Pharmacologic inhibition of ketohexokinase prevents fructose-induced metabolic dysfunction

Abstract

Objective: Recent studies suggest that excess dietary fructose contributes to metabolic dysfunction by promoting insulin resistance, de novo lipogenesis (DNL), and hepatic steatosis, thereby increasing the risk of obesity, type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), and related comorbidities. Whether this metabolic dysfunction is driven by the excess dietary calories contained in fructose or whether fructose catabolism itself is uniquely pathogenic remains controversial. We sought to test whether a small molecule inhibitor of the primary fructose metabolizing enzyme ketohexokinase (KHK) can ameliorate the metabolic effects of fructose.

Methods: The KHK inhibitor PF-06835919 was used to block fructose metabolism in primary hepatocytes and Sprague Dawley rats fed either a high-fructose diet (30% fructose kcal/g) or a diet reflecting the average macronutrient dietary content of an American diet (AD) (7.5% fructose kcal/g). The effects of fructose consumption and KHK inhibition on hepatic steatosis, insulin resistance, and hyperlipidemia were evaluated, along with the activation of DNL and the enzymes that regulate lipid synthesis. A metabolomic analysis was performed to confirm KHK inhibition and understand metabolite changes in response to fructose metabolism in vitro and in vivo. Additionally, the effects of administering a single ascending dose of PF-06835919 on fructose metabolism markers in healthy human study participants were assessed in a randomized placebo-controlled phase 1 study.

Results: Inhibition of KHK in rats prevented hyperinsulinemia and hypertriglyceridemia from fructose feeding. Supraphysiologic levels of dietary fructose were not necessary to cause metabolic dysfunction as rats fed the American diet developed hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis, which were all reversed by KHK inhibition. Reversal of the metabolic effects of fructose coincided with reductions in DNL and inactivation of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP). We report that administering single oral doses of PF-06835919 was safe and well tolerated in healthy study participants and dose-dependently increased plasma fructose indicative of KHK inhibition.

Conclusions: Fructose consumption in rats promoted features of metabolic dysfunction seen in metabolic diseases such as T2D and NASH, including insulin resistance, hypertriglyceridemia, and hepatic steatosis, which were reversed by KHK inhibition.

Keywords: Fructose; Insulin resistance; KHK; Metabolic disease; NAFLD.

Figure 1

PF-06835919 blocked fructose metabolism and ChREBP activation in hepatocytes. (A) Graphical representation of fructose catabolism integrated in the glycolytic pathway. KHK phosphorylation of fructose leads to rapid accumulation of fructose-derived glycolytic metabolites, augmenting glycolysis and activating the lipogenic transcription factor ChREBP. (B) Inhibition of F1P generation in primary human and rat hepatocytes treated with PF-06835919 and fructose. Data points were calculated from 6 independent experiments for both human and rat hepatocytes. (C-H) Primary rat hepatocytes were incubated with 10 mM [¹³C₆]-fructose +/− 30 μM PF-06835919 and the enrichment of fructose derived carbons in glycolytic metabolites was measured. Intracellular (C) [¹³C₆]-F1P, (D) [¹³C₃]-DHAP, (E) [¹³C₃]-pyruvate, (F) [¹³C₃]-lactate, (G) [¹³C₃]-G6P, (H) [¹³C₆]-G6P, (I) [¹³C₃]-malate, and (J) [¹³C₂]-citrate were measured from 3 biological replicates. The data are represented as mean +/− standard error of the mean. Representative images showing ChREBP cellular localization (K) from rat hepatocytes treated overnight with fructose or glucose +/− 30 μM PF-06835919. Green = ChREBP and blue = DAPI/nuclei. (L) Percent change in nuclear ChREBP compared to 5 mM of glucose and DMSO as measured by nuclear/cytoplasmic staining ratio (n = 5/group). (M) ChREBP target gene expression was measured in rat hepatocytes incubated overnight with 10 mM of fructose +/− PF-06835919. The data are mean +/− SEM calculated from the mean from four separate experiments with 3 biological replicates in each. Statistical significance was determined by two-way ANOVA and Bonferroni's test for multiple comparisons. ∗∗∗∗p < 0.0001. Glucose-6-phosphate, G6P; fructose-6-phosphate, F6P; fructose-1,6-bisphosphate, F1,6P; fructose-1-phosphate, F1P; dihydroxyacetone phosphate, DHAP; glyceraldehyde, GA; glyceraldehyde-3-phosphate, GA3P; carbohydrate response element-binding protein, ChREBP; oxaloacetic acid, OAA; tricarboxylic acid, TCA; pyruvate kinase, Pklr; acetyl-CoA carboxylase 1, Acc1; ATP-citrate synthase, Acly; fatty acid synthase, Fasn; aldolase B, AldoB.

