THE VIP/VPAC2R PATHWAY INHIBITS BODY FAT MASS ACCUMULATION WHILE MAINTAINING LEAN BODY MASS IN A MURINE DIO MODEL OF OBESITY BY MODULATING METABOLISM

Background: Gastric dysmotility and gastric slow wave dysrhythmias have been well documented in patients with diabetes. However, little is known on the effect of hyperglycemia on small intestine motility such as intestinal slow waves, due to limited options in measuring its activity. Moreover, food intake and digestion process have been reported to alter the small intestine motility in normal rats, but their roles in that of diabetic rats remains unknown. This study aimed to explore the effect of hyperglycemia on small intestinal myoelectrical activity (IMA) and responses to various meals in diabetic and normal rats.
Methods: IMA was recorded via chronically implanted serosal electrodes in the proximal small intestine in rats with diabetes induced by high-fat diet feeding followed by a low dose of streptozotocin (STZ, 30 mg/kg) and normal rats. The percentage of normal slow wave (% NSW) and dominant frequency (DF) were assessed from IMA under various conditions. Oral glucose tolerance test (OGTT, 20% glucose, 1 g/kg) was performed and blood was collected via the tail vein at baseline and 15, 30, 60, 90, 120, 180 min after glucose administration for the measurement of blood glucose. The correlations of blood glucose with NSW and DF were determined. Regular laboratory chow, high-fat diet, and small or large nutrient liquid meal were used to explore IMA responses to different meals in diabetic and normal rats.
Results: (1) Compared with a postprandial increase in DF in normal rats (40.3±1.4 vs. 42.2±0.7 cycles/min (CPM), vs. Fasting, P=0.001, N=6), diabetic rats showed a blunted postprandial response in DF (39.4±3.7 vs. 39.9±3.1 vs. Fasting, P>0.05, N=8) after a regular chow. However, no difference was found in % NSW between diabetic and normal rats in both fasting and fed states; (2) In the fasting state, % NSW was correlated with the blood glucose level in diabetic rats (r=-0.817, P<0.001, Fig.1a) as well as HbA1C (r=-0.871, P=0.005). After glucose administration, the increase in blood glucose was correlated with a decrease in % NSW (r=-0.647, P<0.001, Fig.1b). (3) % NSW in diabetic rats during the 30-min postprandial state was not altered after a meal, either liquid or solid, regular or high-fat diet, small or large meal, suggesting an absence of gastric-small intestinal reflex.
Conclusions: In type 2 diabetic rats, the regularity of intestinal slow waves is negatively correlated with the blood glucose level in both fasting and fed states. Diabetic rats exhibit a blunted postprandial response in intestinal slow waves compared with normal rats. There seems to be a lack of gastric-small intestinal reflex upon food ingestion in diabetic rats. (This study was supported by an NIH grant, R01DK107754)

Obesity is associated with elevated intestinal nutrient absorption and excessive accumulation of lipids in the liver, adipose tissue, skeletal muscle, and other organs, which contributes to metabolic diseases such as type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular desease, and certain types of cancer. The effect of obesity on intestinal lipid metabolism is currently unclear. We previously demonstrated that the obese phenotype and its associated insulin resistance and NAFLD were ameliorated in mice deficient in the intestinal hormone neurotensin (NT) by inhibiting small intestinal fat absorption and preserving the activity of AMPK, an enzyme that plays a key role as a master regulator of cellular energy homeostasis. However, how NT/AMPK signaling regulates this process remains unknown. The purpose of the current study was to evaluate the genes related to small intestinal lipid absorption in the context of obesity and the regulation of these genes by NT/AMPK signaling. Methods. NT wild type (WT) (Nt ^+/+) and knockout (KO) (Nt ^-/-) mice, fed standard control diet (CD, 10% kcal from fat) or high fat diet (HFD, 60% kcal from fat) were used. i) Total RNA was isolated from mouse jejunal mucosal scrapings and RNAseq analysis performed to profile gene expression; ii) Jejunal crypts were isolated for 2-D monolayer culture; total RNA or protein was isolated for qPCR and western blot analyses, respectively, to confirm gene or protein expression. Results.RNAseq analysis of female mice fed CD or HFD for 28 weeks showed that genes involved in lipid absortion (Fabp1, Fabp2, Cd36, Alpi, and Plin2) were upregulated (FDR <0.05) in Nt ^+/+ mice fed HFD vs. CD; interestingly, these alterations were not noted in Nt ^-/- mice fed HFD vs. LFD; qPCR or western blot further confirmed these results. Concurrently, phosphorylation of AMPKa (p-AMPKa) was decreased in HFD-fed Nt^+/+ mice, which was rescued by NT deficiency; consistently, palmitic acid (PA) treatment decreased p-AMPKa and increased FABP1 and FABP2 protein expression. Conclusions. These findings suggest that HFD increases small intestinal lipid absorption by upregulating FABP1 and FABP2 expression. HFD feeding or PA treatment decreases p-AMPKa activity, suggesting that AMPK mediates HFD-upregulated FABP1 and FABP2 expression. NT deficiency preserves AMPK signaling and prevents HFD-upregulated FABP1 and FABP2 levels, thus reducing increased lipid absorption. NT signaling may represent a therapeutic target to inhibit intestinal lipid absorption associated with obesity.

