatal day 12; but when undernourishment continues into adulthood, only males continue to show decreased leptin levels. If nutritional rehabilitation is implemented early enough in males, serum leptin levels recover and nearly reach levels found in control adult males. Undernutrition also has a long term effect on body weight in both sexes, but nutritional rehabilitation leads to some degree of body weight recovery varying with sex and the age at which rehabilitation was implemented. Undernutrition seems to affect different developmental processes in males than in females, with males being more vulnerable than females in so far as long-term effects on serum leptin levels.     

高脂喂养大鼠肝脏的NF-κBp65表达与胰岛素抵抗的相关性

目的探讨高脂饲料喂养大鼠肝脏NF-κBp65蛋白的表达与胰岛素抵抗的关系。方法采用高脂饲料喂养建立胰岛素抵抗大鼠模型,并用正常血糖-高血浆胰岛素钳夹技术评估。应用Western blotting方法检测大鼠肝脏中NF-κBp65蛋白的表达。结果①高脂饲料组大鼠的葡萄糖输注率明显低于基础饲料组[GIR60~120（0.76±0.28vs4.26±0.70）mg/（kg.min）,P〈0.01]。②高脂饲料组大鼠肝脏NF-κBp65蛋白的表达明显高于基础饲料组（A值118.48±1.45vs68.13±4.84,P〈0.01）。③高脂胰岛素抵抗大鼠肝脏NF-κBp65蛋白表达与GIR60-120（r=-0.993,P=0.000）和ISI（r=-0.773,P=0.009）负相关。结论高脂诱导的胰岛素抵抗大鼠肝脏NF-κB的激活可能是产生肝脏和全身胰岛素抵抗的根源。     

Long-Term Consumption of High-Fat Diet in Rats: Effects on Microglial and Astrocytic Morphology and Neuronal Nitric Oxide Synthase Expression

Obesity in humans is associated with cognitive decline and elevated risk of neurodegenerative diseases of old age. Variations of high-fat diet are often used to model these effects in animal studies. However, we previously reported improvements in markers of memory and learning, as well as larger hippocampi and higher metabolite concentrations in Wistar rats fed high-fat, high-carbohydrate diet (HFCD, 60 % energy from fat, 28 % from carbohydrates) for 1 year; this diet leads to mild ketonemia (Setkowicz et al. in PLoS One 10:e0139987, 2015). In the present study, we follow up on this cohort to assess glial morphology and expression of markers related to gliosis. Twenty-five male Wistar rats were kept on HFCD and twenty-five on normal chow. At 12 months of age, the animals were sacrificed and processed for immunohistochemical staining for astrocytic (glial fibrillary acidic protein), microglial (Iba1), and neuronal (neuronal nitric oxide synthetase, nNOS) markers in the hippocampus. We have found changes in immunopositive area fraction and cellular complexity, as studied by a simplified Sholl procedure. To our knowledge, this study is the first to apply this methodology to the study of glial cells in HFCD animals. GFAP and Iba1 immunoreactive area fraction in the hippocampi of HFCD-fed rats were decreased, while the mean number of intersections (an indirect measure of cell complexity) was decreased in GFAP-positive astrocytes, but not in Iba1-expressing microglia. At the same time, nNOS expression was lowered after HFCD in both the cortex and the hippocampus.     

Microarray Analysis of Gene Expression in the Supraoptic Nucleus of Normoosmotic and Hypoosmotic Rats

1. Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin (OT) gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the OT and VP magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), in order to detect novel genes in this system that might be involved in osmoregulation in the MCNs.2. For this purpose, we used Affymetrix oligonucleotide arrays to analyze the expression of specific genes in laser microdissected rat SONs, and their changes in expression during chronic hypoosmolality. We identified over 40 genes that had three-fold or more greater expression in the SON versus total hypothalamus, and that also changed more than two fold in expression as a result of the chronic hypoosmolar treatment. These genes contained both novel as well as genes previously known to be present in the SON. All of the raw data for the genes that are expressed in the SON and altered by hypoosmolality can be found on the following NINDS website URL address: http://data.ninds.nih.gov/Gainer/Publications     

MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.     

Possible Role of Triacylglycerol-Rich Lipoproteins in the Down-Regulation of Adipose Obese mRNA Expression in Rats Re-Fed a High-Fat Diet

