MRNA expression levels of leptin-related substances can be modified in risperidone-injected rats

Inflammation related to chronic kidney disease (CKD) is an important clinical problem. We recently determined that hepatocyte nuclear factor 1α (HNF1α) was upregulated in the livers of chronic renal failure (CRF) rats—experimental model of CKD. Considering that the promoter region of gene encoding C-reactive protein (CRP) contains binding sites for HNF1α and that the loss-of-function mutation in the Hnfs1α leads to significant reduction in circulating CRP levels, we hypothesized that HNF1α can activate the Crp in CRF rats. Here, we found coordinated upregulation of genes encoding CRP, interleukin-6 (IL-6), HNF1α, and HNF4α in the livers and white adipose tissue (WAT) of CRF rats, as compared to the pair-fed and control animals. This was accompanied by elevated serum levels of CRP and IL-6. CRP and HNFs’ mRNA levels correlated positively with CRP and HNFs’ protein levels in the liver and WAT. Similar upregulation of the Crp, Il-6, and Hnfs in the liver and WAT and increased serum CRP and IL-6 concentrations were found in lipopolysaccharide (LPS)-induced systemic inflammation in rats. Moreover, silencing HNF1α in HepG2 cells by small interfering RNA led to decrease in CRP mRNA levels. Our results suggests that (a) HNFs act in concert with IL-6 in the upregulation of CRP production by the liver and WAT, leading to an increase in circulating CRP concentration in CRF rats and (b) CRF-related inflammation plays an important role in the upregulation of genes that encode HNFs and CRP in the liver and WAT of CRF rats.

The study examined the effect of L-tryptophan on white adipose tissue and its potential to enhance the effectiveness of fasting-dietary obesity correction. Forty-eight Wistar rats were divided into four groups: I ? control; II ? animals that received a high-calorie diet for 12 weeks; III ? rats that after 12 weeks of a high-calorie diet received a balanced diet for 4 weeks; IV ? rats that after 12 weeks of a high-calorie diet received a balanced diet with the addition of L-tryptophan (80 mg/kg body weight) for 4 weeks. The high-calorie diet contained 45% fat, 9% protein, and 31% carbohydrate. Physiological, morphological, histomorphometric, biochemical, and biophysical methods were used in the experiment. A high-calorie diet induced obesity and structural alterations in white adipose tissue in the rats. Dietary L-tryptophan accelerated restoration of adipocyte composition, decreased connective tissue, improved white adipose tissue vascularization, and normalized visceral fat mass and lipid metabolism markers. L-tryptophan also contributed to a reduction in the disturbance of the bioelectric properties of the white adipose tissue in obesity. This may indicate restoration of adipocyte membrane integrity, adipose tissue hydration, and microstructural organization. The presented information may be applied clinically in the comprehensive treatment of obesity.

Objective To explore the effect and mechanism of omega 3-polyunsaturated fatty acid(ω3-PUFA) dietary intervention on mitochondrial function of white adipose tissue in adult rats with postnatal early overfeeding. Methods An overfed animal model by adjusting litter size was developed for the study of neonatal overfeeding. The litter size was adjusted to 3 male rats per litter(small litter, SL group) and 10 pups per litter(normal litter, NL group). After weaning(week 3), the pups were fed standard chow or ω3-PUFA diet(SL-FO) until postnatal weeks 13. Food intake, body weight, and rectal temperature of rats were measured regularly, and energy metabolism of animals was monitored in week 13. During week 3 and 13, subcutaneous adipose tissue was collected. Inguinal preadipocytes of mice were isolated and induced to differentiate, and 50 μmol/L eicosapentaenoicacid(EPA) was administered for 48 h at the late stage of differentiation. The mRNA and protein expression levels of mitochondrial related genes, mitochondrial copy number, and oxygen consumption rate of adipocytes were detected in adipose tissue and adipocytes. Results By the 3rd week, the body weight, food intake, and fat cell area in SL group were higher than those in NL group while the body temperature was lower until to 13 weeks. By the 13th week, the O2 consumption, CO2 output, and heat production of rats in SL group were lower than those in NL group. Meanwhile, the expressions of mitochondrial function related genes such as uncoupling protein 1(UCP1), carnitine palmitoyltransferase 1(CPT1), SIRT1, and mitochondrial biosynthesis regulatory gene peroxisome proliferator-activated receptor coativator-1 (PGC1α) in adipose tissue by the 3rd and 13th week were significantly reduced(P<0.05). After weaning, ω3-PUFA diet significantly reduced weight gain in SL rats, increased UCP1 protein expression, restored energy metabolism level and mitochondrial function related gene expression. In vitro intervention of EPA increased the mitochondrial copy number, the mRNA and protein expression levels of mitochondrial biosynthesis and functional genes, as well as the mitochondrial basic oxygen consumption rate(P<0.05). Conclusion ω3-PUFA improves postnatal overfeeding-induced impairment of the mitochondrial function and biosynthesis of subcutaneous white adipose tissue in rats, which may be an important mechanism for fish oil diet to inhibit the early over-nutrition program and restore the thermogenic metabolism. Key words: Omega 3-PUFA; Postnatal overfeeding; White fat; Mitochondria