Background: Obese people are 80 times more likely to develop diabetes which results from altered glucose homeostasis. The first and most important step in glucose homeostasis is the intestinal absorption of glucose via Na-glucose co-transport (SGLT1) in the brush border membrane (BBM) of villus cells. In both genetic (Zucker) and high-fat diet (HFD) induced obese rats, SGTL1 was stimulated in villus cell BBM secondary to an increase in the affinity of the co-transporter for glucose without change in the number of co-transporters in the BBM. The altered affinity was mediated by phosphorylation of the co-transporter. Adipose-derived secretome (ADS) affects many physiological processes. Further, exosomes (EXs) in ADS are also known to affect physiological functions. However, whether EXs from obese ADS may regulate SGLT1 in villus cells during obesity is not known. Hypothesis: Exosomes from obese ADS uniquely regulates SGLT1 in intestinal epithelial cells during obesity. Aim: Determine the mechanism of regulation of SGLT1 by EXs from obese ADS in intestinal epithelial cells. Methods: ADS was prepared from visceral fat of obese (OZR) and lean Zucker (LZR) rats. EXs were isolated from the ADS using the Total Exosome Isolation Reagent (Invitrogen). Rat small intestinal epithelial cells (IEC-18 cells) grown to confluence in 24 well-plates were treated on day 4 with EXs. Phlorizin-sensitive Na-dependent ³H-O-methyl glucose uptake was performed for SGLT1 activity. Na-K-ATPase activity was determined as P_i released. Western blots for SGLT1 and serine-phosphorylation of SGLT1 protein were performed. Results: EXs from obese-ADS, stimulated SGLT1 in IEC-18 cells (1430±138 pmol.mg protein.2 min in lean and 4393±234 obese treated, n=3, p<0.05). Obese-ADS derived EXs treatment diminished Na/K-ATPase activity in IEC-18 cells (26.6±1.4 nmol P_i.mg protein.min in lean treated and 11.5±1.3 obese treated, n=3, p<0.05). Preliminary kinetics indicated that the mechanism of stimulation of SGLT1 by obese-ADS EXs is secondary to an increase in the affinity (1/K_m) for glucose. Western blot studies revealed that SGLT1 protein was unaltered in both groups. However, serine-phosphorylation of SGLT1 protein was significantly increased in IEC-18 cells treated with Obese EXs. Conclusions: EXs from obese-ADS stimulated SGLT1 in intestinal epithelial cells. The mechanism of stimulation of SGLT1 by EXs from obese-ADS was secondary to an increase in the affinity of the co-transporter for glucose without a change in the number of co-transporters in the BBM. The increased affinity of SGLT1 by EXs from obese-ADS was mediated by serine phosphorylation. This regulation of SGLT1 by EXs from obese-ADS is identical to that seen in vivo in the obese intestines. Therefore, it is likely that EXs from obese-ADS mediates the stimulation of SGLT1 at the BBM of intestinal villus cells during obesity.

