EXOSOMES FROM HIGH FAT DIET INDUCED RAT MODEL OF OBESITY MEDIATE THE REGULATION OF NA-GLUCOSE CO-TRANSPORT (SGLT1) IN INTESTINAL EPITHELIAL CELLS.

Background: Obese people are 6 times more likely to develop Type-2 Diabetes, which results from altered glucose homeostasis. The critical first step in maintaining glucose homeostasis is glucose absorption via Na-glucose co-transport (SGLT1, SLC5A1) at the brush border membrane (BBM) of intestinal villus cells. SGLT1 has been shown to be stimulated in villus cells from high fat diet (HFD) fed obese rats secondary to an increase in the affinity of the co-transporter for glucose without change in the number of co-transporters. Further, adipose derived secretome (ADS) from HFD adipocytes have also been shown to stimulate SGLT1 in intestinal epithelial cells (IEC) by similar mechanisms. Exosomes (EXs), secreted by adipocytes into ADS, have been known to affect many physiological functions. However, it is not known whether EXs isolated from HFD-ADS obesity specifically regulate SGLT1 in intestinal epithelial cells. Hypothesis: EXs from HFD-ADS uniquely regulate SGLT1 in IEC in obesity. Aim: Determine the mechanism of regulation of SGLT1 by EXs from HFD-ADS in IEC in obesity. Methods: ADS was prepared from visceral fat of HFD and control chow diet (CD) fed rats. EXs were isolated from 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 release. Western blot and immunoprecipitation studies were performed for SGLT1 protein expression and phosphorylation respectively with specific antibodies. Results: EXs from HFD-ADS stimulated SGLT1 in IEC-18 cells (458±49 pmol/mg protein●2 min in CD EXs and 893±54 HFD EXs treated, n=3, p<0.05). HFD-ADS derived EXs treatment diminished Na/K-ATPase activity in IEC-18 cells (19.9±1.3 nmol/mg protein●min in CD EXs treated and 9.3±1.0 HFD EXs treated, n=3, p<0.05). Kinetic studies showed that the mechanism of stimulation of SGLT1 by HFD EXs is secondary to an increase in the affinity (1/K_m) for glucose. Western blot analysis revealed that SGLT1 protein expression was unaltered in both groups. However, SGLT1 phosphorylation was stimulated in IEC-18 cells treated with EXs from HFD-ADS. Conclusions: SGLT1 was stimulated by EXs from HFD induced obese ADS in intestinal epithelial cells secondary to an increase of the affinity of the co-transporter for glucose without change in the number of co-transporters in the BBM. The increase in affinity of SGLT1 by EXs from HFD induced obese ADS was mediated by phosphorylation. The mechanism of stimulation of SGLT1 by EXs from HFD-ADS is identical to that seen in vivo in the obese intestine. Therefore, it is likely the stimulation of SGLT1 is mediated by EXs from HFD-ADS at the BBM of intestinal villus cells during obesity.