MOTS-c regulates pancreatic alpha and beta cell functions in vitro.

The aim of this study is to determine the influence of the mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c) peptide on pancreatic cell physiology. Moreover, in this study, we examined the changes in MOTS-c secretion and expression under different conditions. Our experiments were conducted using laboratory cell line cultures, specifically the INS-1E and αTC-1 cell lines, which represent ß and α pancreatic cells, respectively. As the pancreas is an endocrine organ, we also tested its hormone regulation capabilities. Furthermore, we assessed the secretion of MOTS-c after incubating the cells with glucose and free fatty acids. Additionally, we examined key cell culture parameters such as cell viability, proliferation, and apoptosis. The results obtained from this study show that MOTS-c has a significant impact on the physiology of pancreatic cells. Specifically, it lowers insulin secretion and expression in INS-1E cells and enhances glucagon secretion and expression in αTC-1 cells. Furthermore, MOTS-c affects cell viability and apoptosis. Interestingly, insulin and glucagon affect the MOTS-c secretion as well as glucose and free fatty acids. These experiments clearly show that MOTS-c is an important regulator of pancreatic metabolism, and there are numerous properties of MOTS-c yet to be discovered.

Differential expression profile between amygdala and blood during chronic lithium treatment in a rat model of depression - a pilot study.

Lithium is a mood stabilizer widely used in the pharmacotherapy of bipolar disorder and treatment­resistant depression. Taking into account dysregulated inflammatory activity in depression and the immunomodulatory role of lithium, we hypothesized that genes associated with inflammatory responses may be potential biomarkers of lithium action. We aimed to compare gene expression changes between the brain and the periphery after chronic lithium administration in an animal model of depression. Depressive behavior was induced by chronic mild stress protocol for 4 weeks. After 2 weeks, rats started to receive lithium (study group) or water (reference group). The control group were rats not exposed to stress. Amygdala, hippocampus, frontal cortex and peripheral blood were analyzed using whole transcriptome expression microarrays. Changes were confirmed with qPCR and ELISA assay. After 2 weeks of lithium administration, we observed significant changes in gene expression between amygdala and peripheral blood. Logistic regression analysis determined Alox15 expression as a predictor of lithium status, as its expression was tissue­specific and increased in amygdala and decreased in blood. Analysis of serum ALOX15 protein revealed its upregulation after two­week lithium administration. Our study suggests that lithium may have therapeutic potential in depressive behaviors. These results indicate immunomodulatory effect of lithium and that Alox15 may be a new potential marker of chronic lithium treatment.

Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression.

INTRODUCTION: Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation. MATERIALS AND METHODS: As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223-3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13. RESULTS: We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223-3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223-3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes. CONCLUSIONS: These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223-3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223-3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.

Changes in MOTS-c Level in the Blood of Pregnant Women with Metabolic Disorders.

MOTS-c peptide is a member of the group of mitochondria-derived peptides (MDP). It is a product of the open reading frame in the 12S RNA gene. Due to its features and functions in the body, this peptide is classified as a hormone. The first publications indicated that this hormone improves insulin sensitivity and lowers body weight in obese animals. This suggests that it may be an important peptide in maintaining the body's energy homeostasis. The aim of our work was to investigate the potential role of MOTS-c peptide during pregnancy, which is a condition prone to metabolic disorders. The research covered healthy, obese women and women with thyroid disorders. The obtained results indicated an increase in the concentration of MOTS-c in the blood of mothers and newborns in the obese group as compared to the healthy control group and a corresponding decrease in the concentration of this peptide in mothers and newborns in the group with hypothyroidism compared to the obese group. Moreover, we also observed a strong positive correlation between the concentration of MOTS-c in maternal blood and in umbilical cord blood. In summary, the MOTS-c peptide shows changes in blood concentration in various physiological states and may, in the future, become an important tool in the fight against metabolic diseases such as obesity or type 2 diabetes.

