Palmitic acid induces guanylin gene expression through the Toll-like receptor 4/nuclear factor-κB pathway in rat macrophages.

Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.

Influence of food texture on energy metabolism and adiposity in male rats.

NEW FINDINGS: What is the central question of this manuscript? What is the effect of food texture on fat accumulation, lipogenesis and proinflammatory factors in the adipose tissue and on energy balance in male rats? What is the main finding and its importance? Calorie intake and fat accumulation in rats fed soft pellets ad libitum increased, but their body weight did not. The data suggest that, even when BMI is normal, frequent consumption of soft food may contribute to the development of lifestyle-related diseases. ABSTRACT: Dietary factors such as food texture are known to affect feeding behaviour and energy metabolism. We recently found that rats fed soft pellets (SPs) on a 3 h restricted feeding schedule showed glucose intolerance, insulin resistance with disruption of insulin signalling, and hyperplasia of pancreatic ß-cells, even though there were no differences in energy intake and body weight between rats fed control pellets (CPs) and rats fed SPs. We investigated the effect of food texture on fat accumulation, lipogenesis and proinflammatory factors in the mesenteric fat, as well as on energy balance in male rats fed CPs or SPs. We used 7-week-old Wistar rats that were randomly divided into two groups, ad libitum fed either CPs or SPs for 27 weeks. Body weight and calorie intake were monitored once a week throughout the experiment. The calorie intake, lipogenesis and fat accumulation of the rats fed SPs increased, whereas their body weight did not. Additionally, SP rats used their fat mainly as a source of energy and increased their energy expenditure. Our data suggest that the habit of frequently eating soft food causes visceral fat accumulation without an increase in body weight. Further investigations using soft-textured foods could lead to the development of appropriate interventions for non-overweight patients with lifestyle-related diseases.

Guanylin-Guanylyl cyclase-C signaling in macrophages regulates mesenteric fat inflammation induced by high-fat diet.

Guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), are primarily present in the intestine and maintain homeostasis in body fluids. Recently, rats whose macrophages overexpress Gn and GC-C were found to be resistant to diet-induced obesity. Considering that obesity is strongly related to a chronic inflammatory state in white adipose tissues, it is possible that Gn-GC-C macrophages contribute to the regulation of inflammation. In the present study, we investigated the inflammatory state of mesenteric fat in rats transgenic for both Gn and GC-C (double-transgenic [dTg] rats) by evaluating the levels of cyclic guanosine monophosphate (cGMP), a second messenger of Gn-GC-C, cGMP-dependent protein kinase (PKG), and phosphorylated vasodilator-stimulated phosphoprotein (VASP), a target protein of PKG. The levels of cGMP in dTg rats was higher than in WT rats fed the same diet. Although there were no significant differences in levels of PKG and phosphorylated VASP between WT and dTg rats fed a standard diet (STD), these levels in dTg rats fed a high fat diet (HFD) were markedly increased compared with levels in HFD WT rats. Furthermore, mRNA levels of proinflammatory factors in mesenteric fat were lower in HFD dTg rats than in HFD WT rats and were similar to levels in STD WT and dTg rats. These results indicate that the Gn-GC-C system in macrophages regulates the cGMP-PKG-VASP pathway and controls obesity through the downregulation of proinflammatory factors.

Coinjection of CCK and leptin reduces food intake via increased CART/TRH and reduced AMPK phosphorylation in the hypothalamus.

CCK and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important for elucidating the mechanisms by which energy balance is maintained. We found here that coadministration of subthreshold CCK and leptin, which individually have no effect on feeding, dramatically reduced food intake in rats. Phosphorylation of AMP-activated protein kinase (AMPK) in the hypothalamus significantly decreased after coinjection of CCK and leptin. In addition, coadministration of these hormones significantly increased mRNA levels of anorectic cocaine- and amphetamine-regulated transcript (CART) and thyrotropin-releasing hormone (TRH) in the hypothalamus. The interactive effect of CCK and leptin on food intake was abolished by intracerebroventricular preadministration of the AMPK activator AICAR or anti-CART/anti-TRH antibodies. These findings indicate that coinjection of CCK and leptin reduces food intake via reduced AMPK phosphorylation and increased CART/TRH in the hypothalamus. Furthermore, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of CCK and leptin to reduce food intake. Food intake reduction induced by coinjection of CCK and leptin was blocked in midbrain-transected rats. Therefore, the neural pathway from hindbrain to hypothalamus plays an important role in transmitting the anorectic signals provided by coinjection of CCK and leptin. Our findings give further insight into the mechanisms of feeding and energy balance.

