Effect of Tempeh and Daidzein on Calcium Status, Calcium Transporters, and Bone Metabolism Biomarkers in Ovariectomized Rats.

Menopause marks a critical life stage characterized by hormonal changes that significantly impact bone health, leading to a heightened susceptibility to bone fractures. This research seeks to elucidate the impact of daidzein and tempeh on calcium status, calcium transporters, and bone metabolism in an ovariectomized rat model. Forty female Wistar rats, aged 3 months, participated in a two-phase experiment. The initial phase involved inducing a calcium deficit, while the second phase comprised dietary interventions across five groups: Sham (S) and Ovariectomy (O) with a standard diet, O with bisphosphonate (OB), O with pure daidzein (OD), and O with tempeh (OT). Multiple parameters, encompassing calcium levels, calcium transporters, bone histopathology, and serum bone metabolism markers, were evaluated. The findings revealed that the OT group showcased heightened levels of bone turnover markers, such as pyridinoline, C-telopeptide of type I collagen, bone alkaline phosphatase, and procollagen type I N-terminal propeptide, in contrast to S and O groups, with statistical significance (p < 0.05). Histopathologically, both the OD and OT groups exhibited effects akin to the OB group, indicating a decrease in the surface area occupied by adipocytes in the femoral bone structure, although statistically non-equivalent, supporting the directionally similar trends. Although TRPV5 and TRPV6 mRNA expression levels in the jejunum and duodenum did not display statistically significant differences (p > 0.05), the OD and OT groups exhibited increased expression compared to the O group. We hypothesized that obtained results may be related to the effect of isoflavones on estrogen pathways because of their structurally similar to endogenous estrogen and weak estrogenic properties. In conclusion, the daily consumption of pure daidzein and tempeh could potentially improve and reinstate calcium status, calcium transport, and bone metabolism in ovariectomized rats. Additionally, isoflavone products demonstrate effects similar to bisphosphonate drugs on these parameters in ovariectomized rats.

GIP_HUMAN [22-51] Peptide Encoded by the Glucose-Dependent Insulinotropic Polypeptide (GIP) Gene Suppresses Insulin Expression and Secretion in INS-1E Cells and Rat Pancreatic Islets.

GIP_HUMAN [22-51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22-51] in ApoE-/- mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22-51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22-51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22-51] suppressed insulin (Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22-51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22-51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22-51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22-51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22-51] suppresses insulin expression and secretion in pancreatic ß cells without affecting ß cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22-51] on insulin secretion are glucose-dependent.

Changes in metabolic and hormonal profiles during transition period in dairy cattle - the role of spexin.

BACKGROUND: This study aimed to evaluate spexin as a novel blood marker and to describe the relationship of this peptide with selected biochemical metabolites measured during the transition period in dairy cows. Additionally, mRNA expression of the spexin gene as well as spexin receptors - galanin receptor type 2 and galanin receptor type 3, was investigated in several bovine tissues. Blood samples were collected at weekly intervals starting at 21 days before the estimated parturition day until 21 days in milk to determine concentrations of spexin, nonesterified fatty acids, ß-hydroxybutyrate acid, total and active ghrelin, progesterone, glucose, insulin, IGF-I, triglycerides, cholesterol, leptin, corticosterone and 17-ß-estradiol as well as the activity of aspartate transaminase, alkaline phosphatase and gamma-glutamyl transferase. RESULTS: Spexin concentration decreased from 21 d before parturition to calving day and next it rose during the first 14 d of lactation. The lowest concentration of spexin was recorded on the calving day and it differed from the mean level of this peptide before parturition as well as postpartum. Moreover, differences were observed between mean spexin concentrations before and after calving. Spexin levels were moderately negatively correlated with NEFA (r = - 0.39) and total ghrelin contents (r = - 0.41), weakly correlated with BHBA (r = - 0.35) while they showed a moderate positive relationship with progesterone concentrations (r = 0.42). Moreover, we detected that mRNA expression of GALR2, GALR3 and SPX is present in various bovine tissues (kidney, bowel, rumen, spinal cord, lung, skeletal muscle, liver, heart, fat and spleen). CONCLUSION: A negative correlation between spexin concentration and NEFA, BHBA and total ghrelin contents as well as a positive relationship with levels of progesterone, metabolites and hormones, which are key players in the dairy cow transition period, may confirm an important function of this peptide in metabolism regulation. Thus measurement of spexin concentration could provide useful supplementary information for dairy cow herd health monitoring.

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.

Alteration of active and repressive histone marks during adipogenic differentiation of porcine mesenchymal stem cells.

