RNA sequencing unravels novel L cell constituents and mechanisms of GLP-1 secretion in human gastric bypass-operated intestine.

AIMS/HYPOTHESIS: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. METHODS: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo. RESULTS: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. CONCLUSIONS/INTERPRETATION: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.

CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion.

AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

Decreased insulin secretion and glucose clearance in exocrine pancreas-insufficient pigs.

The effect of exocrine pancreatic function on the glucose-mediated insulin response and glucose utilization were studied in an exocrine pancreas-insufficient (EPI) pig model. Five 10-week-old EPI pigs after pancreatic duct ligation and 6 age-matched, non-operated control pigs were used in the study. Blood glucose, plasma insulin and C-peptide concentrations were monitored during meal (MGTT), oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. Upon post-mortem examination, the pancreatic remnants of the EPI pigs showed acinar fibrotic atrophy but normal islets and ß-cell morphology. The EPI pigs displayed increased fasting glucose concentrations compared with control animals (6.4 ± 0.4 versus 4.8 ± 0.1 mmol l(-1) , P < 0.0001) but unchanged insulin concentrations (2.4 ± 0.6 versus 2.1 ± 0.2 pmol l(-1) ). During the OGTT and IVGTT, the EPI pigs showed slower, impaired glucose utilization, with the disruption of a well-timed insulin response. Plasma C-peptide concentrations confirmed the delayed insulin response during the IVGTT in EPI pigs. Oral pancreatic enzyme supplementation (PES) of EPI pigs improved glucose clearance during IVGTT [AUC(glucose) 1295 ± 70 mmol l(-1) × (120 min) in EPI versus 1044 ± 32 mmol l(-1) × (120 min) in EPI + PES, P < 0.0001] without reinforcing the release of insulin [AUC(C-peptide) 14.4 ± 3.8 nmol l(-1) × (120 min) in EPI versus 6.4 ± 1.3 nmol l(-1) × (120 min) in EPI + PES, P < 0.002]. The results suggest the existence of an acino-insular axis regulatory communication. The presence of pancreatic enzymes in the gut facilitates glucose utilization in an insulin-independent manner, indicating the existence of a gut-derived pancreatic enzyme-dependent mechanism involved in peripheral glucose utilization.

The effects of Calorie restriction and Bariatric surgery on Circulating Proneurotensin levels.

CONTEXT: Proneurotensin (pNT) is associated with obesity and T2D, but the effects of Roux-en-Y gastric bypass (RYGB) on postprandial pNT levels are not well studied. OBJECTIVE: Assess effects of RYGB versus very low-energy diet (VLED) on pNT levels in response to mixed-meal tests (MMT), and long-term effects of RYGB on fasting pNT.Study participants: Cohort 1: Nine normoglycemic (NG) and ten T2D patients underwent MMT before and after VLED, immediately post-RYGB and six weeks post-RYGB. Cohort 2: Ten controls with normal weight and ten patients with obesity and T2D, who underwent RYGB or vertical sleeve gastrectomy (VSG), were subjected to MMTs and GIP infusions pre-surgery and three months post-surgery. GLP-1 infusions were performed in normal weight participants. Cohort 3: Fasting pNT was assessed pre-RYGB (n=161), two months post-RYGB (n=92) and 1-year post-RYGB (n=118) in NG and T2D patients. pNT levels were measured using ELISA. RESULTS: Reduced fasting and postprandial pNT were evident after VLED and immediately following RYGB. Reintroduction of solid food post-RYGB increased fasting and postprandial pNT. Prior to RYGB, all patients lacked a meal response in pNT, but this was evident post-RYGB/VSG. GIP- or GLP-1 infusion had no effect on pNT levels. Fasting pNT were higher 1-year post-RYGB regardless of glycemic status. CONCLUSION: RYGB causes a transient reduction in pNT as a consequence of caloric restriction. The RYGB/VSG-induced rise in postprandial pNT is independent of GIP and GLP-1 and higher fasting pNT are maintained one year post-surgically.

Metabolic remission precedes possible weight regain after gastric bypass surgery.

