Resistant starch can improve insulin sensitivity independently of the gut microbiota

Background Obesity-related diseases, including type 2 diabetes and cardiovascular disease, have reached epidemic proportions in industrialized nations, and dietary interventions for their prevention are therefore important. Resistant starches (RS) improve insulin sensitivity in clinical trials, but the mechanisms underlying this health benefit remain poorly understood. Because RS fermentation by the gut microbiota results in the formation of physiologically active metabolites, we chose to specifically determine the role of the gut microbiota in mediating the metabolic benefits of RS. To achieve this goal, we determined the effects of RS when added to a Western diet on host metabolism in mice with and without a microbiota. Results RS feeding of conventionalized mice improved insulin sensitivity and redressed some of the Western diet-induced changes in microbiome composition. However, parallel experiments in germ-free littermates revealed that RS-mediated improvements in insulin levels also occurred in the absence of a microbiota. RS reduced gene expression of adipose tissue macrophage markers and altered cecal concentrations of several bile acids in both germ-free and conventionalized mice; these effects were strongly correlated with the metabolic benefits, providing a potential microbiota-independent mechanism to explain the physiological effects of RS. Conclusions This study demonstrated that some metabolic benefits exerted by dietary RS, especially improvements in insulin levels, occur independently of the microbiota and could involve alterations in the bile acid cycle and adipose tissue immune modulation. This work also sets a precedent for future mechanistic studies aimed at establishing the causative role of the gut microbiota in mediating the benefits of bioactive compounds and functional foods. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0230-5) contains supplementary material, which is available to authorized users.

Tissue analyses. Adipose tissues, livers, ileum, colon and cecal tissues and contents were frozen in liquid nitrogen. Visceral adipose tissue was dissected as described by Caesar et al. (2). A portion of subcutaneous and visceral adipose tissue was fixed in RNA Later (Ambion, Life Technologies, Grand Island, NY). All samples were stored at -80°C until further use. Gene expression analyses were performed as previously described (3)  Primer sequences are provided in Table A.
Microbial community analysis. Gut microbiota composition was assessed by 16S rRNA gene sequencing of fecal samples as previously described (7). Genomic DNA was extracted from feces using a QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) with a bead-beating step.
Amplicon sequencing of the fecal microbiota was done at the University of Minnesota Genomics Center, as described in the supplemental methods. Briefly, the V5-V6 region of the 16S rRNA gene was PCR-enriched using the primer pair 784F (5'-RGGATTAGATACCC-3') and The resulting PCR products were quantified by PicoGreen (Life Technologies). A subset of the amplicon libraries was spot-checked on a Bioanalyzer High-Sensitivity DNA Chip (Agilent Technologoies) for correct amplicon size. Next, samples were normalized to 2nM and pooled together. The total volume of the libraries was reduced by SpeedVac and amplicons were sizeselected at 420 bp +/-20% using the Caliper XT (Perkin Elmer). Next, library pools were cleanedup by 1.8X AMPureXP beads (Beckman Coulter) and eluted in water. The final pool was quantified by PicoGreen and normalized to 2 nM for input into Illumina MiSeq (v3 Kit) to produce 2x300 bp sequencing products. Clustering was done at 10 pM with a 5% spike of PhiX.
The sequences used for analysis can be found in the MG-RAST database (8) (Table B). After addition of 430 μL of 70% acetonitrile and 10 μL of 10% formic acid, the mixtures were cleaned up by phospholipid-depletion solid-phase extraction on a HybridSPE®-Phospholipid 96-well plate (50 mg/2 mL; Sigma-Aldrich) using the same procedure as described in (13). The flow-through fractions were collected and then dried under a gentle nitrogen flow. The residues were dissolved in 200 μL of 50% methanol. Ten-μL aliquots were injected. For the analysis of the high abundance bile acids, the samples were diluted 25 times and then re-injected. The UPLC-MRM/MS analyses were performed using a Dionex UltiMate 3400 RSLC system (Amsterdam, The Netherlands) coupled to an AB Sciex 4000 QTRAP mass spectrometer (Concord, ON, Canada) equipped with a TurboIon electrospray ionization (ESI) source and operated in the negative ion multiple-reaction monitoring (MRM) mode. UPLC separation was carried out on a Waters BEH C18 UPLC column (2.1 x 150 mm, 1.7 μm) with water-acetonitrile-0.01% formic acid as the mobile phase for binary gradient elution using the same UPLC and MRM/MS parameters and operating procedures as described in (9).
Concentrations of the detected bile acids (Table B) were calculated with internal calibration from their linearly regressed standard curves prepared using the authentic compounds of 46 bile acids. Since taurohyodeoxycholic acid and tauroursodeoxycholic acid were not resolved by UPLC-MRM/MS, the total amount of these two compounds in each sample was reported as the concentration of tauroursodeoxycholic acid. The 14 D-labeled bile acids were used as IS for their corresponding non-D-labeled forms. For the bile acids without their D-labeled analogues, chenodeoxycholic-D4 acid was used as the common IS for quantitation of the unconjugated species; tauro-chenodeoxycholic-D4 acid was used as the common IS for quantitation of the taurineconjugated species; glyco-deoxycholic-D4 acid was used as the common IS for quantitation of the glycine-conjugated species. Since there were no standard compounds available for glyco-ωmuricholic acid (MCA), glyco-α-MCA, glyco-β-MCA or glyco-allocholic acid, the identities of these compounds, which shared the same MRM transitions as those of their isomeric glycocholic acid, were deduced from the recorded chromatographic retention times by comparison of the retention times of their corresponding unconjugated and taurine-conjugated species on the acquired UPLC-MRM/MS profiles. The concentrations of glyco-ω-MCA, glyco-α-MCA, glyco-β-MCA, and glyco-allocholic acid were calculated from the calibration curve of glycocholic acid. In the analysis, the lower limits of quantification were 0.005 nmole/g.  TDHCA  Taurodehydrocholic acid  THCA  Taurohyocholic acid  THDCA+TUDCA  Taurohyodeoxycholic and Tauroursodeoxycholic acid*  TLCA  Taurolithocholic acid  TwMCA Tauro-ω-muricholic acid UCA Ursocholic acid UDCA Ursodeoxycholic acid* wMCA ω-muricholic acid *Deuterium-labeled bile acids used as internal standards for quantification (all D4, except for Taurodeoxycholic-D6 acid).