30-Day spexin treatment of mice with diet-induced obesity (DIO) and type 2 diabetes (T2DM) increases insulin sensitivity, improves liver functions and metabolic status.

Spexin (SPX) is a 14 aa peptide discovered in 2007 using bioinformatics methods. SPX inhibits food intake and regulates lipid, and carbohydrate metabolism. Here, we evaluate the ability of SPX at improving metabolic control and liver function in obese and type 2 diabetic animals. The effects of 30 days SPX treatment of mice with experimentally induced obesity (DIO) or type 2 diabetes (T2DM) on serum glucose and lipid levels, insulin sensitivity and hormonal profile (insulin, glucagon, adiponectin, leptin, TNF alpha, IL-6 and IL-1ß) are characterized. In addition, alterations of hepatic lipid and glycogen contents are evaluated. We report that SPX decreases body weight in healthy and DIO mice, and reduces lipid content in all three animal groups. SPX improves insulin sensitivity in DIO and T2DM animals. In addition, SPX modulates hormonal and metabolic profile by regulating the concentration of adiponectin (concentration increase) and leptin (concentration decrease) in the serum blood of DIO and T2DM mice. Lastly, SPX decreases lipid content as well as IL-6 and TNF-α protein levels in liver of DIO and T2DM mice, and reduces IL-6 and TNF-alpha concentrations in the serum derived from T2DM mice. Based on our results, we conclude that SPX could be involved in the development of obesity and type 2 diabetes mellitus and it can be further evaluated as a potential target for therapy of DIO and T2DM.

The Role of Peptide Hormones Discovered in the 21st Century in the Regulation of Adipose Tissue Functions.

Peptide hormones play a prominent role in controlling energy homeostasis and metabolism. They have been implicated in controlling appetite, the function of the gastrointestinal and cardiovascular systems, energy expenditure, and reproduction. Furthermore, there is growing evidence indicating that peptide hormones and their receptors contribute to energy homeostasis regulation by interacting with white and brown adipose tissue. In this article, we review and discuss the literature addressing the role of selected peptide hormones discovered in the 21st century (adropin, apelin, elabela, irisin, kisspeptin, MOTS-c, phoenixin, spexin, and neuropeptides B and W) in controlling white and brown adipogenesis. Furthermore, we elaborate how these hormones control adipose tissue functions in vitro and in vivo.

The Long-Term Effects of High-Fat and High-Protein Diets on the Metabolic and Endocrine Activity of Adipocytes in Rats.

The increasing prevalence of overweight and obesity and the rising awareness of their negative consequences are forcing researchers to take a new view of nutrition and its consequences for the metabolism of whole organisms as well as the metabolism of their individual systems and cells. Despite studies on nutrition having been carried out for a few decades, not many of them have focused on the impacts of these diets on changes in the metabolism and endocrine functions of isolated adipocytes. Therefore, we decided to investigate the effects of the long-term use (60 and 120 days) of a high-fat diet (HFD) and of a high-protein diet (HPD) on basic metabolic processes in fat cells-lipogenesis, lipolysis, and glucose uptake-and endocrine function, which was determined according to the secretion of adipokines into the incubation medium. Our results proved that the HPD diet improved insulin sensitivity, increased the intracellular uptake of glucose (p < 0.01) and its incorporation into lipids (p < 0.01) and modulated the endocrine function of these cells (decreasing leptin secretion; p < 0.01). The levels of biochemical parameters in the serum blood also changed in the HPD-fed rats. The effects of the HFD were inverse, as expected. We observed a decrease in adiponectin secretion and a diminished rate of lipogenesis (p < 0.01). Simultaneously, the secretion of leptin and resistin (p < 0.01) from isolated adipocytes increased. In conclusion, we noted that the long-term use of HPD and HFD diets modulates the metabolism and endocrine functions of isolated rat adipocytes. We summarize that an HFD had a negative effect on fat tissue functioning, whereas an HPD had positive results, such as increased insulin sensitivity and an improved metabolism of glucose and lipids in fat tissue. Moreover, we noticed that negative metabolic changes are reflected more rapidly in isolated cells than in the metabolism of the whole organism.