OBJECTIVE: Some patients regain weight to a variable extent from 1 year after Roux-en-Y gastric bypass surgery (RYGB), though rarely reaching preoperative values. The aim of the present study was to investigate whether, when, and to what extent metabolic remission occurs. METHODS: Fasting metabolite and lipid profiles were determined in blood plasma collected from a nonrandomized intervention study involving 148 patients before RYGB and at 2, 12, and 60 months post RYGB. Both short-term and long-term alterations in metabolism were assessed. Anthropometric and clinical variables were assessed at all study visits. RESULTS: This study found that the vast majority of changes in metabolite levels occurred during the first 2 months post RYGB. Notably, thereafter the metabolome started to return toward the presurgical state. Consequently, a close-to-presurgical metabolome was observed at the time when patients reached their lowest weight and glucose level. Lipids with longer acyl chains and a higher degree of unsaturation were altered more dramatically compared with shorter and more saturated lipids, suggesting a systematic and reversible lipid remodeling. CONCLUSIONS: Remission of the metabolic state was observed prior to notable weight regain. Further and more long-term studies are required to assess whether the extent of metabolic remission predicts future weight regain and glycemic deterioration.

GPR162 is a beta cell CART receptor.

Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knockdown (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding, and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

The role of CART in islet biology.

Cocaine- and amphetamine-regulated transcript (CART) is mostly known for its appetite regulating effects in the central nervous system. However, CART is also highly expressed in the peripheral nervous system as well as in certain endocrine cells. Our group has dedicated more than 20 years to understand the role of CART in the pancreatic islets and in this review we summarize what is known to date about CART expression and function in the islets. CART is expressed in both islet cells and nerve fibers innervating the islets. Large species differences are at hand and CART expression is highly dynamic and increased during development, as well as in Type 2 Diabetes and certain endocrine tumors. In the human islets CART is expressed in alpha cells and beta cells and the expression is increased in T2D patients. CART increases insulin secretion, reduces glucagon secretion, and protects against beta cell death by reducing apoptosis and increasing proliferation. It is still not fully understood how CART mediates its effects or which receptors that are involved. Nevertheless, CART is endowed with several properties that are beneficial in a T2D perspective. Many of the described effects of CART resemble those of GLP-1, and interestingly CART has been found to potentiate some of the effects of GLP-1, paving the way for CART-based treatments in combination with GLP-1-based drugs.

Overexpressed beta cell CART increases insulin secretion in mouse models of insulin resistance and diabetes.

Impaired beta cell function and beta cell death are key features of type 2 diabetes (T2D). Cocaine- and amphetamine-regulated transcript (CART) is necessary for normal islet function in mice. CART increases glucose-stimulated insulin secretion in vivo in mice and in vitro in human islets and CART protects beta cells against glucotoxicity-induced cell death in vitro in rats. Furthermore, beta cell CART is upregulated in T2D patients and in diabetic rodent models as a consequence of hyperglycaemia. The aim of this study was to assess the impact of upregulated beta cell CART on islet hormone secretion and glucose homeostasis in a transgenic mouse model. To this end, mice with beta cell-specific overexpression of CART (CARTtg mice) were generated. CARTtg mice challenged by aging, high fat diet feeding or streptozotocin treatment were phenotyped with respect to in vivo and in vitro insulin and glucagon secretion, glucose homeostasis, and beta cell mass. In addition, the impact of adenoviral overexpression of CART on insulin secretion was studied in INS-1 832/13 cells. CARTtg mice had a normal metabolic phenotype under basal conditions. On the other hand, with age CARTtg mice displayed increased insulin secretion and improved glucose elimination, compared with age-matched WT mice. Furthermore, compared with WT controls, CARTtg mice had increased insulin secretion after feeding a high fat diet, as well as lower glucose levels and higher insulin secretion after streptozotocin treatment. Viral overexpression of CART in INS-1 832/13 cells resulted in increased glucose-stimulated insulin secretion. Together, these results imply that beta cell CART acts to increase insulin secretion when beta cell function is challenged. We propose that the increase in beta cell CART is part of a compensatory mechanisms trying to counteract the hyperglycaemia in T2D.

Obesity-associated Blunted Subcutaneous Adipose Tissue Blood Flow After Meal Improves After Bariatric Surgery.