The purpose of this study was to investigate the effects of 8 weeks of high-intensity interval training (HIIT) and vitamin D3 supplementation on Chemokine (C-C motif) Ligand 5 (CCL-5) and C-C motif chemokine receptor 5 (CCR5) in the white adipose tissue (WAT) of male rats with type 2 diabetes (T2DM). The experimental study involved 40 male Wistar rats divided into 5 groups (n=8). These groups were healthy control (HC), diabetic control (DC), diabetic+HIIT (DHIIT), diabetic+vitamin D3 (DD3), and diabetic+HIIT+vitamin D3 (DHIITD3). The rats completed 8 weeks of HIIT, consisting of 12 sessions lasting 1 minute each at an intensity of 90-95% of their maximum running speed. Additionally, the rats were administered a weekly dose of 10,000 IU/kg of vitamin D3 for 8 weeks. The levels of CCL-5 (P<0.001) and CCR5 (P=0.003) were found to be higher in the DC group as compared to the HC group. However, when HIIT training and vitamin D3 were administered together, there was a decrease in CCL-5 (P=0.001) and CCR5 (P<0.001) in the DHIITD3 group (P=0.001). Similarly, vitamin D3 alone reduced CCR5 levels in the DD3 group (P< 0.001). Also, the decrease of CCR5 in the DD3 group was higher than in the DHIIT group (P=0.022), and the DHIITD3 group was higher than in the DHIIT group (P<0.001), but there was no difference between the DD3 and DHIITD3 groups (P≥0.05). The results indicate that combining HIIT training with vitamin D3 has a greater effect on reducing the expression of CCL-5 and CCR5 in the white adipose tissue of rats with type 2 diabetes induced by streptozotocin (STZ) and a high-fat diet (HFD), compared to the effects of each one alone. It is recommended that the study be conducted by measuring the variables involved in the mechanisms and the changes in CCL-5 and CCR5.

The objective was to assess the effect of a new, highly selective beta 3-adrenergic agonist, CL-316,243 (CL) (J. D. Bloom, M. D. Dutia, B. D. Johnson, A. Wissner, M. G. Burns, E. E. Largis, J. A. Dolan, and T. H. Claus., J. Med. Chem. 35: 3081, 1992), on energy balance and brown and white adipose tissues (BAT and WAT, respectively) in young rats eating a high-fat diet to induce obesity. Chronic treatment with CL increased body temperature and 24-h energy expenditure, mainly by increasing resting metabolic rate. Food intake was not altered but carcass fat was reduced. Interscapular BAT was markedly hypertrophied, with three- to fourfold increases in the content of uncoupling protein (UCP) and cytochrome oxidase. Quantitative immunoelectron microscopy of interscapular BAT of CL-treated rats showed smaller mitochondria with an unchanged total amount of UCP per mitochondrion. The relative frequency of the four major cell types in BAT (mature brown adipocytes, preadipocytes, interstitial cells, endothelial cells) was not altered. The CL-induced hypertrophy differed from that induced by chronic stimulation by endogenous norepinephrine (as in cold-adaptation) in absence of hyperplasia (there was a slightly reduced DNA content), absence of an increase in the thyroxine (T4) 5'-deiodinase activity, and absence of a selective increase in UCP concentration. WAT depots weighed less and had fewer cells (lower DNA content) in the CL-treated rats. Some multilocular adipocytes appeared in these normally almost exclusively unilocular WAT depots (mesenteric, inguinal, epididymal, retroperitoneal). We conclude that CL not only promotes BAT mitochondrial proliferation and thermogenesis and overall energy expenditure and leanness, but also retards the development of WAT hyperplasia during the early stage of diet-induced obesity.

