Epigenetics, 1-Carbon Metabolism, and Homocysteine During Dysbiosis

Abstract

Although a high-fat diet (HFD) induces gut dysbiosis and cardiovascular system remodeling, the precise mechanism is unclear. We hypothesize that HFD instigates dysbiosis and cardiac muscle remodeling by inducing matrix metalloproteinases (MMPs), which leads to an increase in white adipose tissue, and treatment with lactobacillus (a ketone body donor from lactate; the substrate for the mitochondria) reverses dysbiosis-induced cardiac injury, in part, by increasing lipolysis (PGC-1α, and UCP1) and adipose tissue browning and decreasing lipogenesis. To test this hypothesis, we used wild type (WT) mice fed with HFD for 16 weeks with/without a probiotic (PB) in water. Cardiac injury was measured by CKMB activity which was found to be robust in HFD-fed mice. Interestingly, CKMB activity was normalized post PB treatment. Levels of free fatty acids (FFAs) and methylation were increased but butyrate was decreased in HFD mice, suggesting an epigenetically governed 1-carbon metabolism along with dysbiosis. Levels of PGC-1α and UCP1 were measured by Western blot analysis, and MMP activity was scored via zymography. Collagen histology was also performed. Contraction of the isolated myocytes was measured employing the ion-optic system, and functions of the heart were estimated by echocardiography. Our results suggest that mice on HFD gained weight and exhibited an increase in blood pressure. These effects were normalized by PB. Levels of fibrosis and MMP-2 activity were robust in HFD mice, and treatment with PB mitigated the fibrosis. Myocyte calcium-dependent contraction was disrupted by HFD, and treatment with PB could restore its function. We conclude that HFD induces dysbiosis, and treatment with PB creates eubiosis and browning of the adipose tissue.

Keywords: DNMT; MMP; REDD1; butyrate; epigenetics; eubiosis.

FIGURE 1

(A) Body weight of WT mice on a HFD with and without the probiotic treatment at the beginning (0 week) and after 16 weeks of treatment. (B) The mean arterial pressure (MAP, mmHg) was measured by tail-cuff and 16 weeks. *, p < 0.05 when compared with WT or WT + HFD + PB; n = 5.

FIGURE 2

Multi-organ damage measured by estimating serum levels of: (A) Representative agarose gel electrophoresis of CK MM (marker of skeletal muscle injury); CK MB (marker of cardiac muscle injury) and CK BB (marker of neuronal injury); (B) Relative intensity of CK MB in the serum of different groups of mice. *, p < 0.05 when compared with WT or WT + HFD + PB; n = 5.

FIGURE 3

Dysbiosis induced by feeding a HFD: Serum levels of (A) free fatty acid (FFA); and (B) butyrate; and (C) global DNA methylation in blood borne cells. The levels were measured spectroscopically in a microplate and colorimetric methods. *, p < 0.05 when compared with WT or WT + HFD + PB; n = 5.

FIGURE 4

Representative Western blot analysis of DNMT, BHMT, and PEMT (A); Musclin, DAAM1, DAAM2, and REDD1 (B); PGC1a, TFAM, UCP1, DRP1 (C), in LV tissue of WT mice on HFD with and without the treatment with PB. The bar graph represents the intensity in arbitrary unit from n = 5 in each group. *, p < 0.05 compared with WT or the WT plus HFD plus PB.

FIGURE 5

Zymography activity of MMP-2: (A) Representative 8%-gelatin gel zymography on cardiac tissue homogenates from WT mice on HFD with and without the treatment of probiotic. (B) The relative scan intensity (arbitrary unit) of MMP-2 activity. *, p < 0.05 when compared with WT or WT + HFD + PB; n = 5.

FIGURE 6

Representative Histological analysis of collagen staining by trichrome blue in the heart of WT on a HFD with and without the treatment with probiotic. Blue color represents collagen. The arrow indicates the perivascular fibrosis.

FIGURE 7

Representation of single myocyte contraction and calcium (Ca²⁺) transients as measured by Ion-Optic. Calcium transience was detected by FURA-2. The fluorescence ratio of FURA-2 and binding to calcium was recorded. Calcium ratio at cell lengthening and the duration of lengthening was measured. Myocytes were stimulated at 1 Hz. The myocyte lengthening (micron, μM) was determined as contraction in cardiomyocyte from WT mice on HFD treated with and without the probiotic, as previously reported by our group (Mishra et al., 2010).

FIGURE 8

Representative scan of cardiac Echocardiography of WT mice on a HFD treated with and without probiotic. Bar graph represents the ejection fraction of the heart of mice treated with a HFD and with and without probiotic. *, p < 0.05 when compared with WT or WT + HFD + PB; n = 5.