Neuropeptide B promotes proliferation and differentiation of rat brown primary preadipocytes.

Neuropeptide B (NPB) is reported to regulate energy homeostasis and metabolism via the NPBWR1 and NPBWR2 receptors in various tissues. However, the molecular mechanisms triggered from their interaction are not well investigated in brown adipose tissue. In this study, we specifically analyzed the role of NPB in controlling brown adipogenesis in rat brown preadipocytes. We first detected the expression of NPBWR1 and NPB on mRNA and protein level in brown preadipocytes and observed that NPB increased viability and proliferation of preadipocytes. Moreover, NPB stimulated expression of adipogenic genes (Prdm16, Ucp1) and suppressed the expression of antiadipogenic preadipocyte factor 1 (Pref1) during the differentiation process. Altogether, this led to an increase in intracellular lipid accumulation during preadipocyte differentiation, coupled with an increase in adrenaline-induced oxygen consumption mediated by NPB. Furthermore, Ucp1 expression stimulated by NPB was attenuated by blockade of p38 kinase. In summary, we conclude that NPB promotes proliferation and differentiation of rat brown preadipocytes via p38-dependent mechanism and plays an important role in controlling brown adipose tissue formation.

Transcriptome Changes in Three Brain Regions during Chronic Lithium Administration in the Rat Models of Mania and Depression.

Lithium has been the most important mood stabilizer used for the treatment of bipolar disorder and prophylaxis of manic and depressive episodes. Despite long use in clinical practice, the exact molecular mechanisms of lithium are still not well identified. Previous experimental studies produced inconsistent results due to different duration of lithium treatment and using animals without manic-like or depressive-like symptoms. Therefore, we aimed to analyze the gene expression profile in three brain regions (amygdala, frontal cortex and hippocampus) in the rat model of mania and depression during chronic lithium administration (2 and 4 weeks). Behavioral changes were verified by the forced swim test, open field test and elevated maze test. After the experiment, nucleic acid was extracted from the frontal cortex, hippocampus and amygdala. Gene expression profile was done using SurePrint G3 Rat Gene Expression whole transcriptome microarrays. Data were analyzed using Gene Spring 14.9 software. We found that chronic lithium treatment significantly influenced gene expression profile in both mania and depression models. In manic rats, chronic lithium treatment significantly influenced the expression of the genes enriched in olfactory and taste transduction pathway and long non-coding RNAs in all three brain regions. We report here for the first time that genes regulating olfactory and taste receptor pathways and long non-coding RNAs may be targeted by chronic lithium treatment in the animal model of mania.

Genes involved in glucocorticoid receptor signalling affect susceptibility to mood disorders.

OBJECTIVES: In mood disorders chronic stress contributes to decreased glucocorticoid receptor signalling in the brain and resistance in the periphery. We hypothesised that aberrant glucocorticoid receptor function may result from genetic predisposition and that decreased GR signalling in the brain correlates with the expression of genes regulating GR complex formation. METHODS: We performed the association analysis of 698 patients: 490 patients with bipolar disorder and 208 patients with major depressive disorder and 564 control subjects. We genotyped 11 variants using TaqMan assays. Gene expression in the brain tissue was done in male Wistar rats after chronic mild stress protocol. The SRSF5 serum concentration was performed using ELISA. Data were analysed in Statistica and GraphPad. RESULTS: We found an association of STIP1 and SRSF5 variants with major depressive disorder and BAG1 variant with bipolar disorder. Gene expression analysis in a rat model of depression confirmed significant changes in the expression of SRSF5, BAG1, and FKBP4 in the brain. For SRSF5, we observed significantly increased expression in the serum of depressed females and male rats exposed to chronic stress. CONCLUSIONS: Our results indicate the involvement of genes associated with GR function, SRSF5, BAG1, and FKBP4 with susceptibility to mood disorders.

