The Metagenomic Composition and Effects of Fecal-Microbe-Derived Extracellular Vesicles on Intestinal Permeability Depend on the Patient's Disease.

The composition and impact of fecal-microbe-derived extracellular vesicles (EVs) present in different diseases has not been analyzed. We determined the metagenomic profiling of feces and fecal-microbe-derived EVs from healthy subjects and patients with different diseases (diarrhea, morbid obesity and Crohn's disease (CD)) and the effect of these fecal EVs on the cellular permeability of Caco-2 cells. The control group presented higher proportions of Pseudomonas and Rikenellaceae_RC9_gut_group and lower proportions of Phascolarctobacterium, Veillonella and Veillonellaceae_ge in EVs when compared with the feces from which these EVs were isolated. In contrast, there were significant differences in 20 genera between the feces and EV compositions in the disease groups. Bacteroidales and Pseudomonas were increased, and Faecalibacterium, Ruminococcus, Clostridium and Subdoligranum were decreased in EVs from control patients compared with the other three groups of patients. Tyzzerella, Verrucomicrobiaceae, Candidatus_Paracaedibacter and Akkermansia were increased in EVs from the CD group compared with the morbid obesity and diarrhea groups. Fecal EVs from the morbid obesity, CD and, mainly, diarrhea induced a significant increase in the permeability of Caco-2 cells. In conclusion, the metagenomic composition of fecal-microbe-derived EVs changes depending on the disease of the patients. The modification of the permeability of Caco-2 cells produced by fecal EVs depends on the disease of the patients.

Oleic acid regulates the circadian rhythm of adipose tissue in obesity.

The effect of oleic acid (OA) on the regulation of the circadian rhythm present in human visceral (VAT) and subcutaneous (SAT) adipose tissue from patients with morbid obesity has not been analyzed yet. VAT and SAT explants from patients with morbid obesity were incubated with OA to analyze the circadian regulation of clock and other genes related to lipid metabolism (SREBP-1c, FAS, LPL and CPT1), and their association with baseline variables and the improvement of these patients after bariatric surgery. There were significant differences in amplitude and acrophase in VAT with respect to SAT. In VAT, body weight negatively correlated with BMAL1 and CRY1 amplitude, and REVERBα acrophase; body mass index (BMI) negatively correlated with REVERBα acrophase; and waist circumference negatively correlated with PER3 acrophase. In SAT, BMI negatively correlated with CLOCK amplitude, and CLOCK, REVERBα and CRY2 MESOR; and waist circumference negatively correlated with PER3 amplitude and acrophase. A greater short-term improvement of body weight, BMI and waist circumference in patients with morbid obesity after bariatric surgery was associated with a lower CRY1 and CRY2 amplitude and an earlier PER1 and PER3 acrophase in SAT. OA produced a more relevant circadian rhythm and increased the amplitude of most clock genes and lipid metabolism-related genes. OA regulated the acrophase of most clock genes in VAT and SAT, placing CLOCK/BMAL1 in antiphase with regard to the other genes. OA increased the circadian rhythmicity, although with slight differences between adipose tissues. These differences could determine its different behavior in obesity.

Morbid Obesity in Women Is Associated with an Altered Intestinal Expression of Genes Related to Cancer Risk and Immune, Defensive, and Antimicrobial Response.

BACKGROUND: Little is known about the relation between morbid obesity and duodenal transcriptomic changes. We aimed to identify intestinal genes that may be associated with the development of obesity regardless of the degree of insulin resistance (IR) of patients. MATERIAL AND METHODS: Duodenal samples were assessed by microarray in three groups of women: non-obese women and women with morbid obesity with low and high IR. RESULTS: We identified differentially expressed genes (DEGs) associated with morbid obesity, regardless of IR degree, related to digestion and lipid metabolism, defense response and inflammatory processes, maintenance of the gastrointestinal epithelium, wound healing and homeostasis, and the development of gastrointestinal cancer. However, other DEGs depended on the IR degree. We mainly found an upregulation of genes involved in the response to external organisms, hypoxia, and wound healing functions in women with morbid obesity and low IR. CONCLUSIONS: Regardless of the degree of IR, morbid obesity is associated with an altered expression of genes related to intestinal defenses, antimicrobial and immune responses, and gastrointestinal cancer. Our data also suggest a deficient duodenal immune and antimicrobial response in women with high IR.