Brown adipose tissue (BAT) plays an important role in whole body metabolism and with appropriate stimulus could potentially mediate weight gain and insulin sensitivity. Although imaging techniques are available to detect subsurface BAT, there are currently no viable methods for continuous acquisition of BAT energy expenditure. Microwave (MW) radiometry is an emerging technology that allows the quantification of tissue temperature variations at depths of several centimeters. Such temperature differentials may be correlated with variations in metabolic rate, thus providing a quantitative approach to monitor BAT metabolism. In order to optimize MW radiometry, numerical and experimental phantoms with accurate dielectric properties are required to develop and calibrate radiometric sensors. Thus, we present for the first time, the characterization of relative permittivity and electrical conductivity of brown (BAT) and white (WAT) adipose tissues in rats across the MW range 0.5–10 GHz. Measurements were carried out in situ and post mortem in six female rats of approximately 200 g. A Cole–Cole model was used to fit the experimental data into a parametric model that describes the variation of dielectric properties as a function of frequency. Measurements confirm that the dielectric properties of BAT (εr = 14.0–19.4, σ = 0.3–3.3 S m−1) are significantly higher than those of WAT (εr = 9.1–11.9, σ = 0.1–1.9 S m−1), in accordance with the higher water content of BAT.

We have previously shown that rats made obese by lesion of ventromedial hypothalamus (VMH) nuclei, demonstrate an hyper-responsiveness to insulin with regard to whole-body glucose utilization one week after injury. This is mainly due to an increased glucose uptake in white adipose tissue. Six weeks after the lesion, glucose utilization in white adipose tissue returns to normal values. These modifications in insulin responsiveness could be mediated by altered activity and/or concentration of intracellular insulin effectors. In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats. These results indicate the existence of an increased expression of Glut 4 and lipogenic enzymes in white adipose tissue of VMH-lesioned rats which decreased with time and were parallel to glucose utilization determined in vivo.

Corticosterone — product of 11-β-hydroxysteroid dehydrogenase type I (11βHSD1) stimulates adipocytes differentiation and activates lipogenic enzymes gene expression in white adipose tissue (WAT) of rats. The aim of the study was to examine the effect of chronic food restriction, often practised by obese individuals trying to lose body mass, on: a) 11βHSD1 gene expression, b) expression of genes associated with adipocyte differentiation (PPARg, SREBP-1, adiponectin), and c) expression of genes associated with lipogenesis in WAT of rats. Two-month old rats were divided into a control and a food restricted group obtaining 50% of food consumed by controls for 30 days. mRNA levels of studied genes in perirenal WAT were analysed by real-time PCR. 11βHSD1 and lipogenic enzymes activities were measured by radiometric conversion assay and by spectrophotometric assay respectively. Food restriction caused significant increase of 11βHSD1, PPARg, SREBP1, adiponectin and lipogenic enzymes mRNA levels in perirenal WAT. 11βHSD1 and some lipogenic enzymes activities were also increased by food restriction. The coordinated up-regulation of 11βHSD1, and genes associated with adipocyte differentiation and lipogenesis by food restriction suggests that such nutritional condition shifts WAT metabolism, that would permit this tissue to synthesize and accumulate triacylglycerols immediately after refeeding.

Diversity of SREBP-1 gene expression in rat adipose tissue depots in response to refeeding after food restriction

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

Upregulation of lipogenic enzymes genes expression in white adipose tissue of rats with chronic renal failure is associated with higher level of sterol regulatory element binding protein-1