Effects of Bitter Melon and a Chromium Propionate Complex on Symptoms of Insulin Resistance and Type 2 Diabetes in Rat Models.

Trivalent chromium (Cr) and bitter melon (Momordica charantia L., BM) have been shown to independently interact with the insulin signaling pathway leading to improvements in the symptoms of insulin resistance and diabetes in some animal models and human subjects. The aim of this study was to examine whether the combination of the two nutritional supplements could potentially have additive effects on treating these conditions in high-fat-fed streptozotocin (STZ)-induced diabetic rats. The experiment was conducted with 110 male Wistar rats divided into eleven groups and fed either a control or high-fat diet for 7 weeks. Half of the rats on the high-fat diet were injected with STZ (30 mg/kg body mass) to induce diabetes. The high-fat (HF) diets were then supplemented with a combination of Cr (as chromium(III) propionate complex, Cr3: either 10 or 50 mg Cr/kg diet) and bitter melon (lyophilized whole fruit: either 10 or 50 g/kg diet) for 6 weeks. After termination of the experiment, blood and internal organs were harvested for blood biochemical, hematological, and mineral (Cr) analyses using appropriate analytical methods. It was found that neither Cr(III) nor BM was able to significantly affect blood indices in HF and diabetic rats, but BM tended to improve body mass gain, blood glucose, and LDL cholesterol values, but decreased Cr content in the liver and kidneys of the Cr-co-supplemented type 2 diabetic model of rats. Supplementary Cr(III) had no appreciable effect on glucose and lipid metabolism in high-fat-fed STZ-induced diabetic rats. Supplementary BM fruit powder had some observable effects on body mass of high-fat-fed rats; these effects seem to be dampened when BM was co-administered with Cr. Cr(III) and BM appear to act as nutritional antagonists when both administered in food, probably due to binding of Cr by the polyphenol-type compounds present in the plant material. Graphical Abstract.

Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro-The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo.

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

Short-term administration of spexin in rats reduces obesity by affecting lipolysis and lipogenesis: An in vivo and in vitro study.

The present study aimed to characterize the role of spexin (SPX) in maintaining glucose and lipid homeostasis in vivo in rats with diet-induced obesity. The in vitro effect of spexin on metabolic and endocrine functions of adipocytes isolated from obese rats was also investigated. The in vivo experiment was conducted on rats with diet-induced obesity and administered with SPX for 7 days. Lipid and carbohydrate parameters, liver markers, and hormonal profile were measured. In in vitro studies, adipocytes isolated from obese rats were used. The effect of SPX on lipolysis, lipogenesis, and leptin secretion from fat cells was assessed. The results showed that short-term administration of SPX causes weight loss, increases insulin sensitivity, and improves the metabolic state of obese rats. The in vitro experiments showed that spexin and its receptors, namely galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3), were expressed in various fat depots and in adipocytes from obese rats. We also found that the addition of spexin increased the basal and isoproterenol-stimulated lipolysis and reduced the basal and insulin-stimulated lipogenesis in adipocytes isolated from obese rats. Molecular analysis showed that SPX activated hormone-sensitive lipase (HSL) phosphorylation and upregulated perilipin and HSL mRNA expression. These results suggest that SPX regulates metabolism of obese rats by affecting lipolysis and lipogenesis in adipocytes. Moreover, the present study for the first time demonstrates that SPX modulates leptin synthesis and secretion from isolated adipocytes.

Allergic Inflammation Alters microRNA Expression Profile in Adipose Tissue in the Rat.