Mechanism of muscle atrophy in a normal-weight rat model of type 2 diabetes established by using a soft-pellet diet.

Dietary factors such as food texture affect feeding behavior and energy metabolism, potentially causing obesity and type 2 diabetes. We previously found that rats fed soft pellets (SPs) were neither hyperphagic nor overweight but demonstrated glucose intolerance, insulin resistance, and hyperplasia of pancreatic ß-cells. In the present study, we investigated the mechanism of muscle atrophy in rats that had been fed SPs on a 3-h time-restricted feeding schedule for 24 weeks. As expected, the SP rats were normal weight; however, they developed insulin resistance, glucose intolerance, and fat accumulation. In addition, skeletal muscles of SP rats were histologically atrophic and demonstrated disrupted insulin signaling. Furthermore, we learned that the muscle atrophy of the SP rats developed via the IL-6-STAT3-SOCS3 and ubiquitin-proteasome pathways. Our data show that the dietary habit of consuming soft foods can lead to not only glucose intolerance or insulin resistance but also muscle atrophy.

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet.

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

Possible involvement of melanocortin-4-receptor and AMP-activated protein kinase in the interaction of glucagon-like peptide-1 and leptin on feeding in rats.

Glucagon-like peptide-1 (GLP-1) and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important to elucidate a mechanism to maintain energy balance. In the present study, coadministration of subthreshold GLP-1 and leptin dramatically reduced feeding in rats. Although coadministration of GLP-1 with leptin did not enhance leptin signal transduction in the hypothalamus, it significantly decreased phosphorylation of AMP-activated protein kinase (AMPK). In addition, coadministration of GLP-1 with leptin significantly increased proopiomelanocortin (POMC) mRNA levels. Considering that α-melanocortin stimulating hormone (α-MSH) is derived from POMC and functions through the melanocortin-4-receptor (MC4-R) as a key molecule involved in feeding reduction, the interaction of GLP-1 and leptin on feeding reduction may be mediated through the α-MSH/MC4-R system. As expected, the interaction of GLP-1 and leptin was abolished by intracerebroventricular preadministration of the MC4-R antagonists agouti-related peptide and SHU9119. Taken together, GLP-1 and leptin cooperatively reduce feeding at least in part via inhibition of AMPK following binding of α-MSH to MC4-R.

Endocrine mechanisms responsible for different follicular development during the estrous cycle in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance rats (HAA and LAA strains, respectively) were selected and bred according to the avoidance rate in a shuttle-box task. Although they have clear strain differences in ovarian function, their endocrine mechanisms still remain to be clarified. Differences in female reproductive endocrinology between the strains were investigated by means of measuring the plasma concentration of reproductive hormones during the estrous cycle. LAA rats showed approximately threefold lower basal and surge levels of LH, a more than fourfold lower level of FSH surges and higher levels of inhibin A and inhibin B during the estrous cycle compared with the levels seen in HAA rats. The concentration of estradiol-17ß in the proestrous stage was significantly lower in LAA rats than in HAA rats. Additionally, LH and FSH secretions from primary cultured anterior pituitary cells with or without in vitro GnRH stimulation were lower in the cells derived from LAA rats and, in terms of FSH secretion, were unresponsive to GnRH in contrast to cells derived from HAA rats. Although an increased number of preantral follicles in diestrus were observed in LAA rats, number of hCG-induced ovulation was lower in LAA rats. LAA rats may have much more follicle growth during the early stage of folliculogenesis, but most follicles might not grow into mature follicles. These results strongly suggest that the strain difference in ovarian function of these two Hatano rats is due to the difference in the regulation of hypothalamo-hypophyseal system for gonadotropins secretion.

