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Effect of a Ketogenic Diet on Hepatic Steatosis and Hepatic Mitochondrial Metabolism in Nonalcoholic Fatty Liver Disease

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Effect of a Ketogenic Diet on Hepatic Steatosis and Hepatic Mitochondrial Metabolism in Nonalcoholic Fatty Liver Disease Effect of a ketogenic diet on hepatic steatosis and hepatic mitochondrial metabolism in nonalcoholic fatty liver disease Panu K. Luukkonena,b,c, Sylvie Dufoura,d, Kun Lyue, Xian-Man Zhanga,d, Antti Hakkarainenf,g, Tiina E. Lehtimäkif, Gary W. Clinea,d, Kitt Falk Petersena,d, Gerald I. Shulmana,d,e,1,2, and Hannele Yki-Järvinenb,c,1,2 aDepartment of Internal Medicine, Yale School of Medicine, New Haven, CT 06520; bMinerva Foundation Institute for Medical Research, Helsinki 00290, Finland; cDepartment of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland; dYale Diabetes Research Center, Yale School of Medicine, New Haven, CT 06520; eDepartment of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520; fDepartment of Radiology, HUS Medical Imaging Center, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland; and gDepartment of Neuroscience and Biomedical Engineering, Aalto University School of Science, 00076 Espoo, Finland Contributed by Gerald I. Shulman, January 31, 2020 (sent for review December 26, 2019; reviewed by Fredrik Karpe and Roy Taylor) Weight loss by ketogenic diet (KD) has gained popularity in after 48 h of caloric restriction (26). We previously showed that management of nonalcoholic fatty liver disease (NAFLD). KD rapidly a hypocaloric, KD induces an ∼30% reduction in IHTG content in reverses NAFLD and insulin resistance despite increasing circulating 6 d despite increasing circulating NEFA (27). nonesterified fatty acids (NEFA), the main substrate for synthesis of While the antisteatotic effect of KD is well-established, the intrahepatic triglycerides (IHTG). To explore the underlying mecha- underlying mechanisms by which it does so remain unclear. KD nism, we quantified hepatic mitochondrial fluxes and their regula- increases plasma NEFA concentrations, the main substrate of IHTG tors in humans by using positional isotopomer NMR tracer analysis. (11). In the liver, NEFA can either be re-esterified into complex Ten overweight/obese subjects received stable isotope infusions of: lipids, such as TGs, or be transported to the mitochondria to be 13 β 13 [D7]glucose, [ C4] -hydroxybutyrate and [3- C]lactate before and metabolized by β-oxidation into acetyl-CoA, which in turn can after a 6-d KD. IHTG was determined by proton magnetic resonance either be irreversibly condensed with oxaloacetate by citrate syn- 1 spectroscopy ( H-MRS). The KD diet decreased IHTG by 31% in the thase to form citrate and enter the TCA cycle for terminal oxi- face of a 3% decrease in body weight and decreased hepatic insulin MEDICAL SCIENCES dation to CO2 (28, 29) or it can enter the ketogenic pathway, resistance (−58%) despite an increase in NEFA concentrations where it is converted into acetoacetate (AcAc) and β-hydroxybutyrate (+35%). These changes were attributed to increased net hydrolysis (β-OHB) (28). These mitochondrial fluxes are tightly regulated of IHTG and partitioning of the resulting fatty acids toward keto- by substrate availability and product inhibition (29), mitochon- + genesis ( 232%) due to reductions in serum insulin concentra- drial redox state (30), and hormones, such as leptin (31) and − − tions ( 53%) and hepatic citrate synthase flux ( 38%), respectively. triiodothyronine (T3) (32). The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state Significance (+167%) and decreased plasma leptin (−45%) and triiodothyronine (−21%) concentrations. These data demonstrate heretofore unde- scribed adaptations underlying the reversal of NAFLD by KD: That is, Ketogenic diet is an effective treatment for nonalcoholic fatty markedly altered hepatic mitochondrial fluxes and redox state to liver disease (NAFLD). Here, we present evidence that hepatic promote ketogenesis rather than synthesis of IHTG. mitochondrial fluxes and redox state are markedly altered during ketogenic diet-induced reversal of NAFLD in humans. carbohydrate restriction | redox | citrate synthase | insulin resistance | Ketogenic diet for 6 d markedly decreased liver fat content and pyruvate carboxylase hepatic insulin resistance. These changes were associated with increased net hydrolysis of liver triglycerides and decreased endogenous glucose production and serum insulin concentra- onalcoholic fatty liver disease (NAFLD) is the most common tions. Partitioning of fatty acids toward ketogenesis increased, Nchronic liver disease and can progress from steatosis to ad- which was associated with increased hepatic mitochondrial vanced liver disease, including liver cirrhosis and hepatocellular – redox state and decreased hepatic citrate synthase flux. These carcinoma (1 3). It is strongly associated with insulin resistance, data demonstrate heretofore undescribed adaptations un- which is characterized by excessive hepatic glucose production and derlying the reversal of NAFLD by ketogenic diet and highlight – compensatory hyperinsulinemia (4 10). In adipose tissue of sub- hepatic mitochondrial fluxes and redox state as potential jects with NAFLD, insulin fails to suppress lipolysis, which leads to treatment targets in NAFLD. increased hepatic delivery of nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG) Author contributions: P.K.L., G.I.S., and H.Y.