Summary The large amount of absorbed dietary lipid after feeding a high-fat diet is mainly transported as triacylglycerol (TG)-rich lipoproteins (TRL) in the post-prandial blood and is subsequently distributed to peripheral tissues including adipose and muscle tissues. An in vivo and an in vitro study were conducted to investigate the possible role of post-prandial TRL after high fat feeding in the regulation of obese (ob) gene expression. Adult male Wistar rats were fasted for 48 h and re-fed either a fat-free/high-carbohydrate diet or a high-fat diet for 2, 4, or 8 h and plasma glucose, insulin, TG, and leptin as well as ob mRNA expression in epididymal fat pads were examined. Rats re-fed the high-fat diet had significantly higher plasma TG (p<0.05) and lower plasma leptin and adipose ob mRNA (p<0.05) than those fed the fat-free/high-carbohydrate diet; however, plasma glucose and insulin concentrations were not significantly different between the two groups. Plasma lipid analysis found large amount of TRL in rats fed with high-fat diet; however, only very small amount of the TRL was found in rats fed with fat-free/high-carbohydrate diet. We speculated that TRL might involve in regulation of ob gene expression. To further examine the regulation of TRL on ob mRNA expression, differentiated 3T3-L1 adipocytes were treated with TRL collected from rats fed 5 ml soybean oil by gastric intubations. TRL down-regulated ob mRNA not only in a dose and time dependent manner but also in the presence of insulin in 3T3-L1 adipocytes. These results suggest a possible role of TRL in the down-regulation of adipose ob mRNA expression and may account, at least in part, for the previous observations that short-term high fat feeding resulted in lower plasma leptin.     

The Effects of Metformin and Diet on Plasma Testosterone and Leptin Levels in Obese Men

Objective: The aim of this study was to investigate the effects of combined hypocaloric diet and metformin on circulating testosterone and leptin levels in obese men with or without type 2 diabetes. Research Methods and Procedures: Twenty obese men with type 2 diabetes (mean body mass index [BMI]: 35.5 ± 1.1 kg/m²) and 20 nondiabetic obese men were enrolled in the study. We measured serum follicle‐stimulating hormone, luteinizing hormone (LH), total testosterone (TT), free testosterone (FT), sex‐hormone‐binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), and plasma leptin levels before and 3 months after metformin treatment. Both groups were placed on a hypocaloric diet and 850 mg of metformin taken orally twice daily for 3 months. Results: Metformin and hypocaloric diets led to decreases in BMI and waist and hip circumferences in both groups. A significant decrease in TT levels in the diabetic group and FT levels in the control group was found, whereas follicle‐stimulating hormone, LH, and DHEAS levels were not changed significantly. A significant increase in SHBG levels was observed in the control group but not in the patient group. Leptin levels also decreased after treatment in both groups. Decreased testosterone levels were not correlated to changes in waist and hip circumference, waist‐to‐hip ratio, BMI, and levels of fasting blood glucose, leptin, SHBG, or DHEAS in the diabetic group. However, a decrease in FT was correlated to changes in the levels of SHBG (r = ?0.71, p = 0.001) and LH (r = 0.80, p = 0.001) but not to other parameters. Discussion: We conclude that metformin treatment combined with a hypocaloric diet leads to reduced FT levels in obese nondiabetic men and to reduced TT levels in obese men with type 2 diabetes. Increased SHBG levels may account for the decrease in FT levels in the former group.     

Impact of High-Fat Diet and Early Stress on Depressive-Like Behavior and Hippocampal Plasticity in Adult Male Rats

During development, the brain goes through fundamental processes, including organization of neural networks and plasticity. Environmental interventions may change initial brain programming, leading to long-lasting effects and altering the susceptibility to psychopathologies, including depression disorder. It is known that depression is a psychiatric disorder with a high prevalence worldwide, including high rates among adolescents. In this study, we evaluated whether social isolation in the prepubertal period and chronic use of high-fat diet (HFD) may induce depressive-like behavior in male adult rats. We also investigated hippocampal plasticity markers and neurotransmitter systems. We found both social isolation and HFD induced a depressive-like behavior in the forced swimming task. Moreover, chronic HFD reduced synaptic markers in hippocampus, demonstrated by reductions in βIII-tubulin (neuronal marker), PSD-95, SNAP-25, and neurotrophin-3. The HFD group also presented decreased glutamatergic and GABAergic receptors subunits. On the other hand, stress affected hippocampal brain-derived neurotrophic factor (BDNF) signaling pathways, and increased expression of subunit of the NMDA receptor (NR2A). Both factors (stress and diet) decreased GR in the hippocampus without affecting plasma corticosterone at basal levels. Interactions between early stress and HFD access were observed only in the BNDF receptor (tropomyosin receptor kinase B; TrkB) and synaptophysin. In summary, these findings showed that a brief social isolation and chronic HFD, during a sensitive developmental period, cause depressive-like behavior in adulthood. The mechanisms underlying these behavioral effects may involve changes in the levels of synaptic proteins in hippocampus: HFD consumption appears to affect synaptic markers, while social isolation affected BDNF signaling more significantly.     

SHR与WKY大鼠肾脏组织基因表达差异分析

为了观察SHR与WKY大鼠肾脏组织的基因表达的差异,用T11G、AP6引物对12周龄的SHR与WKY大鼠肾脏mRNA进行了差异显示,得到了一条在SHR大鼠肾脏组织中高表达的差异显示条带,进一步分析证明这一差异显示条带含有170 bp,与GenBank 收录的已知的核苷酸序列的同源性低于80%,可能为未知基因的cDNA片段,并定位于SHR大鼠的肾小管上皮细胞内.     

Differential Responses of Hepatic Endoplasmic Reticulum Stress and Inflammation in Diet-Induced Obese Rats with High-Fat Diet Rich in Lard Oil or Soybean Oil

Scopes

To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding.

