Mechanisms underlying the resistance to diet-induced obesity in germ-free mice

Abstract

The trillions of microbes that colonize our adult intestines function collectively as a metabolic organ that communicates with, and complements, our own human metabolic apparatus. Given the worldwide epidemic in obesity, there is interest in how interactions between human and microbial metabolomes may affect our energy balance. Here we report that, in contrast to mice with a gut microbiota, germ-free (GF) animals are protected against the obesity that develops after consuming a Western-style, high-fat, sugar-rich diet. Their persistently lean phenotype is associated with increased skeletal muscle and liver levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream targets involved in fatty acid oxidation (acetylCoA carboxylase; carnitine-palmitoyltransferase). Moreover, GF knockout mice lacking fasting-induced adipose factor (Fiaf), a circulating lipoprotein lipase inhibitor whose expression is normally selectively suppressed in the gut epithelium by the microbiota, are not protected from diet-induced obesity. Although GF Fiaf-/- animals exhibit similar levels of phosphorylated AMPK as their wild-type littermates in liver and gastrocnemius muscle, they have reduced expression of genes encoding the peroxisomal proliferator-activated receptor coactivator (Pgc-1alpha) and enzymes involved in fatty acid oxidation. Thus, GF animals are protected from diet-induced obesity by two complementary but independent mechanisms that result in increased fatty acid metabolism: (i) elevated levels of Fiaf, which induces Pgc-1alpha; and (ii) increased AMPK activity. Together, these findings support the notion that the gut microbiota can influence both sides of the energy balance equation, and underscore the importance of considering our metabolome in a supraorganismal context.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

GF mice are protected against diet-induced obesity. (A) Adult male C57BL/6J mice were conventionalized 3 weeks before they were switched to a high-fat Western diet. Initial weight was recorded (25.5 ± 0.4 and 26.6 ± 0.7 g for GF and conventionalized mice, respectively). Weight gain was monitored weekly for 8 weeks and compared with GF mice (n = 5 per group). (B) Response to acute fat loading in GF and conventionalized mice maintained on a low-fat chow diet. Olive oil (400 μl) was administered by gavage to mice that had fasted overnight. Serum triglycerides levels were measured at the indicated time points (n = 5 per group). (C) Locomotor activity was recorded in low-fat chow-fed GF and conventionalized mice over a 3-day period and then again after they had been on a Western diet for 8 weeks (n = 4 per group). Mean values ± SE are plotted. ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001 compared with GF.

Fig. 2.

The gut microbiota suppresses AMPK activity in the gastrocnemius muscle of mice consuming a Western diet. (A) Immunoblotting of protein lysates from gastrocnemius muscle harvested from 15-week-old male GF or conventionalized C57BL/6J mice fed a Western diet for 5 weeks before death. Representative results from two mice per group are shown. (B) Quantification of the results shown in A (n = 4 per group). Data are expressed relative to actin. (C) Effects of the gut microbiota on Cpt activity in freeze-clamped gastrocnemius muscle samples from the mice studied in A and B (n = 5 per group). ∗, P < 0.05 compared with GF; and ∗∗, P < 0.01.

Fig. 3.

The gut microbiota suppresses AMPK activity in liver. (A) Immunoblotting of protein lysates from liver samples obtained from 15-week-old GF or conventionalized male mice fed a Western diet for 5 weeks before they were killed. Representative results from two mice per group are shown. (B) Quantification of the results shown in A (n = 4 per group). Data are expressed relative to actin. Effects of the gut microbiota on glycogen levels (C) and glycogen synthase activity (D) in freeze-clamped livers (n = 5 per group). ∗∗, P < 0.01 compared with GF; and ∗∗∗, P < 0.001.

Fig. 4.

GF Fiaf−/− mice are not protected against diet-induced obesity and have lower expression of Pgc-1α and genes involved in fatty acid oxidation in their gastrocnemius muscles. (A) qRT-PCR assays of Fiaf expression in the distal small intestines and livers of GF and conventionalized wild-type male mice maintained on a low-fat diet since weaning or given a high-fat Western diet for 8 weeks before being killed. Mean values ± SE are plotted. n = 5 mice per group. ∗∗, P < 0.01, compared with GF mice on the chow diet; Ψ, P < 0.05 compared with GF mice on the Western diet. (B) GF Fiaf-deficient mice become obese on a Western diet. Eight- to 10-week-old GF male wild-type and Fiaf−/− mice were switched to the Western diet and their body weights monitored weekly for 5 weeks (n = 5 per group). (C) Epididymal fat-pad weights of the mice shown in B after 5 weeks on the Western diet. (D) qRT-PCR assays of gastrocnemius muscle RNAs prepared from GF Fiaf−/− mice and wild-type littermates on the Western diet (n = 6 per group). Mean values ± SE are plotted. ∗, P < 0.05 compared with wild-type animals; ∗∗, P < 0.01.