An Isolated Dose of Extra-Virgin Olive Oil Produces a Better Postprandial Gut Hormone Response, Lipidic, and Anti-Inflammatory Profile that Sunflower Oil: Effect of Morbid Obesity.

INTRODUCTION: This study evaluates the effects of 25 mL of three types of oils [extra-virgin olive oil (EVOO), olive oil (OO), and sunflower oil (SO)] on postprandial (3 h) satiety markers and variables related to metabolic status and inflammation in non-obese patients (n = 6) and in those with morbid obesity (n = 6), before and 1 year after Roux-en-Y gastric by-pass (RYGB). METHODS AND RESULTS: After EVOO intake, serum acylated ghrelin decreases and GLP1 increases more than with OO and SO. EVOO causes a higher increase of insulin and lower postprandial hypertriglyceridemia and free fatty acid levels than with OO and SO. EVOO decreases TNFα and IL6 expression in peripheral blood mononuclear cells, with OO inducing intermediate effects and SO inducing an increase of these proinflammatory markers. These results are observed in non-obese patients and in those with morbid obesity after RYGB. However, patients with morbid obesity before RYGB show a profound alteration of this response. CONCLUSION: EVOO produces more beneficial effects than OO, which has lower amounts of minor components, and SO, which has PUFA as its main component. RYGB produces an improvement in the metabolic response to all three types of oils in patients with morbid obesity.

Jejunal Insulin Signalling Is Increased in Morbidly Obese Subjects with High Insulin Resistance and Is Regulated by Insulin and Leptin.

Little is known about the jejunal insulin signalling pathways in insulin resistance/diabetes states and their possible regulation by insulin/leptin. We study in jejunum the relation between insulin signalling and insulin resistance in morbidly obese subjects with low (MO-low-IR) or with high insulin resistance (MO-high-IR), and with type 2 diabetes treated with metformin (MO-metf-T2DM)), and the effect of insulin/leptin on intestinal epithelial cells (IEC). Insulin receptor substrate-1 (IRS1) and the catalytic p110ß subunit (p110ß) of phosphatidylinositol 3-kinase (PI3K) were higher in MO-high-IR than in MO-low-IR. The regulatory p85α subunit of PI3K (p85α)/p110ß ratio was lower in MO-high-IR and MO-metf-T2DM than in MO-low-IR. Akt-phosphorylation in Ser473 was reduced in MO-high-IR compared with MO-low-IR. IRS1 and p110-ß were associated with insulin and leptin levels. The improvement of body mass index (BMI) and HOMA-IR (homeostasis model assessment of insulin resistance index) after bariatric surgery was associated with a higher IRS1 and a lower p85α/p110ß ratio. IEC (intestinal epithelial cells) incubation with a high glucose + insulin dose produced an increase of p85α and p110ß. High dose of leptin produced an increase of IRS1, p85α and p110ß. In conclusion, despite the existence of insulin resistance, the jejunal expression of genes involved in insulin signalling was increased in MO-high-IR. Their expressions were regulated mainly by leptin. IRS1 and p85α/p110ß ratio was associated with the evolution of insulin resistance after bariatric surgery.

The changes in the transcriptomic profiling of subcutaneous adipose tissue after bariatric surgery depend on the insulin resistance state.

BACKGROUND: The changes that are produced in the gene expression of subcutaneous adipose tissue after Roux-en-Y gastric bypass are not yet fully known. OBJECTIVE: To identify the changes in the subcutaneous adipose tissue gene expression of morbidly obese women with low insulin resistance (MO-low-IR) and high insulin resistance (MO-high-IR) to find a relationship with measured obesity-related co-morbidities. SETTING: A university hospital. METHODS: Subcutaneous adipose tissue samples were assessed by microarray analysis before and 2 years after Roux-en-Y gastric bypass in MO-low-IR and MO-high-IR patients. RESULTS: There is a group of shared differentially expressed genes (DEG) in both MO-low-IR and MO-high-IR, also there is a group of exclusive DEG in MO-low-IR and another group in MO-high-IR. In MO-high-IR, the downexpressed DEG are related to the regulation of transcription and are involved in the pathways related to cytokine-cytokine receptor interaction, cancer, phosphatidylinositol 3-kinase-protein kinase B signaling, human T-lymphotropic virus I infection, chemokine signaling, and Janus kinase/signal transducers and activators of transcription signaling. In MO-low-IR, the overexpressed DEG are related to carbohydrate metabolic processes, the downexpressed DEG to the glycosaminoglycan metabolic process and regulation of translation, and the pathways are related to phosphatidylinositol 3-kinase-protein kinase B signaling and metabolic pathways. The fold change of DEG mainly correlates with the percentage of change (Δ) of waist, Δhip, Δglucose, and Δtriglycerides. These DEG were mainly related to cancer, inflammation/immune regulation, metabolic pathways, ribonucleic acid/deoxyribonucleic acid regulation, virus infection, and regulation of cellular proliferation. CONCLUSIONS: This study suggests a potential association between high insulin resistance and the expression of genes related to cancer and chronic immune activation/inflammation.