CONTEXT: Glucose-dependent insulinotropic peptide (GIP) and meal ingestion increase subcutaneous adipose tissue (SAT) perfusion in healthy individuals. The effects of GIP and a meal on visceral adipose tissue (VAT) perfusion are unclear. OBJECTIVE: Our aim was to investigate the effects of meal and GIP on VAT and SAT perfusion in obese individuals with type 2 diabetes mellitus (T2DM) before and after bariatric surgery. METHODS: We recruited 10 obese individuals with T2DM scheduled for bariatric surgery and 10 control individuals. Participants were studied under 2 stimulations: meal ingestion and GIP infusion. SAT and VAT perfusion was measured using 15O-H2O positron emission tomography-magnetic resonance imaging at 3 time points: baseline, 20 minutes, and 50 minutes after the start of stimulation. Obese individuals were studied before and after bariatric surgery. RESULTS: Before bariatric surgery the responses of SAT perfusion to meal (P = .04) and GIP-infusion (P = .002) were blunted in the obese participants compared to controls. VAT perfusion response did not differ between obese and control individuals after a meal or GIP infusion. After bariatric surgery SAT perfusion response to a meal was similar to that of controls. SAT perfusion response to GIP administration remained lower in the operated-on than control participants. There was no change in VAT perfusion response after bariatric surgery. CONCLUSION: The vasodilating effects of GIP and meal are blunted in SAT but not in VAT in obese individuals with T2DM. Bariatric surgery improves the effects of a meal on SAT perfusion, but not the effects of GIP. Postprandial increase in SAT perfusion after bariatric surgery seems to be regulated in a GIP-independent manner.

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects.

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.

GK-rats respond to gastric bypass surgery with improved glycemia despite unaffected insulin secretion and beta cell mass.

Roux-en-Y gastric bypass (RYGB) is the most effective treatment for morbid obesity and results in rapid remission of type 2 diabetes (T2D), before significant weight loss occurs. The underlying mechanisms for T2D remission are not fully understood. To gain insight into these mechanisms we used RYGB-operated diabetic GK-rats and Wistar control rats. Twelve adult male Wistar- and twelve adult male GK-rats were subjected to RYGB- or sham-operation. Oral glucose tolerance tests (OGTT) were performed six weeks after surgery. RYGB normalized fasting glucose levels in GK-rats, without affecting fasting insulin levels. In both rat strains, RYGB caused increased postprandial responses in glucose, GLP-1, and GIP. RYGB caused elevated postprandial insulin secretion in Wistar-rats, but had no effect on insulin secretion in GK-rats. In agreement with this, RYGB improved HOMA-IR in GK-rats, but had no effect on HOMA-ß. RYGB-operated GK-rats had an increased number of GIP receptor and GLP-1 receptor immunoreactive islet cells, but RYGB had no major effect on beta or alpha cell mass. Furthermore, in RYGB-operated GK-rats, increased Slc5a1, Pck2 and Pfkfb1 and reduced Fasn hepatic mRNA expression was observed. In summary, our data shows that RYGB induces T2D remission and enhanced postprandial incretin hormone secretion in GK-rats, without affecting insulin secretion or beta cell mass. Thus our data question the dogmatic view of how T2D remission is achieved and instead point at improved insulin sensitivity as the main mechanism of remission.

Metabolic Effects of Gastric Bypass Surgery: Is It All About Calories?

Bariatric surgery is an efficient method to induce weight loss and also, frequently, remission of type 2 diabetes (T2D). Unpaired studies have shown bariatric surgery and dietary interventions to differentially affect multiple hormonal and metabolic parameters, suggesting that bariatric surgery causes T2D remission at least partially via unique mechanisms. In the current study, plasma metabolite profiling was conducted in patients with (n = 10) and without T2D (n = 9) subjected to Roux-en-Y gastric bypass surgery (RYGB). Mixed-meal tests were conducted at baseline, after the presurgical very-low-calorie diet (VLCD) intervention, immediately after RYGB, and after a 6-week recovery period. Thereby, we could compare fasted and postprandial metabolic consequences of RYGB and VLCD in the same patients. VLCD yielded a pronounced increase in fasting acylcarnitine levels, whereas RYGB, both immediately and after a recovery period, resulted in a smaller but opposite effect. Furthermore, we observed profound changes in lipid metabolism following VLCD but not in response to RYGB. Most changes previously associated with RYGB were found to be consequences of the presurgical dietary intervention. Overall, our results question previous findings of unique metabolic effects of RYGB and suggest that the effect of RYGB on the metabolite profile is mainly attributed to caloric restriction.