Maternal traits during lactation period reduce the anxiety-related behavior in male offspring: Results from a fostering study in Hatano rats.

The Hatano strains of the Sprague Dawley rats have been selectively bred to create high- (HAA) and low- (LAA) active avoidance variants. We previously reported that HAA rats display more anxiety-related behavior than LAA rats, but whether this strain difference is affected by postnatal environmental factors remains unclear. In this study, we performed in- and cross-fostering between the HAA and LAA strains and investigated the effect of postnatal maternal traits on the emotional responses in each strain of the male offspring. We evaluated the effect of the fostering treatment on the emotional responses of the male offspring using the elevated plus maze test. The male LAA offspring reared by HAA dam showed higher anxiety-related behavior than those reared by LAA dam. Next, we quantified and typed various maternal behavior under the in- and cross-fostering conditions during the lactation period using a snapshot sampling method. This method allowed us to evaluate potential maternal traits that may influence the emotional responses of the offspring observed in our first experiment. We found that HAA dams showed long-term resting without offspring and offspring arrangement compared with LAA dams. These findings suggest that postnatal environmental factors may alter anxiety-related behavior in the male LAA offspring and that less direct contact with their offspring during the lactation period may induce anxiety-related behavior in male offspring.

Interleukin-15 derived from Guanylin-GC-C-expressing macrophages inhibits fatty acid synthase in adipocytes.

Recently we found that guanylin (Gn) and its receptor, guanylyl cyclase C (GC-C), are uniquely expressed in the mesenteric macrophages of some diet-resistant rats and that double-transgenic (dTg) rats overexpressing Gn and GC-C in macrophages demonstrate reduced fatty acid synthase and fat accumulation in fat tissue even when fed a high-fat diet (HFD). Lipid accumulation and fatty acid synthase mRNA levels in cocultured dTg rat adipocytes and macrophages were reduced compared with those in adipocytes cultured with WT rat macrophages. Here, we investigated whether Interleukin-15 (IL-15) derived from Gn-GC-C-expressing macrophages regulates lipid accumulation in adipocytes. IL-15 inhibited fatty acid synthase and lipid accumulation via STAT5 in cultured adipocytes. IL-15 mRNA and protein levels in the mesenteric fat of HFD-fed dTg rats were significantly higher than those of HFD-fed WT rats. Phosphorylated STAT5 levels in the mesenteric fat of HFD-fed dTg rats were increased compared with those of HFD-fed WT rats. In addition, the mRNA level of fatty acid synthase in the mesenteric fat was lower in HFD-fed dTg rats than in HFD-fed WT rats. These results support the hypothesis that IL-15 secreted from Gn-GC-C-expressing macrophages contributes to the inhibition of fatty acid synthase and lipid accumulation in adipocytes, leading to obesity resistance.

Guanylyl cyclase C and guanylin reduce fat droplet accumulation in cattle mesenteric adipose tissue.

Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.

Role of the neural pathway from hindbrain to hypothalamus in the regulation of energy homeostasis in rats.

Recent evidence suggests that neural pathways from the hindbrain to the hypothalamus are important for informing the hypothalamus of the body's condition with regard to energy metabolism. Here we examined energy metabolism in rats with transections of the midbrain that severed the neural pathway from the hindbrain to the hypothalamus, and then investigated the levels of various molecules associated with control of energy metabolism in these rats. Food intake and body weight were higher in the midbrain-transected rats than in sham-operated rats. In addition, the midbrain-transected rats showed insulin resistance and hyperleptinemia. Furthermore, the hypothalamic mRNA levels of anorectic proopiomelanocortin and cocaine- and amphetamine-related transcript were significantly lower in midbrain-transected rats than in sham-operated rats. Our findings elucidate the mechanisms of food intake and energy balance from the perspective of multifactorial regulatory systems that underlie functions such as neurohormonal integration.

Role of the neural pathway from hindbrain to hypothalamus in interaction of GLP1 and leptin in rats.