Tissue-Specific Phenotype and Activation of iNKT Cells in Morbidly Obese Subjects: Interaction with Adipocytes and Effect of Bariatric Surgery.

BACKGROUND: The immune response of visceral adipose tissue (VAT) in obesity, in particular the role of invariant natural killer T (iNKT) cells, has not yet been fully elucidated. OBJECTIVE: To characterize iNKT cells and its activation status in VAT and peripheral blood mononuclear cells (PBMC) in morbidly obese subjects (MO), and to analyze their association with metabolic parameters. SUBJECTS AND METHODS: Twenty non-obese and 20 MO subjects underwent Roux-en-Y gastric bypass (RYGB) and were studied before and 6 months after RYGB. VAT and PBMC were obtained. RESULTS: A decrease in VAT iNKT cells from MO was found, however, not in PBMC. Visceral adipocytes from MO presented increased CD1d expression (p = 0.032). MO presented an increase in early activated CD69+ iNKT cells in PBMC before RYGB (p < 0.001), but not after RYGB nor in VAT, and an increase in later activated CD25+ iNKT in VAT (p = 0.046), without differences in PBMC. The co-expression of early and later markers (CD69+CD25+) in iNKT cells was increased in MO in VAT (p = 0.050) and PBMC (p = 0.006), decreasing after RYGB (p = 0.050). CD69+ iNKT and CD69+CD25+ iNKT cells in PBMC after RYGB correlated negatively with glucose, insulin, and insulin resistance levels. CONCLUSIONS: There is a tissue-specific phenotype and activation of iNKT cells in VAT in morbid obesity, which could be involved in VAT immunometabolism dysregulation. Also, the increase in CD1d expression could be to offset the lack of VAT iNKT cells.

Dietary fatty acids modulate adipocyte TNFa production via regulation of its DNA promoter methylation levels.

The factors regulating TNF alpha (TNFa) levels could be considered therapeutic targets against metabolic syndrome development. DNA methylation is a potent regulator of gene expression and may be associated with protein levels. In this study we investigate whether the effect of dietary fatty acids on TNFa released from adipocytes might be associated with modifications of the TNFa promoter DNA methylation status. A group of rats was assigned to three diets with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of visceral adipose tissues were taken for adipocyte isolation, in which released TNFa levels were measured, and for methylation and expression studies. In addition, 3 T3-L1 cells were treated with palmitic, oleic and linoleic acids, with and without 5-Azacitydine (5-AZA). After treatments, cells and supernatants were included in the same analyses as rat samples. TNFa promoter methylation levels, gene expression and secretion were different according to the diets and fatty acid treatments associated with them. Cells treated with 5-AZA displayed higher TNFa levels than in the absence of 5-AZA, without differences between fatty acids. According to our results, dietary fatty acid regulation of adipocyte TNFa levels may be mediated by epigenetic modifications of the TNFa promoter DNA methylation levels.

Changes in SCD gene DNA methylation after bariatric surgery in morbidly obese patients are associated with free fatty acids.

