Role of ghrelin isoforms in the mitigation of hepatic inflammation, mitochondrial dysfunction, and endoplasmic reticulum stress after bariatric surgery in rats.

BACKGROUND/OBJECTIVES: Bariatric surgery improves nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain elusive. We evaluated the potential role of ghrelin isoforms in the amelioration of hepatic inflammation after sleeve gastrectomy and Roux-en-Y gastric bypass (RYGB). SUBJECTS/METHODS: Plasma ghrelin isoforms were measured in male Wistar rats (n = 129) subjected to surgical (sham operation, sleeve gastrectomy, or RYGB) or dietary interventions [fed ad libitum a normal (ND) or a high-fat diet (HFD) or pair-fed diet]. The effect of acylated and desacyl ghrelin on markers of inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in primary rat hepatocytes under palmitate-induced lipotoxic conditions was assessed. RESULTS: Plasma desacyl ghrelin was decreased after sleeve gastrectomy and RYGB, whereas the acylated/desacyl ghrelin ratio was augmented. Both surgeries diminished obesity-associated hepatic steatosis, CD68+- and apoptotic cells, proinflammatory JNK activation, and Crp, Tnf, and Il6 transcripts. Moreover, a postsurgical amelioration in the mitochondrial DNA content, oxidative phosphorylation (OXPHOS) complexes I and II, and ER stress markers was observed. Specifically, following bariatric surgery GRP78, spliced XBP-1, ATF4, and CHOP levels were reduced, as were phosphorylated eIF2α. Interestingly, acylated and desacyl ghrelin inhibited steatosis and inflammation of palmitate-treated hepatocytes in parallel to an upregulation of OXPHOS complexes II, III, and V, and a downregulation of ER stress transducers IRE1α, PERK, ATF6, their downstream effectors, ATF4 and CHOP, as well as chaperone GRP78. CONCLUSIONS: Our data suggest that the increased relative acylated ghrelin levels after bariatric surgery might contribute to mitigate obesity-associated hepatic inflammation, mitochondrial dysfunction, and ER stress.

Targeted disruption of the iNOS gene improves adipose tissue inflammation and fibrosis in leptin-deficient ob/ob mice: role of tenascin C.

BACKGROUND/OBJECTIVES: Obesity is related to a dynamic extracellular matrix (ECM) remodeling, which involves the synthesis and degradation of different proteins, such as tenascin C (TNC) in the adipose tissue (AT). Given the functional relationship between leptin and inducible nitric oxide synthase (iNOS), our aim was to analyze the impact of the absence of the iNOS gene in AT inflammation and ECM remodeling in ob/ob mice. SUBJECTS/METHODS: The expression of genes involved in inflammation and ECM remodeling was evaluated in 10-week-old male double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes as well as in ob/ob mice classified into three groups [control, leptin-treated (1 mg kg-1 day-1) and pair-fed]. RESULTS: Leptin deficiency increased inflammation and fibrosis in AT. As expected, leptin treatment improved the obesity phenotype. iNOS deficiency in ob/ob mice improved insulin sensitivity, AT inflammation, and ECM remodeling, as evidenced by lower AT macrophage infiltration and collagen deposition, a downregulation of proinflammatory and profibrogenic genes Tnf, Emr1, Hif1a, Col6a1, Col6a3, and Tnc, as well as lower circulating TNC levels. Interestingly, leptin upregulated TNC expression and release in 3T3-L1 adipocytes, and iNOS knockdown in 3T3-L1 fat cells produced a significant decrease in basal and leptin-induced Tnc expression. CONCLUSIONS: Ablation of iNOS in leptin-deficient mice improved AT inflammation and ECM remodeling-related genes, attenuating fibrosis, and metabolic dysfunction. The activation of iNOS by leptin is necessary for the synthesis and secretion of TNC in adipocytes, suggesting an important role of this alarmin in the development of AT inflammation and fibrosis.

Role of aquaporin-7 in ghrelin- and GLP-1-induced improvement of pancreatic ß-cell function after sleeve gastrectomy in obese rats.

BACKGROUND/OBJECTIVES: Glycerol is a key metabolite for lipid accumulation in insulin-sensitive tissues as well as for pancreatic insulin secretion. We examined the role of aquaporin-7 (AQP7), the main glycerol channel in ß-cells, and AQP12, an aquaporin related to pancreatic damage, in the improvement of pancreatic function and steatosis after sleeve gastrectomy in diet-induced obese rats. SUBJECTS/METHODS: Male Wistar obese rats (n=125) were subjected to surgical (sham operation and sleeve gastrectomy) or dietary (pair-fed to the amount of food eaten by sleeve-gastrectomized animals) interventions. The tissue distribution and expression of AQPs in the rat pancreas were analyzed by real-time PCR, western blotting and immunohistochemistry. The effect of ghrelin isoforms and glucagon-like peptide 1 (GLP-1) on insulin secretion, triacylglycerol (TG) accumulation and AQP expression was determined in vitro in RIN-m5F ß-cells. RESULTS: Sleeve gastrectomy reduced pancreatic ß-cell apoptosis, steatosis and insulin secretion. Lower ghrelin and higher GLP-1 concentrations were also found after bariatric surgery. Acylated and desacyl ghrelin increased TG content, whereas GLP-1 increased insulin release in RIN-m5F ß-cells. Sleeve gastrectomy was associated with an upregulation of AQP7 together with a normalization of the increased AQP12 levels in the rat pancreas. Interestingly, ghrelin and GLP-1 repressed AQP7 and AQP12 expression in RIN-m5F ß-cells. AQP7 protein was negatively correlated with intracellular lipid accumulation in acylated ghrelin-treated cells and with insulin release in GLP-1-stimulated ß-cells. CONCLUSIONS: AQP7 upregulation in ß-cells after sleeve gastrectomy contributes, in part, to the improvement of pancreatic steatosis and insulin secretion by increasing intracellular glycerol used for insulin release triggered by GLP-1 rather than for ghrelin-induced TG biosynthesis.