A characteristic spatial distribution of the main chromatin fractions is observed in most mammalian cell nuclei, with euchromatin localized in the interior and heterochromatin at the nuclear periphery. It has been shown that interactions of heterochromatin with the nuclear lamina are necessary to establish this conventional architecture. Adipocytes are specific cells in which a reduction in lamin A/C expression is observed. We hypothesize that the loss of lamin A/C during adipogenic differentiation of mesenchymal stem cells (MSCs) may be associated with the reorganization of the main classes of chromatin in the nucleus. Thus, in this study, we examine the abundance and nuclear distribution of selected heterochromatin (H3K9me3, H3K27me3 and H4K20me3) and euchromatin (H4K8ac, H3K4me3 and H3K9ac) histone marks during in vitro adipogenesis, using the pig as a model organism. We found that not only did the expression of lamin A/C decrease in our differentiation system, but so did the expression of lamin B receptor (LBR). The level of two heterochromatin marks, H3K27me3 and H4K20me3, increased during differentiation, while no changes were observed for H3K9me3. The levels of two euchromatin histone marks, H4K8ac and H3K9ac, were significantly higher in adipocytes than in undifferentiated cells, while the level of H3K4me3 did not change significantly. The spatial distribution of all the examined histone marks altered during in vitro adipogenesis. H3K27me3 and H4K20me3 moved towards the nuclear periphery and H3K9me3 localized preferentially in the intermediate part of adipocyte nuclei. The euchromatin marks H3K9ac and H3K4me3 preferentially occupied the peripheral part of the adipocyte nuclei, while H4K8ac was more evenly distributed in the nuclei of undifferentiated and differentiated cells. Analysis of the nuclear distribution of repetitive sequences has shown their clustering and relocalization toward nuclear periphery during differentiation. Our study shows that dynamic changes in the abundance and nuclear distribution of active and repressive histone marks take place during adipocyte differentiation. Nuclear reorganization of heterochromatin histone marks may allow the maintenance of the nuclear morphology of the adipocytes, in which reduced expression of lamin A/C and LBR is observed.

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.

Effects of Ovariectomy and Sex Hormone Replacement on Numbers of Kisspeptin-, Neurokinin B- and Dynorphin A-immunoreactive Neurons in the Arcuate Nucleus of the Hypothalamus in Obese and Diabetic Rats.

KNDy neurons co-expressing kisspeptin (KP), neurokinin B (NKB) and dynorphin A (DYN A) in the arcuate nucleus of the hypothalamus (ARC) are key regulators of reproduction. Their activity is influenced by metabolic and hormonal signals. Previously, we have shown that orchidectomy alters the KP-, NKB-, and DYN A-immunoreactivity in the high-fat diet-induced (HFD) obesity and diabetes type 2 (DM2) models. Considering the potential sex difference in the response of KNDy neurons, we have hypothesized that ovariectomy (OVX) and post-ovariectomy replacement with estradiol (OVX+E2) or estradiol and progesterone (OVX+E2+P4) will also affect these neurons in HFD and DM2 females. Thus, each of these treatment protocols were employed for control, HFD, and DM2 groups of rats leading to nine experimental conditions within which we have determined the number of KP-, NKB-, or DYN-immunoreactive (-ir) neurons and assessed the metabolic and hormonal profiles of the animals. Accordingly: (1) no effects of group and surgery were observed on the number of KP-ir neurons; (2) the overall number of NKB-ir neurons was higher in the OVX+E2+P4 and OVX+E2 animals compared to OVX; (3) overall, the number of DYN A-ir neurons was higher in DM2 vs. control group, and surgery had an effect on the number of DYN A-ir neurons; (4) the metabolic and hormonal profiles were altered in HFD and DM2 animals compared to controls. Current data together with our previously published results indicate sex-specific differences in the response of KNDy neurons to DM2.

Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells.

Phoenixin (PNX) is a recently discovered neuropeptide which modulates appetite, pain sensation and neurons of the reproductive system in the central nervous system. PNX is also detectable in the circulation and in peripheral tissues. Recent data suggested that PNX blood levels positively correlate with body weight as well as nutritional status suggesting a potential role of this peptide in controlling energy homeostasis. PNX is detectable in endocrine pancreas, however it is unknown whether PNX regulates insulin biosynthesis or secretion. Using insulin producing INS-1E cells and isolated rat pancreatic islets we evaluated therefore, whether PNX controls insulin expression, secretion and cell proliferation. We identified PNX in pancreatic alpha as well as in beta cells. Secretion of PNX from pancreatic islets was stimulated by high glucose. PNX stimulated insulin mRNA expression in INS-1E cells. Furthermore, PNX enhanced glucose-stimulated insulin secretion in INS-1E cells and pancreatic islets in a time-dependent manner. Stimulation of insulin secretion by PNX was dependent upon cAMP/Epac signalling, while potentiation of cell growth and insulin mRNA expression was mediated via ERK1/2- and AKT-pathway. These results indicate that PNX may play a role in controlling glycemia by interacting with pancreatic beta cells.