Methods and Results

Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively.

Conclusions

Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.     

Modulation in Wistar Rats of Blood Corticosterone Compartmentation by Sex and a Cafeteria Diet

In the metabolic syndrome, glucocorticoid activity is increased, but circulating levels show little change. Most of blood glucocorticoids are bound to corticosteroid-binding globulin (CBG), which liver expression and circulating levels are higher in females than in males. Since blood hormones are also bound to blood cells, and the size of this compartment is considerable for androgens and estrogens, we analyzed whether sex or eating a cafeteria diet altered the compartmentation of corticosterone in rat blood. The main corticosterone compartment in rat blood is that specifically bound to plasma proteins, with smaller compartments bound to blood cells or free. Cafeteria diet increased the expression of liver CBG gene, binding plasma capacity and the proportion of blood cell-bound corticosterone. There were marked sex differences in blood corticosterone compartmentation in rats, which were unrelated to testosterone. The use of a monoclonal antibody ELISA and a polyclonal Western blot for plasma CBG compared with both specific plasma binding of corticosterone and CBG gene expression suggested the existence of different forms of CBG, with varying affinities for corticosterone in males and females, since ELISA data showed higher plasma CBG for males, but binding and Western blot analyses (plus liver gene expression) and higher physiological effectiveness for females. Good cross- reactivity to the antigen for polyclonal CBG antibody suggests that in all cases we were measuring CBG.The different immunoreactivity and binding affinity may help explain the marked sex-related differences in plasma hormone binding as sex-linked different proportions of CBG forms.     

The Effects of Chromium Complex and Level on Glucose Metabolism and Memory Acquisition in Rats Fed High-Fat Diet

Conditions in which glucose metabolism is impaired due to insulin resistance are associated with memory impairment. It was hypothesized that supplemental chromium (Cr) may alleviate insulin resistance in type 2 diabetes and consequently improve memory acquisition, depending upon its source and level. In a complete randomized design experiment, male Wistar rats (n=60; weighing 200-220 g) were fed either normal (8%, normal diet (ND)) or high-fat (40%, high-fat diet (HFD)) diet and supplemented with Cr as either chromium-glycinate (CrGly) or chromium-acetate (CrAc) at doses of 0, 40, or 80 μg/kg body weight (BW) via drinking water from 8 to 20 weeks of age. Feeding HFD induced type 2 diabetes, as reflected by greater glucose/insulin ratio (2.98 vs. 2.74) comparing to feeding ND. Moreover, HFD rats had greater BW (314 vs. 279 g) and less serum (53 vs. 68 μg/L) and brain (14 vs. 24 ng/g) Cr concentrations than ND rats. High-fat diet caused a 32% reduction in expressions of glucose transporters 1 and 3 (GLUTs) in brain tissue and a 27% reduction in mean percentage time spent in the target quadrant and a 38% increase in spatial memory acquisition phase (SMAP) compared with ND. Compared with supplemental Cr as CrAc, CrGly was more effective to ameliorate response variables (i.e., restoration of tissue Cr concentration, enhancement of cerebral GLUTs expressions, and reduction of the glucose/insulin ratio and SMAP) in a dose-response manner, especially in rats fed HFD. Supplemental Cr as CrGly may have therapeutic potential to enhance insulin action and alleviate memory acquisition in a dose-dependent manner, through restoring tissue Cr reserve and enhancing cerebral GLUTs expressions.     

Factors that Affect Pancreatic Islet Cell Autophagy in Adult Rats: Evaluation of a Calorie-Restricted Diet and a High-Fat Diet

Aging may be a risk factor for type 2 diabetes in the elderly. Dietary intervention can affect glucose tolerance in adults, which may be due to body composition and islet cell autophagy. The aim of this study was to determine the effects of various dietary interventions on islet cell autophagy. Pancreatic tissue and blood samples were collected from Sprague Dawley rats (14–16 months old, n = 15 for each group) that received a normal diet (ND), a high-fat diet (HFD), or a calorie-restricted diet (CRD). The body weight (BW), visceral fat, serum lipid levels, fasting serum glucose, insulin levels, and β/α cell area were determined in 14-16-(0-w), 16-18-(8-w), and 18-20(16-w)-month-old rats. Pancreatic islet autophagy (LC3B and LAMP2), AP (Acid Phosphatase) and apoptosis (apoptosis index, AI (TUNEL assay) and cleaved caspase-3) were detected using immunohistochemistry, ELISA and western blot. At 16 weeks, the expressions of LC3B, LAMP2 and AP markedly increased in both the HFD (P<0.01) and CRD (P<0.05) groups; however, an increase in the AI (P<0.05), cleaved caspase-3 and Beclin1 expression and a decrease in the expressions of BCL2 and BCLXL (P<0.05) were observed in only the HFD group. FFA, triglyceride levels, HOMA-IR, insulin levels and glucagon levels were significantly increased in the HFD group but decreased in the CRD group at 16 weeks (P<0.05). The degree of islet cell autophagy was potentially regulated by the levels of FFA and islet cell insulin and glucagon, which may have been due to the effects of Beclin1/BCL2.