Stearoyl CoA Desaturase-1 (SCD) is considered as playing an important role in the explanation of obesity. The aim of this study was to evaluate whether the DNA methylation SCD gene promoter is associated with the metabolic improvement in morbidly obese patients after bariatric surgery. The study included 120 subjects with morbid obesity who underwent a laparoscopic Roux-en Y gastric by-pass (RYGB) and a control group of 30 obese subjects with a similar body mass index (BMI) to that found in morbidly obese subjects six months after RYGB. Fasting blood samples were obtained before and at six months after RYGB. DNA methylation was measured by pyrosequencing technology. DNA methylation levels of the SCD gene promoter were lower in morbidly obese subjects before bariatric surgery but increased after RYGB to levels similar to those found in the control group. Changes of DNA methylation SCD gene were associated with the changes of free fatty acids levels (r = -0.442, p = 0.006) and HOMA-IR (r = -0.249, p = 0.035) after surgery. RYGB produces an increase in the low SCD methylation promoter levels found in morbidly obese subjects. This change of SCD methylation levels is associated with changes in FFA and HOMA-IR.

Jejunal gluconeogenesis associated with insulin resistance level and its evolution after Roux-en-Y gastric bypass.

BACKGROUND: Intestinal gluconeogenesis (GNG) may play an important role in glucose homeostasis, but there is little information about the condition in humans. OBJECTIVES: To study the relationship between intestinal GNG and insulin resistance, its association with the evolution of morbidly obese patients after bariatric surgery, and the effect of insulin and or leptin. SETTING: Regional university hospital, Malaga (Spain). METHODS: Jejunal mRNA expression of genes involved in GNG was analyzed in 3 groups of morbidly obese patients who underwent Roux-en-Y gastric bypass: with low insulin resistance (MO-low-IR), with high insulin resistance (MO-high-IR), and with type 2 diabetes treated with metformin (MO-metf-T2D). Also, intestinal epithelial cells (IEC) from MO-low-IR were incubated with different doses of insulin and or leptin. RESULTS: In MO-high-IR, glutaminase, phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6 Pase), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 α), and sterol regulatory element-binding proteins 1 c (SREBP-1 c) expressions were significantly higher than in MO-low-IR. In MO-metf-T2 D, only PEPCK was significantly lower than in MO-high-IR. In IEC, an incubation with a high glucose and insulin dose produced an increase of PEPCK and SREBP-1 c, and a decrease of glutaminase, fructose 1,6-bisphosphatase (FBPase), and PGC-1 α expression. At high doses of leptin, G6 Pase and FBPase were significantly increased. The improvement of insulin resistance 3 months after bariatric surgery was positively associated with high G6 Pase and FBPase expression. CONCLUSION: mRNA expression of genes involved in GNG is increased in the jejunum of MO-high-IR, and regulated by insulin and or leptin. High mRNA expression of genes involved in GNG is associated with a better evolution of insulin resistance after bariatric surgery.

The pro-/anti-inflammatory effects of different fatty acids on visceral adipocytes are partially mediated by GPR120.

PURPOSE: This study examines whether G-protein coupled receptor 120 (GPR120) is involved in the pro-/anti-inflammatory effects of different types of fatty acids (FAs) in human visceral adipocytes, and whether these effects may be altered in obesity, a state with a chronic inflammation. METHODS: Pro-/anti-inflammatory effects of palmitic, oleic, linoleic and docosahexaenoic acids on human visceral adipocytes were tested in mature adipocytes from non-obese and morbidly obese (MO) subjects. Also, the effects of these FAs were tested when the GPR120 gene was silenced. RESULTS: In adipocytes from non-obese subjects, palmitic and linoleic acids increased TNF-α and IL-6 mRNA expression (p < 0.05), and decreased IL-10 and adiponectin expression (p < 0.05). However, oleic and docosahexaenoic acids (DHA) produced the opposite effect (p < 0.05). In adipocytes from MO subjects, all FAs used increased TNF-α and IL-6 expression (p < 0.05). Palmitic and linoleic acids decreased IL-10 and adiponectin expression (p < 0.05), but oleic acid and DHA did not have significant effects. Only oleic acid increased adiponectin expression (p < 0.05). The effects of FAs on TNF-α, IL-6, IL-10 and adiponectin expression in non-obese and MO subjects were significantly annulled when the GPR120 gene was silenced in visceral adipocytes differentiated from human mesenchymal stem cells. CONCLUSIONS: FAs are capable of directly acting on visceral adipocytes to modulate differently TNF-α, IL-6, IL-10 and adiponectin expression, with a different and greater effect in MO subjects. These effects are largely annulled when GPR120 expression was silenced, which suggests that they could be mediated by GPR120.