The importance of the nuclear positioning of the PPARG gene for its expression during porcine in vitro adipogenesis.

Proper expression of the PPARG gene, which encodes a key transcription factor of adipogenesis, is indispensable in the formation of mature adipocytes. The positioning of a gene within the nuclear space has been implicated in gene regulation. We here report on the significance of the PPARG gene's nuclear positioning for its activity during in vitro adipogenesis in the pig. We used an established system of differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into adipocytes. The differentiation process was carried out for 7 days, and the cells were examined using the 3D DNA/immuno-FISH and RNA/DNA-FISH approaches. PPARG transcript level was measured using real-time PCR, and PPARγ activity was detected with colorimetric assay. Changes in the nuclear location of the PPARG gene were observed when we compared undifferentiated mesenchymal stem cells with mature adipocytes. The gene moved from the nuclear periphery to the nuclear center as its transcriptional activity increased. The RNA/DNA-FISH approach shows that differences in primary transcript production correlated with the allele's nuclear positioning. Transcriptionally active alleles preferentially occupy the central part of the nucleus, while inactive alleles are found on the nuclear periphery. We also show that transcription of PPARG begins with one allele, but that both alleles are active in later stages of differentiation. Our results provide evidence that functionally distinct alleles of the PPARG gene are positioned in different parts of the cell nucleus. This confirms the importance of nuclear architecture to the regulation of PPARG gene transcription, and thus to the fate of the adipose cell.

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.

High-fat diet and type 2 diabetes induced disruption of the oestrous cycle and alteration of hormonal profiles, but did not affect subpopulations of KNDy neurones in female rats.

Apart from the primary metabolic symptoms of obesity and/or diabetes, there are numerous secondary problems, including disruptions of the reproductive system. The KNDy neurones, which express kisspeptin, neurokinin B and dynorphin A and are located in the arcuate nucleus of the hypothalamus (ARC), are important regulators of reproduction. Their functions are highly influenced by metabolic and hormonal status. We have previously shown that, in male rats with experimentally-induced diabetes type 2 (but not with high-fat diet-induced obesity), there are alterations in the number of these cells. In the present study, we hypothesised that a high-fat diet (HFD) and/or diabetes type 2 (DM2) in female rats affect the oestrous cycle, hormonal profiles and the number of kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. Rats were assigned to one of three groups: a control group fed a regular chow diet, a high-fat diet group (HFD) and a diabetic group (DM2), with both of the latter two groups receiving a high calorie diet (50% of energy from lard). The third group was additionally treated with streptozotocin to induce DM2. Their oestrous cycles was monitored and their metabolic and hormonal status were assessed. We found that HFD and DM2 female rats, despite having significant alterations in their metabolic and hormonal profiles, as well as disruptions of the oestrous cycle, showed no changes in the number of the kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. However, slight differences in the rostrocaudal distribution of these neurones among groups were reported. In conclusion, the data from the present study, together with our previously published results in males, indicate sex differences in the response of KNDy neurones to DM2 but not to HFD conditions.

Spexin: A novel regulator of adipogenesis and fat tissue metabolism.

Spexin (SPX, NPQ) is a novel peptide involved in the regulation of energy metabolism. SPX inhibits food intake and reduces body weight. In obese humans, SPX is the most down-regulated gene in fat. Therefore, SPX might be involved in the regulation of lipid metabolism. Here, we study the effects of SPX on lipolysis, lipogenesis, glucose uptake, adipogenesis, cell proliferation and survival in isolated human adipocytes or murine 3T3-L1 cells. SPX and its receptors, GALR2 and GALR3, are present at mRNA and protein levels in murine 3T3-L1 cells and human adipocytes. SPX inhibits adipogenesis and down-regulates mRNA expression of proadipogenic genes such as Pparγ, C/ebpα, C/ebpß and Fabp4. SPX stimulates lipolysis by increasing the phosphorylation of hormone sensitive lipase (HSL). Simultaneously, SPX inhibits lipogenesis and glucose uptake in human adipocytes and murine 3T3-L1 cells. SPX has no effect on murine 3T3-L1 cell proliferation and viability. Moreover, our research showed that the SPX effect on adipocytes metabolism is mediated via GALR2 and GALR3 receptors. SPX is a novel regulator of lipid metabolism in murine 3T3-L1 and human adipocytes.

