Insulin-like peptide 5 is a microbially regulated peptide that promotes hepatic glucose production

Abstract

Objective: Insulin-like peptide 5 (INSL5) is a recently identified gut hormone that is produced predominantly by L-cells in the colon, but its function is unclear. We have previously shown that colonic expression of the gene for the L-cell hormone GLP-1 is high in mice that lack a microbiota and thus have energy-deprived colonocytes. Our aim was to investigate if energy deficiency also affected colonic Insl5 expression and to identify a potential role of INSL5.

Methods: We analyzed colonic Insl5 expression in germ-free (GF), conventionally raised (CONV-R), conventionalized (CONV-D) and antibiotic-treated mice, and also assessed the effect of dietary changes on colonic Insl5 expression. In addition, we characterized the metabolic phenotype of Insl5-/- mice.

Results: We showed that colonic Insl5 expression was higher in GF and antibiotic-treated mice than in CONV-R mice, whereas Insl5 expression in the brain was higher in CONV-R versus GF mice. We also observed that colonic Insl5 expression was suppressed by increasing the energy supply in GF mice by colonization or high-fat feeding. We did not observe any differences in food intake, gut transit or oral glucose tolerance between Insl5-/- and wild-type mice. However, we showed impaired intraperitoneal glucose tolerance in Insl5-/- mice. We also observed improved insulin tolerance and reduced hepatic glucose production in Insl5-/- mice.

Conclusions: We have shown that colonic Insl5 expression is regulated by the gut microbiota and energy availability. We propose that INSL5 is a hormone that could play a role in promoting hepatic glucose production during periods of energy deprivation.

Keywords: Colon; Gut microbiota; Insulin-like peptide 5 (INSL5); Liver.

Figure 1

Colonic Insl5 expression is reduced by the gut microbiota and energy availability. Insl5 expression in colon from (A) Swiss Webster GF and CONV-R mice (n = 4–5), (B) Swiss Webster mice after 3 days of antibiotic treatment (Abx) or control (n = 4–6), (C) Swiss Webster GF and CONV-R mice on embryonal day 18 (E18), postnatal days 1 (P1) and 3 (P3) and during weeks 1–8 of their life (1 w–8 w) (n = 5), (D) Swiss Webster GF and CONV-R mice on a standard chow diet or a high-fat diet (HFD) (n = 4–6), (E)Insl5 expression in colon from C57Bl/6 GF and CONV-R mice (n = 4–5), (F) C57Bl/6 GF, CONV-D (GF mice that were conventionalized with a normal gut microbiota for 1, 3 and 7 days) and CONV-R mice (n = 3–4), and (G) C57Bl/6 GF, B. thetaiotaomicron-colonized and CONV-R mice, (n = 3–4). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. In F, samples were analyzed by one-way ANOVA with post hoc Bonferroni test, where the mean of each test group was compared to the mean of the (GF) control group.

Figure 2

Insl5 expression is detected in the colon and the brain. (A)Insl5 expression in different C57Bl/6 mouse tissues (samples are pooled from n = 3). Si = small intestine; the numbers indicate that the small intestine was divided in eight equal sized pieces labeled 1 (duodenum) to 8 (ileum). Insl5 expression in (B) hypothalamus (n = 13) and (C) brainstem from C57Bl/6 GF and CONV-R mice (n = 5–7). Data are mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3

C57Bl/6 Insl5−/− mice have impaired hepatic glucose production. (A) Oral glucose tolerance test (OGTT) and (B) serum insulin levels after glucose gavage in 12 to 14-week-old C57Bl/6 WT and Insl5−/− mice (n = 7–24). (C) Intraperitoneal glucose tolerance test (IPGTT) and (D) serum insulin levels after glucose injection in Insl5−/− and C57Bl/6 WT mice (n = 10). (E) Insulin tolerance test (ITT) in Insl5−/− and C57Bl/6 WT mice (n = 7). (F) Pyruvate tolerance test (PTT) in Insl5−/− and C57Bl/6 WT mice (n = 9–10). (G) Immunoblot analysis of G6PC in liver tissue from Insl5−/−mice and C57Bl/6 WT mice after a 12 h fast (n = 8–9). (H) G6Pase activity in liver tissue from C57Bl/6 Insl5−/−mice and WT mice after a 12 h fast (n = 6–9). (I) Immunoblot analysis of PEPCK in liver tissue from C57Bl/6 Insl5−/−mice and WT mice after a 12 h fast (n = 8–9). (J) Glycogen levels in liver tissue from Insl5−/−mice and C57Bl/6 WT mice after a 6 and 12 h fast (n = 6–9). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.