Glucagon-like peptide-1 (GLP1) and leptin are anorectic hormones. Previously, we have shown that i.p. coadministration of subthreshold GLP1 with leptin dramatically reduced food intake in rats. In this study, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of GLP1 and leptin in reducing food intake. Food intake reduction induced by coinjection of GLP1 and leptin was blocked in midbrain-transected rats. These findings indicate that the ascending neural pathway from the hindbrain plays an important role in transmitting the anorectic signals provided by coinjection of GLP1 and leptin.

The short-term effects of soft pellets on lipogenesis and insulin sensitivity in rats.

The purpose of the present study was to investigate the short-term effects of a 12-day, soft pellet (SP) diet with a 3-h restricted feeding schedule on caloric intake, body weight, lipid metabolism, and insulin sensitivity. Glucose and insulin levels were measured pre-, mid-, and post-feeding. The SP rats exhibited postprandial hyperglycemia compared to rats fed control pellets (CP). The insulin response of SP rats during a meal was significantly higher than that of CP rats. There were no significant differences in the hepatic triacylglycerol contents and lipogenesis gene mRNA levels of SP and CP rats. However, the hepatocytes of SP rats were slightly hypertrophic. In addition, histological analysis revealed that the pancreases of SP rats had more islet areas than those of CP rats. This study demonstrated that feeding an SP-only diet for 12 days induces glucose intolerance, suggesting that the consumption of absorbable food, like a soft diet, may trigger glucose metabolism insufficiency and lead to life-threatening diseases.

Possible involvement of food texture in insulin resistance and energy metabolism in male rats.

Food texture is known to affect energy metabolism. Although feeding with soft pellets (SP) or via a tube is known to cause increases in body weight, it is unclear how different food textures influence energy metabolism. In this study, we investigated the effects of two different food textures on energy balance and glucose and lipid metabolism in male Wistar rats. The rats were fed SP or control pellets (CP) on a 3-h restricted feeding schedule for 14 weeks and their energy intake, body weight, and energy expenditure were examined. The levels of gastrointestinal hormones, glucose and insulin, were investigated at pre-, mid, and post-feeding. Glucose tolerance and insulin tolerance tests were conducted, and the expressions of molecules involved in the insulin signaling system or lipogenesis in the liver were examined. Histological investigation of pancreatic islets was carried out using anti-insulin and anti-Ki-67 antibodies. Furthermore, the expression in the liver and circulating blood of microRNA-33 (miR-33), which regulates insulin receptor substance 2, was examined. There were no significant differences in energy intake, body weight, or gastrointestinal hormone levels between the SP and CP rats; however, the SP rats showed glucose intolerance and insulin resistance with disruption of insulin signaling. Increases in lipogenic factors and miR-33 expression were also found in the SP rats. The numbers of insulin-positive areas and Ki-67-positive cells of SP rats were significantly increased. This study shows that a soft food texture causes diabetes without obesity, so differences in food texture may be an important factor in type 2 diabetes.

Endocrinological differences between Hatano high- and low-avoidance rats during early two-way avoidance acquisition.

Hatano high (HAA)- and low (LAA)-avoidance rats were selected from Sprague-Dawley rats genetically on the basis of their active avoidance behavior in a shuttle-box test. The purpose of this study was to investigate stress-related alterations of hormones corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP), prolactin, and adrenocorticotropin (ACTH) in the brain and blood during early avoidance acquisition using two lines of Hatano rats. In paraventricular nucleus (PVN) of the hypothalamus, the CRH levels in HAA rats were significantly increased after shuttle-box tasks compared with before the tasks, whereas the CRH levels in LAA rats significantly decreased after shuttle-box tasks compared with before the tasks. In the HAA rats, the CRH and AVP levels in the median eminence decreased after shuttle-box tasks, whereas there were no significant differences in the levels between before and after shuttle-box tasks in LAA rats. The plasma concentrations of ACTH were significantly higher in HAA rats than in LAA rats after shuttle-box tasks. These results show that the response of CRH-ACTH was higher in HAA rats than in LAA rats. This phenotype may be an important reason for the high avoidance rates of shuttle-box tasks in HAA rats. These endocrine differences in early avoidance acquisition may be involved in regulation of their avoidance responses in the shuttle-box task.

SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.