Ketogenic diet-induced reversal of NAFLD in humans

Ketogenic diet is an effective treatment for nonalcoholic fatty liver disease (NAFLD). Here, we present evidence that hepatic mitochondrial fluxes and redox state are markedly altered during ketogenic diet-induced reversal of NAFLD in humans.

Ketogenic diet for 6 d markedly decreased liver fat content and hepatic insulin resistance. These changes were associated with increased net hydrolysis of liver triglycerides and decreased endogenous glucose production and serum insulin concentrations. Partitioning of fatty acids toward ketogenesis increased, which was associated with increased hepatic mitochondrial redox state and decreased hepatic citrate synthase flux. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by ketogenic diet and highlight hepatic mitochondrial fluxes and redox state as potential treatment targets in NAFLD.

ABSTRACT

Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D₇]glucose, [¹³C₄]β-hydroxybutyrate and [3-¹³C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy (¹H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (−58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (−53%) and hepatic citrate synthase flux (−38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (−45%) and triiodothyronine (−21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and can progress from steatosis to advanced liver disease, including liver cirrhosis and hepatocellular carcinoma. It is strongly associated with insulin resistance, which is characterized by excessive hepatic glucose production and compensatory hyperinsulinemia. In adipose tissue of subjects with NAFLD, insulin fails to suppress lipolysis, which leads to increased hepatic delivery of nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). Excess substrate and hyperinsulinemia may stimulate re-esterification and de novo lipogenesis (DNL) of fatty acids, which can further increase IHTG content and overproduction of very low-density lipoprotein (VLDL)-TG into circulation. Together, these features of NAFLD increase the risk of type 2 diabetes and cardiovascular disease.

Since obesity is an important cause of NAFLD, its management is underpinned by weight loss. Recently, low-carbohydrate ketogenic diets (KD) have gained popularity in the treatment of obesity, type 2 diabetes, and NAFLD. While long-term data comparing different weight loss regimens in NAFLD are virtually nonexistent, a low-carbohydrate diet has been reported to induce a threefold greater IHTG loss than a low-fat, high-carbohydrate diet after 48 h of caloric restriction. We previously showed that a hypocaloric, KD induces an ∼30% reduction in IHTG content in 6 d despite increasing circulating NEFA.

While the antisteatotic effect of KD is well-established, the underlying mechanisms by which it does so remain unclear. KD increases plasma NEFA concentrations, the main substrate of IHTG. In the liver, NEFA can either be re-esterified into complex lipids, such as TGs, or be transported to the mitochondria to be metabolized by β-oxidation into acetyl-CoA, which in turn can either be irreversibly condensed with oxaloacetate by citrate synthase to form citrate and enter the TCA cycle for terminal oxidation to CO₂ or it can enter the ketogenic pathway, where it is converted into acetoacetate (AcAc) and β-hydroxybutyrate (β-OHB). These mitochondrial fluxes are tightly regulated by substrate availability and product inhibition, mitochondrial redox state, and hormones, such as leptin and triiodothyronine (T3).

In this study we examined the effects of a short-term KD on hepatic steatosis by assessing IHTG content and liver stiffness by magnetic resonance spectroscopy/elastography (¹H-MRS/MRE) in 10 overweight/obese participants before and after a 6-d KD. In order to examine the effect that a short-term KD diet might have on rates of hepatic mitochondrial fat oxidation and gluconeogenesis, we applied a positional isotopomer NMR tracer analysis (PINTA) method to assess rates of hepatic mitochondrial flux through pyruvate carboxylase (V_PC) relative to citrate synthase flux (V_CS), as well as rates of endogenous glucose, β-OHB, and lactate production by stable isotope infusions of [D₇]glucose, [¹³C₄]β-OHB, and [3-¹³C]lactate, respectively. Finally, in order to gain insights into how these hepatic mitochondrial fluxes might be regulated during a KD, we also assessed some key potential regulators of these mitochondrial fluxes (i.e., hepatic mitochondrial redox state as reflected by plasma [β-OHB]/[AcAc], plasma leptin, and T3 concentrations) in these same subjects (Fig. 1 )

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