Thylakoids promote release of the satiety hormone cholecystokinin while reducing insulin in healthy humans.

OBJECTIVE: The effects of a promising new appetite suppressor named "thylakoids" (membrane proteins derived from spinach leaves) were examined in a single meal in man. Thylakoids inhibit the lipase/colipase hydrolysis of triacylglycerols in vitro and suppress food intake, decrease body-weight gain and raise the satiety hormone cholecystokinin (CCK) in rats, but their effects in man remain unclear. The aim of this study was to investigate whether thylakoids, when added to a test meal, affect appetite regulation and blood parameters in healthy individuals. MATERIAL AND METHODS: In an intervention crossover study, healthy individuals of normal weight (n=11) were offered a high-fat meal with and without the addition of thylakoids. Blood samples were taken 0 (prior to meal), 30, 60, 120, 180, 240, 300 and 360 min after the start of the meal. Blood samples were analysed for satiety and hunger hormones (CCK, leptin and ghrelin), insulin and blood metabolites (glucose and free fatty acids). RESULTS: The CCK level increased, in particular between the 120 min time-point and onwards, the ghrelin level was reduced at 120 min and leptin level increased at 360 min after intake of the thylakoid-enriched meal. The insulin level was reduced, whereas glucose concentrations were unchanged. Free fatty acids were reduced between time-point 120 min and onwards after the thylakoid meal. CONCLUSIONS: The addition of thylakoids to energy-dense food promotes satiety signals and reduces insulin response during a single meal in man.

Thylakoids suppress appetite by increasing cholecystokinin resulting in lower food intake and body weight in high-fat fed mice.

Thylakoids are membranes isolated from plant chloroplasts which have previously been shown to inhibit pancreatic lipase/colipase catalysed hydrolysis of fat in vitro and induce short-term satiety in vivo. The purpose of the present study was to examine if dietary supplementation of thylakoids could affect food intake and body weight during long-term feeding in mice. Female apolipoprotein E-deficient mice were fed a high-fat diet containing 41% of fat by energy with and without thylakoids for 100 days. Mice fed the thylakoid-enriched diet had suppressed food intake, body weight gain and body fat compared with the high-fat fed control mice. Reduced serum glucose, serum triglyceride and serum free fatty acid levels were found in the thylakoid-treated animals. The satiety hormone cholecystokinin was elevated, suggesting this hormone mediates satiety. Leptin levels were reduced, reflecting a decreased fat mass. There was no sign of desensitization in the animals treated with thylakoids. The results suggest that thylakoids are useful to suppress appetite and body weight gain when supplemented to a high-fat food during long-term feeding.

Vagotomy and accompanying pyloroplasty down-regulates ghrelin mRNA but does not affect ghrelin secretion.

In this study, we have examined how the lack of vagus activity affects the long-term secretion of total and active ghrelin. We subjected mice to sham-operation, pyloroplasty or vagotomy and pyloroplasty. The study lasted for 2 weeks, during which body weight development and daily food intake was monitored. At the end of the study, the mice were sacrificed, and serum and fundus were collected. Measurements of total and active serum ghrelin revealed no difference between the surgical groups and sham-operated mice, despite the fact that fundic ghrelin mRNA was down-regulated. The results presented here suggest that the vagus activity is not required for the long-term secretion of neither total nor active ghrelin in mice. They also suggest that fundic ghrelin mRNA expression is affected by pyloroplasty and vagotomy but that this effect does not translate into changes in ghrelin levels in the circulation.

Effects of sucrose, glucose and fructose on peripheral and central appetite signals.

In the Western world, consumption of soft drinks has increased the last three decades and is partly responsible for the epidemic-like increase in obesity. Soft drinks, originally sweetened by sucrose, are now sweetened by other caloric sweeteners, such as fructose. In this study, we investigated the short-term effect of sucrose, glucose or fructose solutions on food intake and body weight in rats, and on peripheral and central appetite signals. Rats received water containing either of the sugars and standard rat chow for two weeks. Rats receiving water alone and standard chow were controls. All rats offered the sugar solutions increased their total caloric intake. The increased caloric intake occurred despite the fact that the rats offered either of the sugar solutions consumed less chow. As a consequence of the increased caloric intake, the sugar-drinking rats had elevated serum levels of free fatty acids, triglycerides and cholesterol. In addition, consuming sugar solutions resulted in increased serum leptin, decreased serum PYY and down-regulated hypothalamic NPY mRNA. Serum ghrelin was increased in rats receiving fructose solution. Moreover, consumption of sucrose or fructose solution resulted in up-regulated hypothalamic CB1 mRNA. Hypothalamic POMC mRNA was down-regulated in rats receiving glucose or fructose. In conclusion, consumption of glucose, sucrose or fructose solution results in caloric overconsumption and body weight gain through activation of hunger signals and depression of satiety signals as well as activation of reward components. The weight-promoting effect of these sugar solutions may possibly be ameliorated by the down-regulation of NPY mRNA and increased serum leptin.