Adipose tissue is a major source of circulating exosomal microRNAs (miRNAs) that are modulators of the immune response in various types of tissues and organs, including airways. Still, no evidence exists if allergic airway inflammation may affect fat tissue inflammation via alterations in the miRNA expression profile. Therefore, we investigated the miRNA expression profile in the adipose tissue upon induced allergic inflammation in the airways in the rat. Brown Norway rats were chronically sensitized to house dust mite extract for seven weeks. Body composition was performed using MiniSpec Plus. The eosinophil count and the total IgE level were determined to confirm the induction of allergic inflammation. MiRNA expression profiling was done using the next-generation sequencing with validation by qPCR. We found that allergic airway inflammation significantly increased fat in adipose tissue, glucose concentration, and the gene expression of adipose tissue-derived proinflammatory peptides (leptin, TNFα). In miRNA-seq analysis, we showed significant differences in the expression of 36 mature miRNAs, three precursors, and two miRNA families in adipose tissue of allergic rats. Two miRNAs-miRNA-151-5p and miRNA-423-3p-showed significantly increased expression in qPCR in adipose tissue and lungs of sensitized animals. Allergic airway inflammation affects fat tissue and alters miRNA expression profile in adipose tissue in the rat.

Neuroinflammatory Gene Expression Pattern Is Similar between Allergic Rhinitis and Atopic Dermatitis but Distinct from Atopic Asthma.

METHODS: In the study, we included 86 children diagnosed with atopic asthma (n = 25), allergic rhinitis (n = 20), and atopic dermatitis (n = 20) and healthy control subjects (n = 21) of Caucasian origin from the Polish population. The blood leukocyte expression of 31 genes involved in neuroinflammatory response (neurotrophins, their receptors, neuropeptides, and histamine signaling pathway) was analysed using TaqMan low-density arrays. The relative expression of selected proteins from plasma was done using TaqMan Protein Assays. Statistical analysis was done using Statistica. RESULTS: Blood expression of 31 genes related to neuroimmune interactions showed significant increase in both allergic diseases, allergic rhinitis and atopic dermatitis, in comparison to the control group. We found 12 genes significantly increased in allergic rhinitis and 9 genes in which the expression was elevated in atopic dermatitis. Moreover, 9 genes with changed expression in atopic dermatitis overlapped with those in allergic rhinitis. Atopic asthma showed 5 genes with altered expression. The peripheral expression of neuroinflammatory genes in the human study was verified in target tissues (nasal epithelium and skin) in a rat model of allergic inflammation. CONCLUSIONS: A common pattern of neuroinflammatory gene expression between allergic rhinitis and atopic dermatitis may reflect similar changes in sensory nerve function during chronic allergic inflammation.

FGF-1 modulates pancreatic ß-cell functions/metabolism: An in vitro study.

Fibroblast growth factor 1 (FGF-1), also known as acidic fibroblast growth factor (aFGF), is a growth factor and signaling protein encoded by the Fgf1 gene. Previous studies have shown that FGF-1 may also participate in the regulation of glucose metabolism, both in healthy organisms and in pathological conditions such as diabetes. Because insulin the main regulator of glucose metabolism is secreted from pancreatic beta cells, we investigated whether FGF-1 directly affects the secretion of this hormone and regulates the metabolism of beta cells and isolated pancreatic islets. By using insulin-producing INS-1E cells and isolated pancreatic islets, we investigated the effect of FGF-1 on cell proliferation, viability, apoptosis, and insulin expression and secretion. Our study showed that FGF1 and fibroblast growth factor receptors (FgfRs: FgfR1, FgfR2, FgfR3, and FgfR4) are present on mRNA level in INS-1E cells and isolated rat pancreatic islets. We also proved that FGF1 stimulates the proliferation of INS-1E beta cells and enhances the viability of these cells and that of isolated pancreatic islet cells, and that ERK1/2 kinase is involved in the regulation of INS-1E cell proliferation. Moreover, we found that FGF1 can stimulate insulin secretion from both INS-1E cells and isolated rat pancreatic islets. Thus, the FGF1 peptide increases cell survival and decreases cell death. The obtained results indicate that FGF1 may play a role in controlling the physiology and metabolism of pancreatic beta cells as well as glycemia.