Methylation patterns of Vegfb promoter are associated with gene and protein expression levels: the effects of dietary fatty acids.

PURPOSE: We have investigated the epigenetic regulation by dietary fatty acids of Vegfb levels in rats' white adipose tissue and 3T3-L1 cells. METHODS: A group of rats were assigned to three diets, each one with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of white adipose tissues were taken for the methylation and expression studies. Additionally, 3T3-L1 cells were treated with palmitic, oleic, and linoleic fatty acids. After treatment, cells were harvested and genetic material was extracted for the analysis of Vegfb levels. RESULTS: We report evidence of changes in the methylation levels of the CpG island at the Vegfb promoter and in the Vegfb expression levels in vivo and in vitro by dietary fatty acid, with the main contribution of the linoleic fatty acid. Vegfb promoter methylation levels were closely related to the Vegfb gene expression. CONCLUSION: According to our results, the regulation of Vegfb gene expression by dietary fatty acids may be mediated, at least in part, by epigenetic modifications on Vegfb promoter methylation. Considering the deep association between angiogenesis and tissue growth, we suggest the nutriepigenetic regulation of Vegfb as a key target in the control of the adipose tissue expansion.

GLP-1 and estrogen conjugate acts in the supramammillary nucleus to reduce food-reward and body weight.

The obesity epidemic continues unabated and currently available pharmacological treatments are not sufficiently effective. Combining gut/brain peptide, GLP-1, with estrogen into a conjugate may represent a novel, safe and potent, strategy to treat diabesity. Here we demonstrate that the central administration of GLP-1-estrogen conjugate reduced food reward, food intake, and body weight in rats. In order to determine the brain location of the interaction of GLP-1 with estrogen, we avail of single-photon emission computed tomography imaging of regional cerebral blood flow and pinpoint a brain site unexplored for its role in feeding and reward, the supramammillary nucleus (SUM) as a potential target of the conjugated GLP-1-estrogen. We confirm that conjugated GLP-1 and estrogen directly target the SUM with site-specific microinjections. Additional microinjections of GLP-1-estrogen into classic energy balance controlling nuclei, the lateral hypothalamus (LH) and the nucleus of the solitary tract (NTS) revealed that the metabolic benefits resulting from GLP-1-estrogen injections are mediated through the LH and to some extent by the NTS. In contrast, no additional benefit of the conjugate was noted on food reward when the compound was microinjected into the LH or the NTS, identifying the SUM as the only neural substrate identified here to underlie the reward reducing benefits of GLP-1 and estrogen conjugate. Collectively we discover a surprising neural substrate underlying food intake and reward effects of GLP-1 and estrogen and uncover a new brain area capable of regulating energy balance and reward.

Effect of Roux-en-Y gastric bypass-induced weight loss on the transcriptomic profiling of subcutaneous adipose tissue.

BACKGROUND: The changes in the transcriptomic profiling of subcutaneous adipose tissue (SAT) when weight loss stabilizes after a Roux-en-Y gastric bypass (RYGB) are still largely unknown. OBJECTIVES: To investigate the changes produced in SAT gene expression of morbidly obese women when their weight loss stabilizes 2 years after RYGB. SETTING: University hospital. METHODS: SAT biopsies of the periumbilical area were taken before and 2 years after RYGB. Gene expression levels were assessed by microarray analysis and significant differences in gene expression were validated by real-time quantitative polymerase chain reaction. The findings were also confirmed in an independent population of morbidly obese women. RESULTS: Microarray analysis revealed that the overexpressed differentially expressed genes have a prominent role in the pathways involved in biosynthetic processes, especially lipid or carboxylic ones (stearoyl-Coenzyme A desaturase-1, fatty acid desaturase-1, fatty acid elongase-6, ATP citrate lyase, fatty acid synthase, lipin-1, monoacylglycerol O-acyltransferase, patatin-like phospholipase domain containing-3, phosphate cytidylyltransferase-2, cholesteryl ester transfer protein, transmembrane 7 superfamily member 2, pyruvate carboxylase, and glycogen synthase 2). Most of the underexpressed differentially expressed genes are related with immune system and inflammation processes (immune responses, response to stress, cell death, regulation of biological quality, immune effector process, the response to endogenous stimulus, and the response to other types of stimulus). CONCLUSION: An improvement of the SAT inflammatory and immune profile and an induction of genes involved in the regulation of lipid metabolism are shown when weight loss stabilizes 2 years after RYGB. Most of the genes shown are clearly linked to obesity and other metabolic disorders.

