THE ROLE OF GLUCAGON-LIKE PEPTIDE 1 RECEPTOR ON REGULATION OF GLUCOSE HOMEOSTASIS IN RESPONSE TO INTESTINAL ELECTRICAL STIMULATION IN TYPE 2 DIABETIC RATS

Introduction: Enteroendocrine cells (EEC) secrete satiety hormones GLP-1 and PYY in response to luminal nutrients through nutrient-sensing GPCRs and are implicated in obesity (OB). We recently identified expression of regulator of G-protein signaling 9 (RGS9), a negative regulator of GPCR signaling, in human EECs and show that it may function to cease GLP-1 and PYY hormone secretion. Whether alterations to gut RGS can influence subsequent energy intake patterns is unknown. We aimed to investigate the role of gut RGS in GPCR-mediated hormone secretion with alterations to energy intake in mice with diet induced obesity (DIO) and transgenic mice overexpressing RGS9 (RGS9-KI).

Methods: Colon and terminal ileum tissue from euthanized mice (lean n=9; DIO n=9) were preserved in RNAlater and prepared for RT-qPCR. Overnight fasted DIO mice received rectal enemas of solutions containing water (Vehicle, n=3), 1,10-Phenanthroline [(1,10-PA), a GLP-1 secretagogue via T2R5 agonism, 5 mM, n=3], or 1,10-PA (5 mM) + CCG-50014 (non-selective RGS inhibitor, 1 mM)(n=4). Mice were placed in individual cages with premeasured food (~20 g) and food intake was measured at 2, 4, and 6 hours (h). In an additional cohort blood plasma was collected at fasting and 2h after food intake (0.8g chow) in WT and RGS9-KI mice. GLP-1 was measured by ELISA. The feeding paradigm test mentioned above was done in overnight fasted WT(n=8) and RGS9-KI mice(n=6).

Results: Compared to lean, DIO mice had elevated ileal mRNA expression of RGS9 (p<0.05)(1A). In DIO mice, 1,10-PA alone was sufficient to decrease energy intake at 2h compared to vehicle(p<0.05)(1B). At 4h, reduction in energy intake was only observed in mice treated with 1,10-PA+CCG-50014 (vs vehicle, p<0.001; vs 1,10-PA, p<0.001). In DIO mice, only treatment with 1,10-PA+CCG-50014 increased GLP-1 secretion (1C). At fasting and 2h post prandial, there was no difference in GLP-1 levels between WT and RGS9-KI mice(1D-E). WT mice displayed an increased trend in GLP-1 (pM) after 2h (p=0.06), whereas a decreased trend in GLP-1 was observed in RGS9-KI mice (p=0.07)(1F). ΔGLP1 levels were reduced in RGS9-KI mice compared to WT (p<0.05)(1G). Cumulative food intake was unchanged between WT and RGS9-KI mice during a 6h period, ΔFI in the 2-4 h time period was significantly increased in RGS9-KI mice compared to WT (P<0.05)(1H-J).

Conclusions: Similar to previous findings in humans, RGS9 is overexpressed in DIO intestine. Non-selective inhibition of RGS in mice, led to decrease food intake. Mice overexpressing RGS9 demonstrate an impaired GLP-1 secretion response to food intake and consume significantly more energy in the 2-4h postprandial time compared to controls. RGS9 inhibition may be a potential target for OB treatment.

