Ketogenic Diet Suppressed T-Regulatory Cells and Promoted Cardiac Fibrosis via Reducing Mitochondria-Associated Membranes and Inhibiting Mitochondrial Function

Abstract

Ketogenic diet (KD) is popular in diabetic patients but its cardiac safety and efficiency on the heart are unknown. The aim of the present study is to determine the effects and the underlined mechanisms of KD on cardiac function in diabetic cardiomyopathy (DCM). We used db/db mice to model DCM, and different diets (regular or KD) were used. Cardiac function and interstitial fibrosis were determined. T-regulatory cell (Treg) number and functions were evaluated. The effects of ketone body (KB) on fatty acid (FA) and glucose metabolism, mitochondria-associated endoplasmic reticulum membranes (MAMs), and mitochondrial respiration were assessed. The mechanisms via which KB regulated MAMs and Tregs were addressed. KD improved metabolic indices in db/db mice. However, KD impaired cardiac diastolic function and exacerbated ventricular fibrosis. Proportions of circulatory CD4⁺CD25⁺Foxp3⁺ cells in whole blood cells and serum levels of IL-4 and IL-10 were reduced in mice fed with KD. KB suppressed the differentiation to Tregs from naive CD4⁺ T cells. Cultured medium from KB-treated Tregs synergically activated cardiac fibroblasts. Meanwhile, KB inhibited Treg proliferation and productions of IL-4 and IL-10. Treg MAMs, mitochondrial respiration and respiratory complexes, and FA synthesis and oxidation were all suppressed by KB while glycolytic levels were increased. L-carnitine reversed Treg proliferation and function inhibited by KB. Proportions of ST2L⁺ cells in Tregs were reduced by KB, as well as the production of ST2L ligand, IL-33. Reinforcement expressions of ST2L in Tregs counteracted the reductions in MAMs, mitochondrial respiration, and Treg proliferations and productions of Treg cytokines IL-4 and IL-10. Therefore, despite the improvement of metabolic indices, KD impaired Treg expansion and function and promoted cardiac fibroblast activation and interstitial fibrosis. This could be mainly mediated by the suppression of MAMs and fatty acid metabolism inhibition via blunting IL-33/ST2L signaling.

Figure 1

Effects of ketogenic diet on cardiac functions, left ventricular size, and collagen contents. Cardiac functions were represented as +dp/dt_max (a), −dp/dt_max (b), E/A ratio (c), LVEF (d) and LVEDd (e). Left ventricular size was represented as LVEDd. Collagen contents were represented as Masson's Trichrome staining (f) quantified as collagen volume fraction (g). KD: ketogenic diet; LVEF: left ventricular ejection fraction; LVEDd: left ventricular end-diastolic diameter.

Figure 2

Ketogenic diet suppressed Treg functions which promoted cardiac fibroblast activation. Effects of ketogenic diet on Treg proportions in whole blood cells (a) and serum Treg cytokines (b, c). Effects of ketogenic diet on Treg proportions in CD4⁺ T cells derived from the spleen (d) and medium Treg cytokines (e, f). Effects of spleen-derived CD4⁺CD25⁺Foxp3⁺ Treg cultured medium from different treatments on activation markers (g) and cell vitality (h). P values were derived either from the t-test (a, d) or the ANOVA followed by SNK for multiple comparisons (b, c, e–h).

Figure 3

Effects of KB on glucose and FA metabolism in Tregs. Effects of KB on extracellular acidification rates (a) and oxygen consumption (b, c) in CD4⁺CD25⁺Foxp3⁺ cells derived from the spleen. Effects of KB on intracellular FA contents (d) and absorption (e) in CD4⁺CD25⁺Foxp3⁺ cells. Effects of KB on key genes involved in FA synthesis and oxidation (f). Effects of KB on CD25⁺Foxp3⁺ cells derived from CD4+ T cells (g). P values were derived either from the ANOVA followed by SNK for multiple comparisons (a–c, f, g) or the t-test (d, e). Basic Resp: basic respiration; Max Resp: maximum respiration; ATP Prod: ATP production.

Figure 4

Effects of KB on MAMs (a, b) and mitochondrial respiratory complex (c) contents and mitochondrial number (d). MAMs: mitochondria-associated endoplasmic reticulum membranes; FASL-4: long-chain fatty-acid CoA synthases-4; Sigma-1R: Sigma-1 receptor; OD: optical density; Ctyc: cytochrome C; CV: complex V; CIII: complexes III; CIV: complexes. Other abbreviations were the same as the above.

Figure 5

Effects of KB on IL-33/ST2L signaling and its role in KB-induced MAMs and Treg alterations. Flow cytometry detected the changes of membrane ST2L expression in CD4⁺CD25⁺Foxp3⁺ Tregs (a). Cultured medium levels of IL-33 and sST2 PBS-/KB-treated Tregs (b, c). Cultured medium levels of IL-33 and sST2 and MAMs in membrane-ST2L-negative/positive Tregs (d, e). Reinforcement of membrane-ST2L expression on Treg MAMs and Treg proportions in CD4⁺ T cells (f, g, h). sST2: soluble ST2; ST2L⁻: ST2L⁻ Tregs; ST2L⁺: ST2L⁺ Tregs; Con: control vector; ST2L: ST2L vector. Levels of IL-33 were detected by ELISA after NP-40 treatment.