Spexin in the physiology of pancreatic islets-mutual interactions with insulin.

Spexin is an interesting peptide, which may play an important role in the regulation of the metabolic homeostasis of an organism. Current knowledge on spexin expression, secretion, and influence on tissues and endocrine glands is very limited. We investigated spexin localization in the endocrine pancreas and measured its in vitro secretion from isolated pancreatic islets at various glucose concentrations, simultaneously monitoring insulin release. Also, gene expression for spexin and insulin was estimated. We found the presence of spexin inside beta cells and an increase in its release from islets after a short term and decrease after a long term following glucose administration. Finally, negative feedback loops between spexin and insulin were found, indicating the presence of multilateral relationships between glucose, insulin, and spexin inside pancreatic islets.

Leptin gene polymorphism affects leptin level in childhood asthma.

BACKGROUND: Leptin may induce inflammation in asthma by activation of Th2 cells. It has also been demonstrated that leptin expression increases upon inflammation and that asthmatic patients show increased serum leptin levels. We hypothesized that the polymorphism in leptin (LEP) and leptin receptor (LEPR) genes is associated with childhood asthma and may affect their serum level. To our knowledge, there are no reports analyzing LEP and LEPR polymorphisms in association with their serum levels in childhood asthma. METHODS: We analyzed 35 subjects: 25 asthmatic pediatric patients and 10 healthy children aged from 6 to 18. The diagnosis of allergic asthma was based on clinical manifestation, lung function, positive skin prick tests and increased immunoglobulin E levels. The polymorphisms were genotyped with use of polymerase chain reaction-restriction fragment length polymorphism method. Serum levels of leptin and leptin receptor were determined using BioVendor enzyme-linked immunosorbent assay kits. Statistical analysis was done with Statistica v.12. RESULTS: We observed that leptin levels were increased in asthmatic subjects as compared to healthy controls and were significantly higher during exacerbation than in the asymptomatic period (P = 0.025). We observed that LEP polymorphism (rs13228377) was associated with higher serum leptin levels in asthma and these two variables had high predictive value for asthma risk (P = 0.007, odds ratio 17.5, predictive accuracy 83.9%). LEPR polymorphisms did not show association with its serum level and asthma risk. CONCLUSION: LEP polymorphism may increase asthma risk via influence on its serum level.

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism.

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpß and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.

Interleukin 4 affects lipid metabolism and the expression of pro-inflammatory factors in mature rat adipocytes.

Chronic low-grade inflammation contributes to diseases associated with fat tissue metabolism such as obesity and diabetes by the disturbed production of adipose tissue proteins, both pro- and anti-inflammatory. Interleukin-4 (IL-4) is one of the main inflammatory cytokines that activates Th2-dependent immune response and its increased expression was observed in the course of diseases characterized by chronic low-grade systemic inflammation such as obesity and asthma. We aimed to investigate if IL-4 may influence lipid metabolism and inflammatory responses in primary mature rat adipocytes. Mature adipocytes were isolated from male Wistar rats in incubated with IL-4 at three concentrations. We measured lipogenesis and lipolysis as well as the expression of selected genes using ddCt method was used to calculate relative gene expression. Protein level in tissue was analyzed using Western blot. Protein concentration in cell medium was analyzed using ELISA. Statistical analysis was done using GraphPad Prism 5 software. In primary cell model, we found that IL-4 stimulated lipogenesis and inhibited lipolysis in mature rat adipocytes. It also stimulated the expression of pro-inflammatory cytokines produced by adipocytes and decreased the expression of anti-inflammatory protein, adiponectin. Moreover, we found increased expression of inflammatory cytokines and transcription factors associated with Th2 response. Our observations suggest that low-grade inflammation enhances fat accumulation and significantly alters adipocyte metabolism.