Branched chain amino acids and carbohydrate restriction exacerbate ketogenesis and hepatic mitochondrial oxidative dysfunction during NAFLD - PubMed (original) (raw)

. 2020 Nov;34(11):14832-14849.

doi: 10.1096/fj.202001495R. Epub 2020 Sep 12.

Marc McLeod 2, Meghan Maguire 1, Rohit Mahar 2, Nathan Kattapuram 1, Christine Zhang 1, Chaitra Surugihalli 1, Vaishna Muralidaran 1, Kruthi Vavilikolanu 1, Clayton E Mathews 2, Matthew E Merritt 2, Nishanth E Sunny 1

Affiliations

Branched chain amino acids and carbohydrate restriction exacerbate ketogenesis and hepatic mitochondrial oxidative dysfunction during NAFLD

Muhammed S Muyyarikkandy et al. FASEB J. 2020 Nov.

Abstract

Mitochondrial adaptation during non-alcoholic fatty liver disease (NAFLD) include remodeling of ketogenic flux and sustained tricarboxylic acid (TCA) cycle activity, which are concurrent to onset of oxidative stress. Over 70% of obese humans have NAFLD and ketogenic diets are common weight loss strategies. However, the effectiveness of ketogenic diets toward alleviating NAFLD remains unclear. We hypothesized that chronic ketogenesis will worsen metabolic dysfunction and oxidative stress during NAFLD. Mice (C57BL/6) were kept (for 16-wks) on either a low-fat, high-fat, or high-fat diet supplemented with 1.5X branched chain amino acids (BCAAs) by replacing carbohydrate calories (ketogenic). The ketogenic diet induced hepatic lipid oxidation and ketogenesis, and produced multifaceted changes in flux through the individual steps of the TCA cycle. Higher rates of hepatic oxidative fluxes fueled by the ketogenic diet paralleled lower rates of de novo lipogenesis. Interestingly, this metabolic remodeling did not improve insulin resistance, but induced fibrogenic genes and inflammation in the liver. Under a chronic "ketogenic environment," the hepatocyte diverted more acetyl-CoA away from lipogenesis toward ketogenesis and TCA cycle, a milieu which can hasten oxidative stress and inflammation. In summary, chronic exposure to ketogenic environment during obesity and NAFLD has the potential to aggravate hepatic mitochondrial dysfunction.

Keywords: fatty liver; hepatic insulin resistance; lipogenesis; liver mitochondria.

© 2020 Federation of American Societies for Experimental Biology.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Figures

FIGURE 1

FIGURE 1

Chronic induction of ketogenesis following carbohydrate restriction and BCAA supplementation. A, Whole body β-hydroxybutyrate turnover rates during fed, food-restricted and insulin stimulated states. B, Fed to food-restricted induction of β-hydroxybutyrate turnover. C, Insulin stimulated suppression of β-hydroxybutyrate turnover. D, Serum β-hydroxybutyrate concentration during fed, food-restricted and insulin stimulated states. E, Fed to food-restricted induction of serum β-hydroxybutyrate concentrations. F, Insulin stimulated suppression of serum β-hydroxybutyrate concentrations. G, Gene expression profile of Hmgcs2, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 and (H) Gene expression profile of Bdh1, 3-hydroxybutyrate dehydrogenase, type 1. Gene expression profiles are relative to the LF fed group. Results (n = 6–9/group) were considered significant at P ≤ .05 following pairwise mean comparisons which are represented by the following alphabets. “a”—LF vs HF; “b”—LF vs HF-Kt; “c”—HF vs HF-Kt

FIGURE 2

FIGURE 2

The ketogenic dietary environment results from the induction of hepatic β-oxidation. RT-qPCR assay profiles of genes involved in hepatic β-oxidation under fed and food-restricted conditions are represented on the left y-axis and the fold change in expression profiles from feeding to food-restriction are represented on the right y-axis. Gene expression profiles are relative to the LF “Fed” group. A, Cpt1a, carnitine palmitoyltransferase 1a (B) Lcad, Long-chain acyl-CoA dehydrogenase (C) Mcad, Medium-chain acyl-CoA dehydrogenase (D) Acox1, acyl-Coenzyme A oxidase 1 and (E) Cd36, CD36 molecule. F, Hepatic acyl-carnitine levels in food-restricted livers; C6, C8, C14, and C16 are hexanoyl- octanoyl-, myristoyl,- and palmitoyl-carnitines, respectively. Results (n = 6–9/group) were considered significant at P ≤ .05 following pairwise mean comparisons which are represented by the following alphabets. “a”—LF vs HF; “b”—LF vs HF-Kt; “c”—HF vs HF-Kt. “#”— indicates statistical significance at P ≤ .05 using a Student’s t test between the means of the “Fed” and “Food-restricted” groups