Na-K-ATPase (NKA) in intestinal epithelial cells provides the favorable transcellular Na gradient required for the functioning of the Na dependent co-transporters at the brush border membrane (BBM). Enzymatic activity of NKA is measured as inorganic phosphate (Pi) release in cellular homogenates while function of NKA in live cells is determined as uptake of ⁸⁶Rb, which is no longer available. In Zucker rat (ZR) model of obesity, Pi release was significantly decreased in obese ZR (OZR) compared to leans (LZR). Similarly, in Sprague Dawley (SD) rat model of high-fat diet (HFD) induced obesity (DIO), Pi release was diminished. While enzymatic activity measured as Pi release is diminished in villus cells during obesity, whether functioning of NKA is affected in live villus cells from OZR and DIO is altered in not known. Further, since radioactive ⁸⁶Rb is no longer available, whether the use of atomic absorption spectroscopy (AAS) as an alternative to ⁸⁶Rb uptake to measure NKA function has also not been established. Hypothesis: NKA functioning is altered in villus cells during obesity as determined by AAS. Aim: Determine obesity mediated alterations in NKA. Methods: SD rats fed with HFD for 6 wks was used as DIO model. Distal ileal villus cells were isolated by Ca chelation. NKA activity was determined on villus cell homogenates as ouabain sensitive Pi release. Live isolated villus cells were used to measure NKA activity as a function of ouabain sensitive Rb uptake using AAS. mRNA expression levels of NKA subunits were determined by real-time quantitative PCR (RT-qPCR) using rat specific primers. Results: Ouabain sensitive Pi release was decreased by 40% in villus cells from DIO (n=3,p<0.05). RT-qPCR demonstrated that the mRNA expression levels of the NKA-β subunit was significantly reduced in DIO (Control: 1.08±0.12; SD-HFD: 0.73±0.03; n=4, p <0.05), whereas the mRNA expression levels of the NKA-α subunit remained unchanged (Control: 1.006±0.05; SD-HFD: 1.02±0.04; n=5). To determine if the use of AAS is a viable alternative to ⁸⁶Rb uptake in measuring NKA function in live villus cells, Rb uptake was measured by AAS and was found to be decreased in villus cells from SD-HFD rats by 53% compared to control (n=3, p<0.05). We have previously demonstrated that Pi release was decreased in OZR. Further, NKA function in live villus cells measured as Rb uptake by AAS was found to be decreased in villus cells from OZR by 57% compared to leans (n=3, p<0.05). Conclusions: Both the enzymatic activity and intact cell function of NKA was decreased in villus cells from diet induced and genetic models of obesity. Preliminary molecular studies suggest that the mechanism is secondary to decreased synthesis of subunits of NKA in obesity. Finally, this study establishes the use of AAS as an ideal method to determine the function of NKA in live intestinal epithelial cells.

Background and aims: GLP-1 is a physiological regulator and neurotransmitter primarily promoting insulin synthesis and reducing glucose. GLP-1 functioned exclusively through GLP-1R, a G protein-coupled glucagon family protein. The activation of GLP-1R plays an important role in controlling glucose metabolism in T2D diabetes. Recently, our studies have demonstrated a remarkable effect of intestine electrical stimulation (IES) on decreasing food intake, body weight and blood glucose in T2D male rats. To elucidate the mechanism that how IES reduced blood glucose in T2D rat, in this study, we assessed the expression and activation of GLP-1R in response to IES in type 2 diabetic rats. Methods: Four-weeks SD male rats were fed with high fat diet for a few weeks and then injected with streptozotocin (35mg/kg) and to induce T2D. After surgical implantation of IES electrodes in the duodenum, rats were randomized to receive 3 hours daily IES (N=8) or sham-SNS (0mA, N=8) for 8 weeks. The IES was performed using parameters of 3mA, 5ms, 0.6s on/ 0.9s off and 40 Hz. At the end of the experiment the tissues of brain, pancreas, proximal and distal intestine were collected. The activation and expression of GLP-1R in each tissue were assessed by Western blot and Immunofluorescence staining. Results: 1) chronic IES reduced food intake at weeks 4 (IES vs Sham, p=0.05), 5 (p=0.05), 6 (p=0.005) and 8 (p=0.02) and reduced body weight at weeks 7 (IES vs Sham, p=0.029) and 8 (p=0.032). 2) Chronic IES decreased the blood glucose level at 30min (IES vs Sham, p=0.028) and 60 min (p=0.023) after oral glucose. Chronic IES also improved insulin sensitivity reflected as a decrease in blood glucose at 30min (IES vs Sham, p=0.01) and 45 min (p=0.03) after insulin injection. 3) Compared with sham-IES, chronic IES elevated GLP-1R protein expression in the pancreas (intensity:0.2±0.02 vs 0.5±0.2, p=0.05), proximal intestine (intensity: 0.6±0.4 vs 1.3±0.6, p=0.03) and nucleus tractus solitarius (intensity: 11±3.2 vs 18.7±4.9, p=0.001). 4) The tissue immunofluorescence staining showed that compared with sham-IES, IES dramatically increased the expression of GLP-1R in Brunner’s gland (intensity: 12±2 vs 29±6, p=0.0001), and the number of mucosa lamina propria lymphocytes (cell: 6±2 vs 19±3, p=0.001) and enteroendocrine cells (cell: 3±1 vs 10±3, p=0.03) in the proximal intestine. Conclusion: Taken together, our data suggested that GLP-1R plays an important role in mediating the glucose homeostasis in response to IES in T2D rat through a gut-brain-pancreas signaling pathway. (This work was supported by an NIH grant, R01DK107754)