Spexin Modulates Functions of Rat Endocrine Pancreatic Cells.

OBJECTIVES: Spexin is a peptide whose action is poorly understood but which is expressed in many tissues. This encouraged us to investigate the potential role of spexin in the regulation of pancreatic secretion. METHODS: Cells/islets were incubated with different concentrations of glucose and spexin to measure insulin secretion. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and BrdU (5-bromo-2'-deoxyuridine) tests were performed to assess the viability and proliferation of pancreatic islets after spexin treatment. Real-time polymerase chain reaction was used to detect messenger RNA expression for insulin, insulin receptor, and Pdx (pancreatic duodenal homeobox-1). RESULTS: Insulin secretion from cultured cells and isolated islets was reduced by spexin at 16 mM glucose level. In obese rats, insulin secretion was decreased after injection with spexin. Spexin treatment showed an increase in cultured cells and pancreatic islets cell viability and proliferation as well as an increase in proliferating cell nuclear antigen protein level. In contrast, a decrease in insulin and Pdx gene expression was found. CONCLUSIONS: The effects of spexin on insulin secretion in vitro and in vivo and also on cells viability and proliferation confirm that this peptide may be strongly involved in the pathogenesis of diabetes or its recovery.

Long-term obestatin treatment of mice type 2 diabetes increases insulin sensitivity and improves liver function.

PURPOSE: Obestatin and ghrelin are peptides encoded by the preproghrelin gene. Obestatin inhibits food intake, in addition to regulation of glucose and lipid metabolism. Here, we test the ability of obestatin at improving metabolic control and liver function in type 2 diabetic animals (type 2 diabetes mellitus). METHODS: The effects of chronic obestatin treatment of mice with experimentally induced type 2 diabetes mellitus on serum levels of glucose and lipids, and insulin sensitivity are characterized. In addition, alterations of hepatic lipid and glycogen contents are evaluated. RESULTS: Obestatin reduced body weight and decreased serum glucose, fructosamine, and ß-hydroxybutyrate levels, as well as total and low-density lipoprotein fractions of cholesterol. In addition, obestatin increased high-density lipoproteins cholesterol levels and enhanced insulin sensitivity in mice with type 2 diabetes mellitus. Moreover, obestatin diminished liver mass, hepatic triglycerides and cholesterol contents, while glycogen content was higher in livers of healthy and mice with type 2 diabetes mellitus treated with obestatin. These changes were accompanied by reduction of increased alanine aminotransferase, aspartate aminotransferase, and gamma glutamyl transpeptidase in T2DM mice with type 2 diabetes mellitus. Obestatin increased adiponectin levels and reduced leptin concentration. Obestatin influenced the expression of genes involved in lipid and carbohydrate metabolism by increasing Fabp5 and decreasing G6pc, Pepck, Fgf21 mRNA in the liver. Obestatin increased both, AKT and AMPK phosphorylation, and sirtuin 1 (SIRT1) protein levels as well as mRNA expression in the liver. CONCLUSION: Obestatin improves metabolic abnormalities in type 2 diabetes mellitus, restores hepatic lipid contents and decreases hepatic enzymes. Therefore, obestatin could potentially have a therapeutic relevance in treating of insulin resistance and metabolic dysfunctions in type 2 diabetes mellitus.

Kisspeptin-10 inhibits proliferation and regulates lipolysis and lipogenesis processes in 3T3-L1 cells and isolated rat adipocytes.

INTRODUCTION: Kisspeptin, which is encoded by the KISS1 gene and acts via GPR54, plays a role in the regulation of reproductive functions. Expression of KISS1 and GRPR54 has been found in peripheral tissues, including adipose tissue, and was shown to be influenced by metabolic status. PURPOSE: We hypothesized that kisspeptin could be involved in regulation of lipid metabolism in the mouse 3T3-L1 cell line and in isolated rat adipocytes. METHODS: First, we characterized expression profiles of KISS1 and GPR54 mRNA and proteins in adipose cells isolated from male rats. Secondly, we studied the effects of kisspeptin-10 on cell proliferation and survival in 3T3-L1 cells. Thirdly, we assessed the rapid action of kisspeptin-10 on lipid metabolism and glucose uptake using 3T3-L1 cells and rat primary adipocytes. Finally, we examined the effects of kisspeptin-10 on the secretion of leptin and adiponectin in rat adipocytes. RESULTS: We have found that: (1) KISS1 and GPR54 were expressed in mouse 3T3-L1 cells and isolated rat adipocytes; (2) kisspeptin-10: (i) inhibited cell proliferation, viability and adipogenesis in 3T3-L1 and decreased expression of PPAR-γ and CEBPß-genes, which are involved in the differentiation processes and adipogenesis; (ii) increased lipolysis in 3T3-L1 cells and rat adipocytes by enhancing expression of periliphin and hormone-sensitive lipase; (iii) modulated glucose uptake and lipogenesis; (iv) stimulated leptin and decreased adiponectin secretion from rat adipocytes. CONCLUSION: Kisspeptin-10 could play a role in the regulation of lipid metabolism in mouse 3T3-L1 cells and rat adipocytes.