Hypothalamic gene expression following ghrelin therapy to gastrectomized rodents.

We investigated whether ghrelin depletion (by gastrectomy surgery) and/or treatment/replacement with the gastric hormone ghrelin alters the expression of key hypothalamic genes involved in energy balance, in a manner consistent with ghrelin's pro-obesity effects. At 2 weeks after surgery mice were treated with ghrelin (12 nmol/mouse/day, sc) or vehicle for 8 weeks. Gastrectomy had little effect on the expression of these genes, with the exception of NPY mRNA in the arcuate nucleus that was increased. Ghrelin treatment (to gastrectomized and sham mice) increased the mRNA expression of orexigenic peptides NPY and AgRP while decreasing mRNA expression of the anorexigenic peptide POMC. Two weeks gavage treatment with the ghrelin mimetic, MK-0677, to rats increased NPY and POMC mRNA in the arcuate nucleus and MCH mRNA in the lateral hypothalamus. Thus, while predicted pro-obesity ghrelin signalling pathways were activated by ghrelin and ghrelin mimetics, these were largely unaffected by gastrectomy.

Enterostatin up-regulates the expression of the beta-subunit of F(1)F(o)-ATPase in the plasma membrane of INS-1 cells.

Exposure to high-fat diet easily promotes overeating while at the same time disrupting insulin secretion and islet function. Enterostatin is a peptide which is secreted from the pancreas in response to high-fat feeding and has been shown to inhibit fat intake as well as insulin secretion in experimental animal models. Until recently, there was no known receptor for enterostatin. In 2002, Berger and co-workers found enterostatin to target the beta-subunit of the F(1)-ATPase in rat brain membranes as well as in a clonal beta-cell line (INS-1). In this study, we found the beta-subunit of F(1)-ATPase to be ectopically expressed in the plasma membrane of INS-1 cells using both immunohistochemistry and Western blotting. Incubation with enterostatin for 60 min resulted in a 3.5-fold increase of the protein expression of the beta-subunit of F(1)-ATPase in the plasma membrane. Furthermore, we found ATP to be able to displace the binding of enterostatin to purified bovine F(1)-ATPase. This reported targeting of enterostatin to the beta-subunit of F(1)-ATPase in insulin cells may provide a link between high-fat intake and islet function.

Effects of GIP on regional blood flow during normoglycemia and hyperglycemia in anesthetized rats.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion, and affects ß-cell turnover. This study aimed at evaluating if some of the beneficial effects of GIP on glucose homeostasis can be explained by modulation of islet blood flow. Anesthetized Sprague-Dawley rats were infused intravenously with different doses of GIP (10, 20, or 60 ng/kg*min) for 30 min. Subsequent organ blood flow measurements were performed with microspheres. In separate animals, islets were perfused ex vivo with GIP (10-6 -10-12  mol/L) during normo- and hyperglycemia and arteriolar responsiveness was recorded. The highest dose of GIP potentiated insulin secretion during hyperglycemia, but had no effect in normoglycemic rats. The highest GIP concentration decreased blood perfusion of whole pancreas, pancreatic islets, duodenum, colon, liver and kidneys. The decrease in blood flow was unaffected by ganglion blockade or adenosine receptor inhibition. In contrast to this, in single perfused islets GIP induced a dose-dependent arteriolar dilation. Thus, high doses of GIP exert a direct dilatory effect on islet arterioles in isolated islets, but induce a generalized vasoconstriction in splanchnic organs, including the whole pancreas and islets, in vivo. The latter effect is unlikely to be mediated by adenosine, the autonomic nervous system, or endothelial mediators.