The expression of genes involved in jejunal lipogenesis and lipoprotein synthesis is altered in morbidly obese subjects with insulin resistance.

The dyslipidemia associated with type 2 diabetes mellitus (T2DM) is an important risk factor for atherosclerotic cardiovascular disease. However, until now little attention has been paid to the role that the intestine might have. The aim of this research was to determine the relation between insulin resistance and intestinal de novo lipogenesis/lipoprotein synthesis in morbidly obese subjects and to study the effect of insulin on these processes. Jejunal mRNA expression of the different genes involved in the intestinal de novo lipogenesis/lipoprotein synthesis was analyzed in three groups of morbidly obese subjects: Group 1 with low insulin resistance (MO-low-IR), group 2 with high insulin resistance (MO-high-IR), and group 3 with T2DM and treatment with metformin (MO-metf-T2DM). In addition, intestinal epithelial cells (IECs) from MO-low-IR were incubated with different doses of insulin/glucose. In Group 2 (MO-high-IR), the jejunal mRNA expression levels of apo A-IV, ATP-citrate lyase (ACLY), pyruvate dehydrogenase (lipoamide) beta (PDHB), and sterol regulatory element-binding protein-1c (SREBP-1c) were significantly higher and acetyl-CoA carboxylase alpha (ACC1) and fatty-acid synthase lower than in Group 1 (MO-low-IR). In Group 3 (MO-metf-T2DM), only the ACLY and PDHB mRNA expressions were significantly higher than in Group 1 (MO-low-IR). The mRNA expression of most of the genes studied was significantly linked to insulin and glucose levels. The incubation of IEC with different doses of insulin and glucose produced a higher expression of diacylglycerol acyltransferase 2, microsomal triglyceride transfer protein, apo A-IV, SREBP-1c, and ACC1 when both, glucose and insulin, were at a high concentration. However, with only high insulin levels, there were higher apo A-IV, PDHB and SREBP-1c expressions, and a lower ACLY expression. In conclusion, the jejunum of MO-high-IR has a decreased mRNA expression of genes involved in de novo fatty-acid synthesis and an increase of genes involved in acetyl-CoA and lipoprotein synthesis. This effect is attenuated by metformin. In addition, the expression of most of the genes studied was found to be regulated by insulin.

FNDC5 could be regulated by leptin in adipose tissue.

INTRODUCTION: Irisin activates the thermogenic function in adipose tissues. However, little is known on the association between human irisin and different cardiometabolic risk factors. We analyse the influence of morbid obesity on irisin levels and its relation with leptin and different cardiovascular risk factors. MATERIAL AND METHODS: We measured the serum irisin level and the fibronectin type III domain containing 5 (FNDC5) expression in adipose tissue from 33 morbidly obese subjects and 12 nonobese subjects. We also studied the effect of leptin on FNDC5 expression. RESULTS: Serum irisin was higher in the nonobese subjects than in morbidly obese subjects, both before (P = 0·043) and after bariatric surgery (P = 0·042). The variable that best explained the serum irisin levels in a multiple linear regression model was the waist-to-hip ratio (WHR) (R(2) = 0·201) (Beta = -0·357, P = 0·046). Those morbidly obese subjects with android-type obesity had lower serum irisin levels than those with gynecoid-type obesity, both before (P = 0·027) and after bariatric surgery (P = 0·006). Only the percentage change in WHR was associated with serum irisin levels after bariatric surgery (r = -0·529, P = 0·005). FNDC5 expression levels in subcutaneous adipose tissue (SAT) were higher in the nonobese than in the morbidly obese subjects (P = 0·042). In SAT explants from nonobese subjects, leptin (20 and 150 ng/mL) produced a decrease in FNDC5 expression (P = 0·009 and P = 0·037, respectively). CONCLUSIONS: We showed decreased serum irisin levels in morbidly obese subjects, related mainly to WHR. FNDC5 expression could be regulated by leptin.