Nonalcoholic fatty liver disease (NAFLD) currently affects about 25% of the adult U.S. population and is characterized by excess hepatic fat accumulation (steatosis) due to the inability of the hepatic mitochondria to metabolize the excess fat under obese conditions; the cause of which remains unknown. We have previously reported that neurotensin (NT), a 13 amino acid peptide hormone released from the gut in response to fat, promotes obesity and hepatic steatosis by increasing fat absorption in the intestine. The main objective of this study was to evaluate the potential role of NT in directly inducing mitochondrial dysfunction and ROS (reactive oxygen species) imbalance in the liver under obese conditions. Methods. Hepatocytes were isolated from male or female: NT wild type (WT) (Nt^+/+) and knockout (KO) (Nt^-/-) mice fed a low fat diet (LFD,10% kcal from fat) or high fat diet (HFD, 60% kcal from fat) for 23-28 weeks; and NT receptor 1 WT (Ntr1^+/+) and KO (Ntr1^-/-) mice fed a normal chow diet. i) Hepatic lipid metabolism pathways, in the presence of exogenous NT and palmitic acid (PA), were analyzed by western blot and qPCR and verified in whole liver extracts. ii) Mitochondrial oxidative phosphorylation (OXPHOS) protein abundance was assessed by western blot. iii) Mitochondrial function and ROS generation were measured by Seahorse - Cell Mito Stress Test and Mitotracker, MitoSOX and CellROX dyes in isolated hepatocytes, and iv) Fat metabolism capacity was assessed by Fatty Acid Oxidation (FAO) Assay-Seahorse and BODIPY staining. Results. Here we show that NT promotes hepatic steatosis under obese conditions by promoting lipid uptake and decreasing mitochondrial FAO. i) Lipid uptake triggered the translocation of PGC1α (the mitochondrial biogenesis protein) to the hepatocyte nucleus, where it transcriptionally upregulated FAO and OXPHOS related genes. NT acting through NTR1/ERK signaling axis inhibited PGC1α transcription. As such, NT treatment decreased ii) OXPHOS complex expression and iii) mitochondrial function in Ntr1^+/+ but not Ntr1^-/- hepatocytes. Furthermore, NT treatment decreased mitochondrial ROS, but not cellular ROS which was increased with lipid overload. iv) Nt^-/- hepatocytes exhibited significantly higher fat metabolism capacity than corresponding Nt^+/+ hepatocytes. Conclusion. NT compromises the mitochondrial ability to efficiently catalyze ingested lipid. Therefore, Nt or Ntr1 KO hepatocytes demonstrate significantly higher fat metabolism capacity than corresponding WT hepatocytes. NT also causes ROS imbalance by decreasing mitochondrial ROS through OXPHOS inhibition, but the lipid overload contributes to oxidative stress by increasing cellular ROS. Importantly, we have uncovered a novel role for NT in causing mitochondrial dysfunction and ROS imbalance in the pathogenesis of NAFLD.

Background. Nonalcoholic fatty liver disease (NAFLD), represent the most common cause of chronic liver disease in Western countries. It is closely correlated with obesity, metabolic syndrome and with risk for developing atherosclerotic cardiovascular disease (CVD). It is widely established that the CV component dictates the patient outcomes more frequently and to a greater extent than does the progression of the liver component and it is the most common cause of death for NAFLD patients. So, it is recommended that statins should be added to therapy in patients at increased risk for developing CV complications. Atorvastatin is a hydrophobic statin indicated for the treatment of hypercholesterolemia, combined hyperlipidemia and NAFLD. Furthermore, the co-activation of FXR and GPBAR1, two members of bile acid-activated receptors family, by the dual ligands BAR502, represent new therapeutic option for the pharmacological treatment of hepatic and metabolic disorders. Aim. To compare the effects of BAR502 and atorvastatin in two rodent model of atherosclerosis and NAFLD to evaluate the efficacy of their combination. Material and methods. Apolipoprotein E deficient mice (ApoE^−/−) and C57BL6 mice were fed with HFD-F alone or with BAR502, or Atorvastatin, or their combination for 14 and 8 weeks respectively and then sacrificed. The aortic rings, aorta and liver were isolated and analyzed. Results. C57BL6 mice on a HFD-F gained significantly more weight than naïve mice, became insulin resistant and developed microvescicular steatosis and ballooning of hepatocytes. Treating mice with BAR502, atorvastatin and particularly with their combination, reversed this pattern, reducing body weight, improving insulin sensitivity and liver histopathology. Transcriptome analysis of the liver revealed a synergic activity of the two agents. The two agents, in ApoE^-/- mice exposure to HFD-F, a standard model of atherosclerosis, decreased body weight gain and, in particular their combination, decreased cholesterol and LDL plasma levels. Combinatorial treatment of BAR502 and atorvastatin also resulted in the reduction of thickness and atheroma area in the thoracic aorta. A systemic anti-inflammatory activity, with the increasing IL10⁺/IL6⁺ macrophages ratio, was exerted by BAR502-atorvastatin combination treatment. The GPBAR1 and FXR expression in the atherosclerotic plaques in rodent and patients aorta, highlighted by IF staining, confirmed the involvement of the two receptors in the atherosclerotic process. Conclusions. The co-activation of FXR and GPBAR1 improves the beneficial and therapeutic effects exerted by atorvastatin on liver hepatic metabolism and atherosclerotic lesions, suggesting BAR502-atorvastatin combination as potential novel treatment for NAFLD and correlated atherosclerotic cardiovascular disease.