Crosstalk between adipokines and myokines in fat browning.

Skeletal muscle is the largest organ determining whole-body insulin sensitivity and metabolic homoeostasis. Adaptive changes of skeletal muscle in response to physical activity include adjustments in the production and secretion of muscle-derived bioactive factors, known as myokines, such as myostatin, IL-4, IL-6, IL-7 and IL-15, myonectin, follistatin-like 1 or leukaemia inhibitory factor. These myokines not only act locally in the muscle in an autocrine/paracrine manner, but also are released to the bloodstream as endocrine factors to regulate physiological processes in other tissues. Irisin, derived from the cleavage of FNDC5 protein, constitutes a myokine that induces myogenesis and fat browning (switch of white adipocytes to brown fat-like cells) together with a concomitant increase in energy expenditure. Besides being a target for irisin actions, the adipose tissue also constitutes a production site of FNDC5. Interestingly, irisin secretion from subcutaneous and visceral fat depots is decreased by long-term exercise training and fasting, suggesting a discordant regulation of FNDC5/irisin in skeletal muscle and adipose tissue. Accordingly, our group has recently reported that the adipokine leptin differentially regulates FNDC5/irisin expression in skeletal muscle and fat, confirming the crosstalk between both tissues. Moreover, irisin secretion and function are regulated by other myokines, such as follistatin or myostatin, as well as by other adipokines, including fibroblast growth factor 21 and leptin. Taken together, myokines have emerged as novel molecular mediators of fat browning and their activity can be modulated by adipokines, confirming the crosstalk between skeletal muscle and adipose tissue to regulate thermogenesis and energy expenditure.

Guanylin and uroguanylin stimulate lipolysis in human visceral adipocytes.

BACKGROUND/OBJECTIVES: Uroguanylin and guanylin are secreted by intestinal epithelial cells as prohormones postprandially and act on the hypothalamus to induce satiety. The impact of obesity and obesity-associated type 2 diabetes (T2D) on proguanylin and prouroguanylin expression/secretion as well as the potential role of guanylin and uroguanylin in the control of lipolysis in humans was evaluated. SUBJECTS/METHODS: Circulating and gastrointestinal expression of proguanylin (GUCA2A) and prouroguanylin (GUCA2B) were measured in 134 subjects. In addition, plasma proguanylin and prouroguanylin were measured before and after weight loss achieved either by Roux-en-Y gastric bypass (RYGB) (n=24) or after a conventional diet (n=15). The effect of guanylin and uroguanylin (1-100 nmol l(-1)) on lipolysis was determined in vitro in omental adipocytes. RESULTS: Circulating concentrations of prouroguanylin, but not proguanylin, were decreased in obesity in relation to adiposity. Weight loss achieved by RYGB increased plasma proguanylin and prouroguanylin. Obese T2D individuals showed higher expression of intestinal GUCA2A as well as of the receptors of the guanylin system, GUCY2C and GUCY2D, in omental adipocytes. The incubation with guanylin and uroguanylin significantly stimulated lipolysis in differentiated omental adipocytes, as evidenced by hormone-sensitive lipase phosphorylation at Ser563, an increase in fatty acids and glycerol release together with an upregulation of several lipolysis-related genes, including AQP3, AQP7, FATP1 or CD36. CONCLUSIONS: Both guanylin and uroguanylin trigger lipolysis in human visceral adipocytes. Given the lipolytic action of the guanylin system on visceral adipocytes, the herein reported decrease of circulating prouroguanylin concentrations in obese patients may have a role in excessive fat accumulation in obesity.

Leptin administration activates irisin-induced myogenesis via nitric oxide-dependent mechanisms, but reduces its effect on subcutaneous fat browning in mice.

