miR-375 prevents high-fat diet-induced insulin resistance and obesity by targeting the aryl hydrocarbon receptor and bacterial tryptophanase (tnaA) gene

Background: Diet manipulation is the basis for prevention of obesity and diabetes. The molecular mechanisms that mediate the diet-based prevention of insulin resistance are not well understood. Here, as proof-of-concept, ginger-derived nanoparticles (GDNP) were used for studying molecular mechanisms underlying GDNP mediated prevention of high-fat diet induced insulin resistance. Methods: Ginger-derived nanoparticles (GDNP) were isolated from ginger roots and administered orally to C57BL/6 high-fat diet mice. Fecal exosomes released from intestinal epithelial cells (IECs) of PBS or GDNP treated high-fat diet (HFD) fed mice were isolated by differential centrifugation. A micro-RNA (miRNA) polymerase chain reaction (PCR) array was used to profile the exosomal miRs and miRs of interest were further analyzed by quantitative real time (RT) PCR. miR-375 or antisense-miR375 was packed into nanoparticles made from the lipids extracted from GDNP. Nanoparticles was fluorescent labeled for monitoring their in vivo trafficking route after oral administration. The effect of these nanoparticles on glucose and insulin response of mice was determined by glucose and insulin tolerance tests. Results: We report that HFD feeding increased the expression of AhR and inhibited the expression of miR-375 and VAMP7. Treatment with orally administered ginger-derived nanoparticles (GDNP) resulted in reversing HFD mediated inhibition of the expression of miR-375 and VAMP7. miR-375 knockout mice exhibited impaired glucose homeostasis and insulin resistance. Induction of intracellular miR-375 led to inhibition of the expression of AhR and VAMP7 mediated exporting of miR-375 into intestinal epithelial exosomes where they were taken up by gut bacteria and inhibited the production of the AhR ligand indole. Intestinal exosomes can also traffic to the liver and be taken up by hepatocytes, leading to miR-375 mediated inhibition of hepatic AhR over-expression and inducing the expression of genes associated with the hepatic insulin response. Altogether, GDNP prevents high-fat diet-induced insulin resistance by miR-375 mediated inhibition of the aryl hydrocarbon receptor mediated pathways over activated by HFD feeding. Conclusion: Collectively our findings reveal that oral administration of GDNP to HFD mice improves host glucose tolerance and insulin response via regulating AhR expression by GDNP induced miR-375 and VAMP7.

A. Depiction of the sucrose gradient purification of ginger-derived nanoparticles (GDNP). B. Electron micrograph of purified GDNP from the red box in panel A. C. GDNP size distribution, as determined using the Nano-sight NS300.

Figure S2. GDNP uptake by small intestine epithelial cells.
A. Scanning image of intestine showed the signals from DIR labeled GDNP. The region of the intestine was used to prepare the section for confocal microscopy shown in panel B. B. GDNP uptake by small intestine epithelial cells. Scale bar as indicated. C. TargetScan screenshot depicting the sequence target of miR-375 in AhR mRNA 3'UTR. Black boxes showing sequence homology between human and mouse AhR. D. Body weight of mice receiving adoptive transfer of nanoparticles for 14 days.  Nano-antisense-miR375 or scrambled miRNA along with exosomes derived from GDNP HFD mice.

Figure S5. miR-375 correlated as biomarker with disease progression
A. Scatter plot depicting the linear correlation between ALT vs miR-375 and AST vs miR-375 levels (human stool exosomes). B. Scatter plot depicting the linear correlation between adiponectin vs miR-375 levels (human stool exosomes).

Figure S6. Uptake of HFD-Exo by liver F4/80 cells and developed the insulin resistance.
A. Scanned organ images demonstrating preferential localization of HFD-Exo to the liver. B. Flow cytometry analysis of PKH-26-labeled exosome uptake by hepatocytes (Albumin + ) and Kupffer cells (F4/80 + ). Percentage of cells summarized at right. C. & D. PKH26-labeled HFD exosomes visualized by confocal microscopy in Kupffer cells/F4/80/purple (yellow arrow; C) and hepatocytes/albumin + /green (D). E. Glucose tolerance (upper) and insulin tolerance (lower) tests for mice receiving the HFD-EXo for 14 days while mice were fed HFD. F. Body weight of mice receiving adoptive transfer of HFD-Exo along with nanoparticles packaged with scramble or miR-375 for 14 days. Student t test (two-tailed) was used to calculate statistical significance. (p value *<0.05).

Figure S7. HFD-Exo induced insulin resistance via AhR.
A. Glucose tolerance test of C57BL/6 and AhR KO mice receiving adoptive transfer of HFD-Exo for 14 days while fed HFD. B. Insulin tolerance test of C57BL/6 and AhR KO mice receiving adoptive transfer of HFD-Exo for 14 days while fed HFD. Student t test (two-tailed) was used to calculate statistical significance. (p value *<0.05; **<0.01).

Figure S8. Plasma levels of free amino acid in HFD-fed mice treated with PBS or GDNP vs control lean mice
A. & B. 2DLC-MS mass spectrometry analysis was performed to analyze levels of plasma amino acids. Data are shown as fold change in measured levels as normalized to levels observed in lean control mice (dotted line) with decreases shown in (A) and increases in (B). C. Glucose uptake by FL83B cells treated with fecal exosomes isolated from HFD mice gavage-given GDNP along with nanoparticles packaged with antisense-miR375 or scramble miRNA for 16 hours followed by 1nM insulin treatment for 1 hour prior to cells were harvested. One-way ANOVA with the Tukey multiple comparison was used to calculate statistical significance. (p value *<0.05).