Background: Colorectal cancer (CRC) affects over 100,000 patients annually and is the third leading cause of cancer-related deaths in the US. Bile acids (BAs), cholesterol-derived surfactants that emulsify dietary lipids to facilitate absorption, are critical mediators of gut physiology and metabolism, partly through affecting their natural receptors, Farnesoid X Receptor (FXR)'s activity. BAs dysregulation is a convergent point of genetic and dietary risk factors of CRC. Recently, a couple of gut microbiome-derived BAs, such as 7-oxo-deoxycholic acid (7-oxo-DCA), have been identified in liver diseases. While the gut microbes responsible for generating 7-oxo-DCA have been identified, yet the impacts of 7-oxo-DCA on intestinal epithelial cells remain largely elusive. Methods & Results: Here we discovered that 7-oxo-DCA elevated in both the serum and cecum samples of genetically mutated APC^min+/- mice, either on a normal chow diet (ND, adenoma model) or on a High-fat diet (HFD, adenocarcinomas model). Moreover, we found that 7-oxo-DCA facilitates human normal and cancer cell lines, HIEC6 and HCT116 cells' growth and migration. Further, 7-oxo-DCA promotes intestinal stem cells' proliferation in intestinal organoids generated from WT and APC^min+/- mice. In addition, our in vivo administration of 7-oxo-DCA in APC^min+/- mice displayed increased total BAs amount, higher gut permeability, and more tumor loads. Mechanistically, we revealed that 7-oxo-DCA is an antagonistic BA of FXR and downregulated FXR signaling in vitro and in vivo. Conclusion: In summary, we uncover the novel role of a microbial BA, 7-oxo-DCA, and its role in promoting intestinal tumorigenesis. The modulation of the BA-FXR axis in the tumor initiation and progression will hasten the development of novel diagnostic and therapeutic tools for CRC.

Background: Vasoactive Intestinal Peptide (VIP) binds with high affinity to its VPAC1 and VPAC2 receptors, thus regulating key physiologic functions centrally and peripherally. Previously, we documented in VIP^-/- and VPAC1^-/- mice a leaner body phenotype and altered calorimetric parameters and metabolic hormones. Past reports described in VPAC2^-/- mice impaired circadian rhythm, reduced food intake, and altered metabolism.
Aims: To study the effects of the VIP/VPAC2 pathway on body phenotype, feeding behavior, energy balance, and metabolism during obesity development using a DIO (diet induced obesity) model in VPAC2^-/- and WT mice.
Methods: Two groups of VPAC2^-/- and wild-type (WT), 8 to 10 weeks old, male mice were placed on a 14-week, 45% high-fat diet (HFD) to induce obesity. Body fat and lean mass were evaluated bi-weekly by Echo-MRI. Feeding and drinking behavior, total energy expenditure (EE), respiratory gases (VO₂ and VCO₂), and physical activity parameters were analyzed by indirect calorimetric using a Sable-Promethion cage system. Plasma levels of metabolic hormones and plasma lipids were assayed during fasting and 1-hour post-feeding conditions respectively by Luminex multiplex and colorimetric assays.
Results: Echo-MRI analysis revealed that HFD-fed VPAC2^-/- mice, compared to HFD-fed WT mice had: a) lower body weight values starting at study week 8 (p< 0.05) and until week 14 (p< 0.001), b) lower percent fat mass starting at study week 6 (p< 0.05) until week 14 (p<0.001), c) higher lean mass from week 8 until week 14 (p<0.01). Indirect calorimetry analysis revealed that VPAC2^-/- mice experienced significant metabolic alterations during the dark cycle with lower Energy Expenditure (p<0.001), lower VO₂ (p<0.001), and lower VCO₂ (p<0.01); as well as during the light cycle with lower VO₂ (p<0.05), and lower RQ (p<0.001). No significant differences were found in food and water intake and physical activity parameters. In VPAC2^-/- mice, fasting plasma concentrations of ghrelin, GLP-1, insulin, and PYY parameters were lower, while leptin and glucagon were higher than HFD fed WT mice; post-feeding plasma concentrations of insulin were higher, whereas post-feeding levels of ghrelin GLP-1 and leptin were lower. In HFD fed VPAC2R-/- mice, fasting plasma concentrations of total cholesterol were higher and triglycerides were lower, whereas post-feeding plasma concentrations of total cholesterol, and triglycerides were lower.
Conclusion: Our data suggests that the VIP/VPAC2R pathway plays a key role during obesity development by inhibiting body fat accumulation while inhibiting lean mass loss. Furthermore, the VIP/VPAC2 pathway regulates metabolism by decreasing respiratory gas, and total energy expenditure, altering plasma metabolic hormone levels, and lowering post-prandial total cholesterol and triglycerides.