Resistin regulates pituitary somatotrope cell function through the activation of multiple signaling pathways.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues and organs, including the pituitary. However, there is no information on whether resistin, as described previously for other adipokines such as leptin and adiponectin, could regulate this gland. Likewise, the molecular basis of resistin actions remains largely unexplored. Here we show that administration of resistin to dispersed rat anterior pituitary cells increased GH release in both the short (4 h) and long (24 h) term, decreased mRNA levels of the receptor of the somatotrope regulator ghrelin, and increased free cytosolic Ca(2+) concentration in single somatotropes. By means of a pharmacological approach, we found that the stimulatory action of resistin occurs through a Gs protein-dependent mechanism and that the adenylate cyclase/cAMP/protein kinase A pathway, the phosphatidylinositol 3-kinase/Akt pathway, protein kinase C, and extracellular Ca(2+) entry through L-type voltage-sensitive Ca(2+) channels are essential players in mediating the effects of resistin on somatotropes. Taken together, our results demonstrate for the first time a regulatory role for resistin on somatotrope function and provide novel insights on the intracellular mechanisms activated by this protein.

Regulation of pituitary cell function by adiponectin.

Adiponectin is a member of the family of adipose tissue-related hormones known as adipokines, which exerts antidiabetic, antiatherogenic, antiinflammatory, and antiangiogenic properties. Adiponectin actions are primarily mediated through binding to two receptors expressed in several tissues, AdipoR1 and AdipoR2. Likewise, adiponectin expression has been detected in adipocytes as well as in a variety of extra-adipose tissues, including the chicken pituitary. Interestingly, adiponectin secretion and adiponectin receptor expression in adipocytes have been shown to be regulated by pituitary hormones. These observations led us to investigate whether adiponectin, like the adipokine leptin, regulates pituitary hormone production. Specifically, we focused our analysis on somatotrophs and gonadotrophs because of the relationship between the control of energy metabolism, growth and reproduction. To this end, the effects of adiponectin on both GH and LH secretion as well as its interaction with major stimulatory regulators of somatotrophs (ghrelin and GHRH) and gonadotrophs (GnRH) and with their corresponding receptors (GHS-R, GHRH-R, and GnRH-R), were evaluated in rat pituitary cell cultures. Results show that adiponectin inhibits GH and LH release as well as both ghrelin-induced GH release and GnRH-stimulated LH secretion in short-term (4 h) treated cell cultures, wherein the adipokine also increases GHRH-R and GHS-R mRNA content while decreasing that of GnRH-R. Additionally, we demonstrate that the pituitary expresses both adiponectin and adiponectin receptors under the regulation of the adipokine. In sum, our data indicate that adiponectin, either locally produced or from other sources, may play a neuroendocrine role in the control of both somatotrophs and gonadotrophs.

Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes.

Ghrelin is a newly discovered peptide that binds the receptor for GH secretagogues (GHS-R). The presence of both ghrelin and GHS-Rs in the hypothalamic-pituitary system, together with the ability of ghrelin to increase GH release, suggests a hypophysiotropic role for this peptide. To ascertain the intracellular mechanisms mediating the action of ghrelin in somatotropes, we evaluated ghrelin-induced GH release from pig pituitary cells both under basal conditions and after specific blockade of key steps of cAMP-, inositol phosphate-, and Ca2+-dependent signaling routes. Ghrelin stimulated GH release at concentrations ranging from 10-10 to 10-6 m. Its effects were comparable with those exerted by GHRH or the GHS L-163,255. Combined treatment with ghrelin and GHRH or L-163,255 did not cause further increases in GH release, whereas somatostatin abolished the effect of ghrelin. Blockade of phospholipase C or protein kinase C inhibited ghrelin-induced GH secretion, suggesting a requisite role for this route in ghrelin action. Unexpectedly, inhibition of either adenylate cyclase or protein kinase A also suppressed ghrelin-induced GH release. In addition, ghrelin stimulated cAMP production and also had an additive effect with GHRH on cAMP accumulation. Ghrelin also increased free intracellular Ca2+ levels in somatotropes. Moreover, ghrelin-induced GH release was entirely dependent on extracellular Ca2+ influx through L-type voltage-sensitive channels. These results indicate that ghrelin exerts a direct stimulatory action on porcine GH release that is not additive with that of GHRH and requires the contribution of a multiple, complex set of interdependent intracellular signaling pathways.