FIGURE 3

FIGURE 3

Impact of the ketogenic diet on the hepatic TCA cycle in mice with NAFLD. A, Mathematical modeling of the biochemical reactions in the TCA cycle. B, Basal and ADP stimulated O2 consumption rates by the isolated mitochondria. C, Rates of pyruvate utilization by the isolated mitochondria; v1 = rate of pyruvate entry into the mitochondrial matrix from the respiration buffer, v2 = pyruvate carboxylation to form oxaloacetate, v3 = formation of acetyl-CoA from pyruvate. D, Condensation of two acetyl-CoA molecules to form a β-hydroxybutyrate (Ketogenesis; v4). E, Rates of biochemical reactions in the hepatic TCA cycle; v5 = condensation of acetyl-CoA and oxaloacetate to form citrate; citrate synthase flux, v6 = formation of α-ketoglutarate from citrate, v7 = formation of succinate from α-ketoglutarate, v8 = reversible conversion of succinate to fumarate, v9 = reversible conversion of fumarate to malate, v10 = formation of oxaloacetate from malate. Calculated rates (n = 6–7/ group) which are significant at P ≤ .05 following pairwise mean comparisons are represented by the following alphabets. “a”—LF vs HF; “b”— LF vs HF-Kt; “c”—HF vs HF-Kt. Calculated rates (n = 6–7/group) which are significant at P ≤ .1 following pairwise mean comparisons are represented by the following alphabets. “d”—LF vs HF; “e”—LF vs HF-Kt; “f”—HF vs HF-Kt

FIGURE 4

FIGURE 4

The ketogenic dietary environment suppressed hepatic de novo lipogenesis. A, Representative 2H NMR spectra of the Folch extracted lipids from the liver of LF, HF and HF-Kt mice. Assignments of marked peaks on the spectrum are as follows: (a) ω−3 methyl, (d) aliphatic chain, (e) α3 aliphatic, (f) monounsaturated allylic (h) All α2 aliphatic (l) sn-1, sn-3 of esterified glycerol, (m) sn-2 of esterified glycerol, (n) olefinic (o) chloroform and (p) pyrazine standard. Dotted lines on pyrazine (p) and methyl (a) peaks are showing the level of deuterium enrichment on the terminal methyl position in each of the sample, which ultimately gives the qualitative information of the de novo lipogenesis. B, Measured enrichment of deuterium in the methyl peaks of lipids, (C) Hepatic de novo lipogenesis and (D) Lipogenic gene expression in the liver. Results (n = 5–6/group) were considered significant at P ≤ .05 following pairwise mean comparisons which are represented by the following alphabets. “a”—LF vs HF; “b”—LF vs HF-Kt; “c”—HF vs HF-Kt

FIGURE 5

FIGURE 5

The metabolic remodeling mediated by the ketogenic environment did not translate to improvements in insulin sensitivity. A, Endogenous glucose production (EGP) during food-restricted (basal) and insulin stimulated states. B, Suppression of EGP by insulin. C, Suppression of plasma FFAs by insulin. D, Hepatic insulin sensitivity index, calculated as the product of fasting EGP and fasting serum insulin. E, Glucose disposal rate, which is a reflection of muscle insulin sensitivity (F). Adipose insulin sensitivity index, calculated as the product of fasting FFAs and fasting serum insulin. G, Phosphorylation rates of AKT in the liver, and their fold increase following insulin stimulation. Results (n = 5–8/group) were considered significant at P ≤ .05 following pairwise mean comparisons which are represented by the following alphabets. “a”—LF vs HF; “b”—LF vs HF-Kt; “c”—HF vs HF-Kt. “*”—indicates statistical significance at P ≤ .05 using a Student’s t test between means of basal and insulin stimulated groups

FIGURE 6

FIGURE 6

Chronic exposure to the ketogenic environment accelerates liver injury during NAFLD. A, Masson’s Trichrome staining of liver tissue following food-restriction. B, Fasting serum Alanine Aminotransferase (ALT) levels. C, Hepatic fibrogenic gene expression profiles after food-restriction. Fed and food-restricted expression profiles of genes involved in the inflammatory on set in the liver. D, Tnfa, Tumor necrosis factor alpha (E) Il1b, Interleukin 1 beta (F) Nlrp3, NLR family, pyrin domain containing 3 and (G) Il6. Interleukin 6. Results (n = 5–8/group) were considered significant at P ≤ .05 following pairwise mean comparisons which are represented by the following alphabets. “a”—LF vs HF; “b”— LF vs HF-Kt; “c”—HF vs HF-Kt. Results which are significant at P ≤ .1 following pairwise mean comparisons are represented by the following alphabets. “d”—LF vs HF; “e”—LF vs HF-Kt; “f”—HF vs HF-Kt. “#”—indicates statistical significance at P ≤ .05 using a Student’s t test between means of the fed and food-restricted groups

FIGURE 7

FIGURE 7

Impact of chronic ketogenic environment on mitochondrial metabolism, lipogenesis, and oxidative stress during NAFLD. A ketogenic environment mediates sustained rates of oxidation of free fatty acids in the liver (1) generating 2-carbon units of acetyl-coA, which are shunted toward ketone synthesis (2) with selective upregulation of biochemical reactions in the TCA cycle (3). This chronic metabolic adaptation by the mitochondrial oxidative machinery occurs simultaneously with the downregulation of acetyl-CoA flux to de novo lipogenesis (4). The sustained induction of mitochondrial oxidative metabolism in the liver provides a favorable environment for the sustained generation of ROS (5) fueling higher rates of inflammation, in turn aggravating hepatic injury during NAFLD

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