Resistin is produced by rat pancreatic islets and regulates insulin and glucagon in vitro secretion.

Resistin participates in the regulation of energy homeostasis, insulin resistance, and inflammation. The potential expression in pancreas, and modulation of the endocrine pancreas secretion by resistin is not well characterized, therefore, we examined it on several levels. We examined the localization of resistin in rat pancreatic islets by immunohistochemistry and immunofluorescence, and the potential presence of resistin mRNA by RT-PCR and protein by Western Blot in these structures. In addition, we studied the regulation of insulin and glucagon secretion by resistin in pancreatic INS-1E ß- and InR-G9 α-cell lines as well as isolated rat pancreatic islets. We identified resistin immunoreactivity in the periphery of rat pancreatic islets and confirmed the expression of resistin at mRNA and protein level. Obtained data indicated that resistin is co-localized with glucagon in pancreatic α-cells. In addition, we found that in vitro resistin decreased insulin secretion from INS-1E cells and pancreatic islets at normal (6 mM) and high (24 mM) glucose concentrations, and also decreased glucagon secretion from G9 cells and pancreatic islets at 1 mM, whereas a stimulation of glucagon secretion was observed at 6 mM glucose. Our results suggest that resistin can modulate the secretion of insulin and glucagon from clonal ß or α cells, and from pancreatic islets.

TRPV6 modulates proliferation of human pancreatic neuroendocrine BON-1 tumour cells.

Highly Ca(2+) permeable receptor potential channel vanilloid type 6 (TRPV6) modulates a variety of biological functions including calcium-dependent cell growth and apoptosis. So far, the role of TRPV6 in controlling growth of pancreatic neuroendocrine tumour (NET) cells is unknown. In the present study, we characterize the expression of TRPV6 in pancreatic BON-1 and QGP-1 NET cells. Furthermore, we evaluate the impact of TRPV6 on intracellular calcium, the activity of nuclear factor of activated T-cells (NFAT) and proliferation of BON-1 cells. TRPV6 expression was assessed by real-time PCR and Western blot. TRPV6 mRNA expression and protein production were down-regulated by siRNA. Changes in intracellular calcium levels were detected by fluorescence calcium imaging (fura-2/AM). NFAT activity was studied by NFAT reporter assay; cell proliferation by bromodeoxyuridine (BrdU), MTT and propidium iodine staining. TRPV6 mRNA and protein are present in BON-1 and QGP-1 NET-cells. Down-regulation of TRPV6 attenuates BON-1 cell proliferation. TRPV6 down-regulation is associated with decreased Ca(2+) response pattern and reduced NFAT activity. In conclusion, TRPV6 is expressed in pancreatic NETs and modulates cell proliferation via Ca(2+)-dependent mechanism, which is accompanied by NFAT activation.

Obestatin stimulates differentiation and regulates lipolysis and leptin secretion in rat preadipocytes.

Obestatin is a 23-amino acid peptide encoded by the ghrelin gene, which regulates food intake, body weight and insulin sensitivity. Obestatin influences glucose and lipid metabolism in mature adipocytes in rodents. However, the role of this peptide in rat preadipocytes remains to be fully understood. The current study characterized the effects of obestatin on lipid accumulation, preadipocyte differentiation, lipolysis and leptin secretion in rat primary preadipocytes. Obestatin enhanced lipid accumulation in rat preadipocytes and increased the expression of surrogate markers of preadipocyte differentiation. At the early stage of differentiation, obestatin suppressed lipolysis. By contrast, lipolysis was stimulated at the late stage of adipogenesis. Furthermore, obestatin stimulated the release of leptin, a key satiety hormone. Overall, the results indicated that obestatin promotes preadipocyte differentiation. Obestatin increased leptin release in preadipocytes, while the modulation of lipolysis appears to depend upon the stage of differentiation.

Neuropeptide B and W regulate leptin and resistin secretion, and stimulate lipolysis in isolated rat adipocytes.

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.