Brandt's voles Lasiopodomys brandtii exhibit large increases in nonshivering thermogenesis to cope with chronic cold exposure, resulting in compensatory hyperphagia and fat mobilization. These physiological events are accompanied by a remarkable reduction in serum leptin levels. However, the role of hypoleptinemia in cold adaptation in this species is still unknown. In the present study, we tested the hypothesis that hypoleptinemia contributes to increases in food intake and brown adipose tissue (BAT) thermogenesis by modifying hypothalamic neuropeptides in cold-exposed Brandt's voles. Adult male voles were transferred to 5 degrees C for 28 days. Accompanied by a decrease in serum leptin levels, hypothalamic agouti-related protein (AgRP) mRNA levels were significantly increased, but there were no changes in the long form of leptin receptor (Ob-Rb), suppressor of cytokine signaling 3 (SOCS3), neuropeptide Y (NPY) mRNA, proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated peptide (CART) mRNA levels in the hypothalamus. When cold-exposed voles were returned to warm (23 degrees C) for 28 days, body mass, food intake, serum leptin, and AgRP mRNA were restored to control levels. Leptin administration in cold-exposed voles decreased food intake as well as hypothalamic AgRP mRNA levels. There were no significant effects of leptin administration on hypothalamic Ob-Rb, SOCS3, NPY, POMC, CART mRNA, and uncoupling protein 1 levels under cold conditions. These results suggest that hypoleptinemia partially contributes to cold-induced hyperphagia, which might involve the elevation of hypothalamic AgRP gene expression.

Perivascular adipose tissue (PVAT) is a complex tissue that is increasingly recognized for its roles in vascular health and disease. The form and function of PVAT are different depending on species and anatomical location, and understanding its cellular and molecular characteristics gives greater insights. We had previously successfully performed single-nucleus RNA sequencing (snRNAseq) on brown fat depots, the thoracic aortic PVAT (taPVAT) and subscapular brown adipose tissue (BAT), from Dahl Salt Sensitive rats. However, the application of the same nuclei isolation method to white adipose tissue (WAT) depots (perivascular and non-perivascular) from the same rat strain resulted in insufficient nuclei capture and low transcript numbers. These challenges were also encountered when processing WAT from cattle. While nuclei isolation methods have been developed and optimized for human and mouse WAT depots, they have not been evaluated across WAT depots from other species, such as cow and rat. Because these latter species are important models for cardiovascular and metabolic diseases, this study aimed to validate and optimize a nuclei isolation protocol for use with WAT from them. Protocols were evaluated based on a) the quantity of nuclei isolated, b) the quality of nuclei determined via microscopic visualization, and c) the total number of detected transcripts and genes following snRNAseq. A modified protocol developed for human WAT, incorporating liquid nitrogen pulverization and Dounce homogenization of flash-frozen tissue, was tested. This protocol, with key modifications for optimization, proved translatable to rat and cow WAT depots to improve nuclei yield (rat retroperitoneal fat, 3,100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue, 2,200 nuclei/mg tissue; cow white fat, 2,050 nuclei/mg tissue). Further analysis via snRNAseq, however, identified limitations. While cow WAT expressed nearly 1,720 median genes/cell, both rat white depots were significantly lower [mesenteric PVAT (mesPVAT), 189; retroperitoneal (RP) fat, 294 median genes/cell], hindering downstream analyses in the rat tissue. These findings suggest that biological differences in adipose depots within and between species pose important challenges for the application of snRNAseq on rat WAT.

Chronological analysis of caloric restriction-induced alteration of fatty acid biosynthesis in white adipose tissue of rats

We examined the effects of a 9-week exercise training (TR) in Wistar male rats, beginning at 4 weeks of age, on the density of endothelial cells (ECs) in epididymal white adipose tissue (WAT) and the mRNA expression of angiogenic factors in adipose tissue stromal vascular fraction (SVF) cells. The number of ECs and mRNA expressions were assessed by lectin staining and real-time reverse transcriptase-polymerase chain reaction, respectively. Compared with control (CR) rats, TR rats gained weight more slowly and had significantly lower final weight of WAT due to the reduction in the size and the number of adipocytes. TR significantly increased the number of ECs per square millimeter and per adipocyte (1.37- and 1.23-fold, respectively) in WAT. This is probably because the number of adipocytes is fewer while the number of ECs is constant in the WAT of TR rats, because the regression line of TR rats for adipocyte number-dependent EC number was shifted toward the left without significant differences in the slopes between groups. TR also induced the upregulation of mRNA expression of vascular endothelial growth factor (Vegf)-A and Vegf-receptor-2 in SVF cells, thereby retaining a constant number of ECs in the WAT.