-J. designed research; P.K.L. recruited partic- (4–11). Excess substrate and hyperinsulinemia may stimulate re- ipants, performed clinical studies and drafted the manuscript; P.K.L., S.D., K.L., and X.-M.Z. esterification and de novo lipogenesis (DNL) of fatty acids, which analyzed plasma samples; A.H. and T.E.L. obtained magnetic resonance imaging data; P.K.L., S.D., K.L., X.-M.Z., A.H., T.E.L., G.W.C., K.F.P., G.I.S., and H.Y.-J. analyzed data; and can further increase IHTG content and overproduction of very low- P.K.L., K.F.P., G.I.S., and H.Y.-J. wrote the paper. – density lipoprotein (VLDL)-TG into circulation (12 16). Together, Reviewers: F.K., Oxford Centre for Diabetes; and R.T., Newcastle University. these features of NAFLD increase the risk of type 2 diabetes and The authors declare no competing interest. cardiovascular disease (1, 2). This open access article is distributed under Creative Commons Attribution-NonCommercial- Since obesity is an important cause of NAFLD, its management NoDerivatives License 4.0 (CC BY-NC-ND). – is underpinned by weight loss (17 22). Recently, low-carbohydrate 1G.I.S. and H.Y.-J. contributed equally to this work. ketogenic diets (KD) have gained popularity in the treatment of 2To whom correspondence may be addressed. Email: [email protected] or obesity, type 2 diabetes, and NAFLD (23–25). While long-term data [email protected]. comparing different weight loss regimens in NAFLD are virtually This article contains supporting information online at https://www.pnas.org/lookup/suppl/ nonexistent, a low-carbohydrate diet has been reported to induce a doi:10.1073/pnas.1922344117/-/DCSupplemental. threefold greater IHTG loss than a low-fat, high-carbohydrate diet First published March 16, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1922344117 PNAS | March 31, 2020 | vol. 117 | no. 13 | 7347–7354 Downloaded by guest on October 1, 2021 In this study we examined the effects of a short-term KD on decreased on the average by 3.0 ± 0.3% from 93.5 ± 5.3 to 90.7 ± hepatic steatosis by assessing IHTG content and liver stiffness by 5.2 kg (P < 0.00001) (Fig. 2D and Table 1). magnetic resonance spectroscopy/elastography (1H-MRS/MRE) in 10 overweight/obese participants before and after a 6-d KD. In KD Decreased IHTG Content. IHTG content decreased by ∼31% order to examine the effect that a short-term KD diet might have from 10.3 ± 2.3 to 7.1 ± 2.0% (P < 0.001) (Fig. 3A)asdetermined on rates of hepatic mitochondrial fat oxidation and gluconeo- by 1H-MRS. Liver stiffness as determined by MRE remained un- genesis, we applied a positional isotopomer NMR tracer analysis changed (2.6 ± 0.1 vs. 2.5 ± 0.1 kilopascals [kPa], before vs. after, (PINTA) method (33–35) to assess rates of hepatic mitochon- P = 0.18) (Fig. 3B). Activities of plasma γ-glutamyltransferase V drial flux through pyruvate carboxylase ( PC) relative to citrate (GGΤ) decreased from 48 ± 10 to 38 ± 7 U/L (P < 0.05) and V synthase flux ( CS), as well as rates of endogenous glucose, alkaline phosphatase (ALP) from 82 ± 8to73± 7U/L(P < 0.05) β-OHB, and lactate production by stable isotope infusions of (Fig. 3), while plasma alanine aminotransferase (ALT) and as- 13 β 13 [D7]glucose, [ C4] -OHB, and [3- C]lactate, respectively. Fi- partate aminotransferase (AST) remained unchanged during the nally, in order to gain insights into how these hepatic mito- diet (Table 1). The AST/ALT ratio increased significantly by chondrial fluxes might be regulated during a KD, we also ∼34% from 0.84 ± 0.09 to 1.13 ± 0.15 (P < 0.05) during the diet assessed some key potential regulators of these mitochondrial (Table 1). fluxes (i.e., hepatic mitochondrial redox state as reflected by β plasma [ -OHB]/[AcAc], plasma leptin, and T3 concentrations) in KD Improved Plasma Glucose, TGs, and Insulin Sensitivity. Fasting SI Appendix these same subjects (Fig. 1 and ,Fig.S1). plasma glucose concentrations decreased by 13% from 112 ± 3 ± P < A Results to 98 3mg/dL( 0.01) (Fig. 4 ), while fasting NEFA concentrations increased by 35% from 0.55 ± 0.02 to 0.74 ± 0.02 The Study Diet Was Ketogenic and Participants Were Compliant. mmol/L (P < 0.001) (Fig. 4B). Plasma TG concentration, which Characteristics of the participants are shown in Table 1. Their in the fasting state reflects predominantly liver-derived VLDL- dietary intakes were assessed by 3-d food records at baseline and A TGs, decreased by 25% from 1.26 ± 0.14 to 0.94 ± 0.10 mmol/L at the end of the 6-d KD (Fig. 1 ). Compliance was verified by P < C measuring plasma ketone bodies (β-OHB and AcAc).
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