Nutritional Interventions with Bacillus coagulans Improved Glucose Metabolism and Hyperinsulinemia in Mice with Acute Intermittent Porphyria

Abstract

Acute intermittent porphyria (AIP) is a metabolic disorder caused by mutations in the porphobilinogen deaminase (PBGD) gene, encoding the third enzyme of the heme synthesis pathway. Although AIP is characterized by low clinical penetrance (~1% of PBGD mutation carriers), patients with clinically stable disease report chronic symptoms and frequently show insulin resistance. This study aimed to evaluate the beneficial impact of nutritional interventions on correct carbohydrate dysfunctions in a mouse model of AIP that reproduces insulin resistance and altered glucose metabolism. The addition of spores of Bacillus coagulans in drinking water for 12 weeks modified the gut microbiome composition in AIP mice, ameliorated glucose tolerance and hyperinsulinemia, and stimulated fat disposal in adipose tissue. Lipid breakdown may be mediated by muscles burning energy and heat dissipation by brown adipose tissue, resulting in a loss of fatty tissue and improved lean/fat tissue ratio. Probiotic supplementation also improved muscle glucose uptake, as measured using Positron Emission Tomography (PET) analysis. In conclusion, these data provide a proof of concept that probiotics, as a dietary intervention in AIP, induce relevant changes in intestinal bacteria composition and improve glucose uptake and muscular energy utilization. Probiotics may offer a safe, efficient, and cost-effective option to manage people with insulin resistance associated with AIP.

Keywords: glucose homeostasis; gut microbiome; hepatic porphyria; insulin resistance; metabolic disease; nutritional intervention; probiotics.

Figure 1

Feeding AIP mice with a B. coagulans-supplemented diet ameliorates glucose tolerance and hyperinsulinemia. (A) GTT curves performed after 5 g/kg i.p. glucose overload to induce severe hyperglycemia. Blood glucose was measured from 0 to 180 min after glucose injection. (B) Box plot showing the total peak area under the curve (AUC) calculated from the 1st to the 3rd hour post-glucose overload. (C) Analysis of the slope was carried out from the glycemic peak at 30 min up to 90 min after carbohydrate loading. (D) Insulin levels were measured in serum from WT and AIP mice with or without treatments. All experimental groups were fasted 14 h before the GTT and insulin measurements. Data are shown as average and standard deviation (SD). * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni post-test.

Figure 2

B. coagulans-supplemented-diet-induced weight loss was accompanied by higher lean/fat ratio. (A) Body weight was measured weekly in mice and normalized for kg of weekly food ingested. (B) Body fat mass (%) and (C) lean/fat mass ratio were assessed using quantitative MRI. Data are shown as mean and standard deviation (SD). * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni post-test.

Figure 3

B. coagulans supplementation slightly modified glucose uptake in the liver and brain of AIP mice, but strongly enhanced glucose absorption in skeletal muscle. (A) [¹⁸F]FDG radioactivity measured ex vivo in mice livers. (B) [¹⁸F]FDG radioactivity measured ex vivo in the brain (right). (C,D) Glut1/2/3 and InsRβ chain analyzed using WB in liver and brain tissues. (E) [¹⁸F]FDG radioactivity measured ex vivo in skeletal muscle. (F) Glut1/4 and InsRβ chain analyzed usign WB in the skeletal muscle. (G) Representative PET/CT in vivo images of the brain (axial plane) and of skeletal muscle (tibial anterior, on top: axial plane; on bottom: coronal plane) performed 1h after [¹⁸F]FDG injection in fasted mice. Ex vivo acquired data were normalized by [¹⁸F]FDG injected volume and tissue weight (g). Values are shown as average and standard deviation (SD). * p < 0.05, *** p < 0.001, one-way ANOVA followed by Bonferroni post-test.

Figure 4

[¹⁸F]FDG uptake and glucose transporters in white and brown adipose tissues. (A) Ex vivo quantification of [¹⁸F]FDG radiotracer measured in white fat pads (mesenteric, gonadal, retroperitoneal, and subcutaneous). (B) Ex vivo quantification of [¹⁸F]FDG radiotracer measured in BAT at RT and after cold stimulus (left). (C) Representative PET/CT in vivo images of [¹⁸F]FDG uptake in BAT (interscapular space and sagittal plane) performed at RT and 4 °C in fasted mice. Data were normalized on [¹⁸F]FDG injected volume and tissue weight (g). Representative images of WBs from Glut1/4 and InsRβ chain assessed in (D) BAT and (E) WAT (gonadal fat) in mice were challenged with cold. Values are shown as mean and standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Bonferroni post-test.

Figure 5

B. coagulans supplementation enhanced hepatic heme content in AIP mice. Bar graph shows heme content as colorimetrically measured in frozen liver samples. Data were normalized by mg of liver protein. ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Bonferroni post-test.

Figure 6

Gut microbiota changes associated with the disease and nutritional supplementation with B. coagulans. (A) Bacterial species not present or with reduced abundance in the gut microbiota of AIP mice when compared to WT that were increased after the probiotic supplementation. (B) Bacterial species highly represented in the gut microbiome in AIP mice, and that were restored upon B. coagulans administration to levels found in control WT animals. (C–F) Bacterial species that changed their presence with age in both WT and AIP mice, but nutritional supplementation with B. coagulans maintained their presence with respect to basal time levels. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Bonferroni post-test.