NEUROTENSIN PROMOTES NONALCOHOLIC FATTY LIVER DISEASE BY COMPROMISING HEPATIC MITOCHONDRIAL FITNESS AND FAT METABOLISM CAPACITY

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.