Resistant starch can improve insulin sensitivity independently of the gut microbiota

Abstract

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.

Keywords: Adipose tissue macrophages; Gut microbiota; Insulin sensitivity; Resistant starch.

Fig. 1

Germ-free C3H mice experienced similar gains in body weight as their conventionalized counterparts when fed a Western diet. Body weight gain in germ-free (GF) and conventionalized (CVZ) male C57BL/6 (B6) mice (a) and GF and CVZ male C3H/HeN (C3H) mice (b) fed a WD. Energy intake measured at week 4 in GF and CVZ B6 mice (c) and GF and CVZ C3H mice (d) fed a WD. **p < 0.01 versus CVZ (two-way ANOVA with repeated measures and Bonferroni post hoc tests). Mean ± SEM. a N = 6–8 mice. b N = 8 mice. c N = 4 cages. d N = 3 cages

Fig. 2

Resistant starches improved insulin sensitivity in C3H mice in the presence of a microbiota. Glycemic response after insulin injection in conventionalized 15-week-old C3H/HeN (C3H) mice fed experimental diets for 7 weeks (a, b) (LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4). Body weights at necropsy (c), body weight gain over 8 weeks of dietary intervention (d), subcutaneous adipose tissue (SAT) weights at necropsy (e), and energy intake (as recorded over a week at week 4) (f) in conventionalized C3H mice fed experimental diets for 8 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 versus WD (one-way ANOVA with Dunnett’s post hoc tests). Mean ± SEM. a, b N = 6–9. c–e N = 8–9. f N = 4 cages

Fig. 3

Resistant starches changed the gut microbiota composition in conventionalized C3H mice fed experimental diets for 8 weeks. a Alpha-diversity index. b Principal Coordinate Analysis plot of β-diversity based on binary Jaccard distance. c Log-fold change in the relative abundance of taxa significantly affected by the dietary intervention (adjusted p value <0.05). LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4. *p < 0.05, **p < 0.01 versus WD (one-way ANOVA with Dunnett’s post hoc tests). Mean ± SEM. N = 8

Fig. 4

Resistant starches improved plasma insulin levels and the index of insulin resistance in conventionalized (CVZ) and germ-free (GF) C3H mice fed experimental diets for 8 weeks. a Plasma insulin levels in 6-h fasted mice. b Plasma glucose levels in 6-h fasted mice. c Index of insulin resistance (IR), also known as HOMA-IR. LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4. *p < 0.05 versus WD fed mice of the same microbial status (one-way ANOVA with Dunnett’s post hoc tests). Microbial status significantly impacted fasting insulinemia and glycemia (analysis of all eight treatments using two-way ANOVA). Mean ± SEM. N = 7–8

Fig. 5

Gut peptide and hormone levels in germ-free (GF) and conventionalized (CVZ) C3H mice fed experimental diets for 8 weeks. a Fasting plasma C-peptides levels. b Proglucagon mRNA expression in the colon. c Fasting plasma glucagon-like peptide 1 (GLP-1) levels. d PYY mRNA expression in the colon. e Fasting plasma peptide YY (PYY) levels. f Fasting plasma ghrelin levels. g Adiponectin mRNA expression in the subcutaneous adipose tissue (SAT). h Fasting plasma adiponectin levels. Mice were fasted for 6 h prior to sampling. LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4. ^# p = 0.07, *p < 0.05 versus WD fed mice of the same microbial status (one-way ANOVA with Dunnett’s post tests). Microbial status significantly impacted C-peptide, GLP-1, PYY, and ghrelin levels, as well as proglucagon mRNA expression (analysis of all eight treatments using two-way ANOVA). Mean ± SEM. N = 7–8, except for c and f where N = 6–8

Fig. 6

Altered expression of macrophage markers in the adipose tissues of C3H mice fed a Western diet supplemented with resistant starches for 8 weeks. a–d F4/80 and CD11c expression in the subcutaneous and visceral adipose tissues (SAT and VAT). e–h Flow cytometry analysis of the stromal vascular fraction (SVF) isolated from the SAT of conventionalized C3H mice. e Percentage of macrophages (F4/80⁺ CD11b⁺). f Percentage of M1 (CD11c⁺) macrophages. g Percentage of M2 (CD206⁺) macrophages. h Ratio of M1/M2, using CD11c as a M1 marker and CD206 as a M2 marker. LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4. ^$ p = 0.11, ^# p = 0.05–0.07, *p < 0.05, ***p < 0.001 versus WD fed mice of the same microbial status (one-way ANOVA with Dunnett’s post hoc tests). Analysis performed after normalization by log-transformation for d (GF data) and f. Microbial status significantly impacted F4/80 expression in SAT (analysis of all eight treatments using two-way ANOVA). Mean ± SEM. a–d N = 5–8. e–h N = 14–15 except for the LFD where N = 6

Fig. 7

Markers of intestinal permeability in germ-free (GF) and conventionalized (CVZ) C3H mice fed experimental diets for 8 weeks. a Gut permeability as assessed by administering 4 kDa FITC-dextran. b Zonula occludens 1 (ZO-1) and c occludin mRNA expression in the ileum. LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4. Mean ± SEM. a N = 6–8. b–c N = 7–8

Fig. 8

Resistant starches restored the cecal bile acid profiles of germ-free (GF) and conventionalized (CVZ) C3H mice fed experimental diets for 8 weeks. Mean bile acid concentration for each dietary treatment (nmol/g of cecal content). Only bile acids with mean concentrations above 1 nmol/g for at least one experimental treatment are shown. LFD low-fat diet, WD Western diet, RS2 WD with resistant starch 2, RS4 WD with resistant starch 4, CA cholic acid, CDCA chenodeoxycholic acid, MCA muricholic acid, HCA hyocholic acid, MuroCA murocholic acid, ACA allocholic acid, LCA lithocholic acid, DCA deoxycholic acid, HDCA hyodeoxycholic acid, ILCA isolitocholic acid, UDCA ursodeoxycholic acid, K keto derivative, T taurine-conjugated species. Complete abbreviation list is available in the supplemental materials and methods. °p < 0.05 versus WD fed mice of the same microbial status (one-way ANOVA with Dunnett’s post hoc tests after normalization by log-transformation when needed). N = 7–8