UNLABELLED: BACKGROUND/OBJETIVES: Obese leptin-deficient ob/ob mice exhibit high adiposity and reduced muscle mass with leptin replacement promoting weight loss and inducing muscle accretion through PGC-1α-dependent mechanisms. Our aim was to analyze in vivo and in vitro the effect of leptin on FNDC5, a novel PGC-1α-dependent myokine that is synthesized and cleaved to form irisin that induces white adipose browning. METHODS/RESULTS: Twelve-week-old male wild-type and ob/ob mice were divided in three groups as follows: control, leptin-treated (1 mg kg(-1) day(-1)) and pair-fed. Leptin administration was associated with increased gastrocnemius weight and cell surface area, higher Pgc1a and Fndc5 transcript levels and a slight increase in circulating irisin. Leptin upregulated Fndc5 expression through nitric oxide (NO)-dependent mechanisms in murine C2C12 myocytes and stimulated both basal and irisin-stimulated myogenesis, as evidenced by increased myocyte cell proliferation, higher myogenin and myonectin transcript levels together with lower mRNA expression of myostatin and dystrophin and the muscle atrophy-related factors MuRF1 and MAFbx. Interestingly, leptin downregulated Fndc5 expression in a NO-independent manner in murine differentiated subcutaneous adipocytes. Furthermore, leptin prevented the irisin-induced upregulation of both brown (Ucp1 and Cidec) and beige (Tmem26) adipocyte-specific genes and the increase in uncoupling protein-1-positive cells. CONCLUSIONS: Taken together, our results provide evidence for a regulatory role of leptin on FNDC5/irisin, favoring muscle accretion but reducing fat browning.

Reduced hepatic aquaporin-9 and glycerol permeability are related to insulin resistance in non-alcoholic fatty liver disease.

BACKGROUND/OBJECTIVES: Glycerol represents an important metabolite for the control of lipid accumulation and hepatic gluconeogenesis. We investigated whether hepatic expression and functionality of aquaporin-9 (AQP9), a channel mediating glycerol influx into hepatocytes, is impaired in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in the context of insulin resistance. SUBJECTS/METHODS: Liver biopsies were obtained from 66 morbid obese patients undergoing bariatric surgery (66% women, mean body mass index (BMI) 46.1±1.0 kg m(-2)) with available liver echography and pathology analysis of the biopsies in this cross-sectional study. Subjects were classified according to normoglycemia (NG), impaired glucose tolerance (IGT) or type 2 diabetes (T2D). Hepatic expression of AQP9 was analyzed by real-time PCR, western blotting and immunohistochemistry, while glycerol permeability (P(gly)) was measured by stopped-flow light scattering. RESULTS: AQP9 was the most abundantly (P<0.0001) expressed aquaglyceroporin in human liver (AQP9>>>AQP3>AQP7>AQP10). Obese patients with T2D showed increased plasma glycerol as well as lower P(gly) and hepatic AQP9 expression. The prevalence of NAFLD and NASH in T2D patients was 100 and 65%, respectively. Interestingly, AQP9 expression was decreased in patients with NAFLD and NASH as compared with those without hepatosteatosis, in direct relation to the degree of steatosis and lobular inflammation, being further reduced in insulin-resistant individuals. The association of AQP9 with insulin sensitivity was independent of BMI and age. Consistent with these data, fasting insulin and C-reactive protein contributed independently to 33.1% of the hepatic AQP9 mRNA expression variance after controlling for the effects of age and BMI. CONCLUSIONS: AQP9 downregulation together with the subsequent reduction in hepatic glycerol permeability in insulin-resistant states emerges as a compensatory mechanism whereby the liver counteracts further triacylglycerol accumulation within its parenchyma as well as reduces hepatic gluconeogenesis in patients with NAFLD.

Short- and long-term changes in gastric morphology and histopathology following sleeve gastrectomy in diet-induced obese rats.

BACKGROUND: Sleeve gastrectomy (SG) has been used as a multipurpose surgical procedure for the treatment of morbid obesity. The aim of the study was to analyze gastric morphology and histology at two different time points after SG in rats. METHODS: Thirty-five male Wistar rats were fed ad libitum during 3 months on a high-fat diet to induce obesity. Subsequently, 25 diet-induced obese rats underwent either SG (n = 12) or a sham operation (n = 13). The remaining ten obese animals encompassed the nonoperated control group (Co). Four weeks postoperatively, 15 rats (n = 5 rats/experimental group) were sacrificed, while the remaining 20 rats were sacrificed after 16 weeks (animals/group; Co = 5, sham = 8, SG = 7) to compare the gastric morphological and histopathological changes over time. Body weight and food intake were regularly recorded. RESULTS: For both time periods, the Co groups exhibited the highest body weight, while the rats undergoing the SG showed the lowest weight gain (P < 0.05). Initially, significant differences (P < 0.005) in food intake relative to body weight were observed between the Co rats and animals undergoing surgery, which disappeared thereafter. The actual total stomach size after both experimental periods in the SG group was similar to that of non- and sham-operated rats mainly due to a forestomach enlargement, which was more pronounced after 16 weeks. Traits of gastritis cystica profunda characterized by gastric foveolae elongation with hyperplasia and cystic dilatation of the glands were observed in the residual stomachs of the sleeve-gastrectomized rats. These findings were mostly observed after 16 weeks of performing the SG, although they were also detected occasionally following 4 weeks postoperatively. No intestinal metaplasia was observed. CONCLUSION: After SG gastric macro- and microscopic changes with functional implications in both the short and long term take place.