Figure 2

PF-06835919 inhibited fructose metabolism in the rats. (A) Hepatic and kidney fructose-1-phosphate (F1P) levels were measured in the rats administered vehicle and increasing doses of PF-06835919 after an acute intravenous fructose bolus (500 mg/kg). Percent inhibition was calculated from vehicle controls from three independent experiments and combined for analysis (n = 8–24/group). (B) Intestinal F1P measured in intestinal tissue samples from the vehicle- and PF-06835919-treated animals orally bolused with fructose (2 g/kg) (n = 6/group). Statistical significance determined by unpaired two-tailed t-test. (C) Urine fructose, (D) plasma fructose, (E) plasma glucose, and enrichment of fructose carbons in (F) [¹³C₃]-glucose, (G) [¹³C₆]-glucose, and (H) [¹³C₆]-sorbitol were measured in the rats administered PF-06835919 (3, 10, and 30 mg/kg) after an oral bolus of a 2 mg/kg 50:50 mixture of ¹³C₆/¹²C₆ fructose. Plasma metabolites were measured in the conscious rats at the indicated time points. n = 5–6 per time point and statistical significance was determined by two-way ANOVA and Tukey's test for multiple comparisons. ∗p < 0.05 for 30 mg/kg, #p < 0.05 for 10 mg/kg, and ˆp < 0.05 for 3 mg/kg at the indicated time points. n = 7–8 per group and statistical significance was determined by ANOVA and Bonferroni's test for multiple comparisons. All of the data represent the mean +/− SEM.

Figure 3

KHK inhibition prevented fructose induced hyperlipidemia, hyperinsulinemia, and steatosis. (A) Body weight of the Sprague Dawley rats fed chow or high-fructose chow with the indicated daily BID dose of PF-06835919 (n = 10/group) for 7 weeks. (B) Perigonadal fat pad mass measured at necropsy (n = 10/group). (C) Day 16 plasma fructose as measured over a 24-hr period in the fructose-fed rats (n = 5/group). (D) Urine fructose content measured from the rats housed in metabolic cages for 24 hrs (n = 10/group). (E) Fasting plasma insulin measured throughout the study (n = 9–10/group). (F) Terminal liver triglycerides (n = 10/group) and (G) osmium PPD-stained histological sample images demonstrating periportal staining. Red arrow = osmium-stained lipid droplet. (H) Weekly fed and (I) fasted plasma triglycerides (n = 10/group). Statistical significance determined by ANOVA and Bonferroni's test for multiple comparisons for B, D, and F. Statistical significance determined by two-way ANOVA and Tukey's test for multiple comparisons of A, C, E, H, and I. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. All the data represent mean +/− SEM.

Figure 4

KHK inhibition suppressed ChREBP and DNL in fructose-fed rats. (A) Hepatic gene expression following a 6-hr fast measured in Sprague Dawley rats fed chow or high fructose diet, and treated with vehicle or 30 mg/kg PF-06835919 for 7 days (n = 6/group). Statistical significance determined by ANOVA and Bonferroni's test for multiple comparisons. ∗p < 0.05 compared to chow and #p < 0.05 compared to vehicle fructose diet. (B–E) Western blotting images and densitometry of hepatic ChREBP, PKLR, and ACC1 normalized to actin (n = 5/group) from the rats fed chow or a fructose diet and treated with PF-06835919 for 7 weeks (n = 5/group). Total terminal liver (F) TG-48:0-FA 16:0 measured by LC-MS/MS from the 7-week study (n = 10/group). Statistical significance determined by ANOVA and Tukey's test for multiple comparisons. (G) Plasma TG-48:0-FA 16:0 measured by LC-MS/MS from weekly plasma samples over the 7-week study (n = 10). Statistical significance was determined by two-way ANOVA and Bonferroni's test for multiple comparisons. (H) D₂O incorporation into plasma palmitate was measured in a separate study with rats fed high fructose diet and treated with the indicated dose of PF-06835919 for two weeks (n = 8/group). Statistical significance determined by ANOVA and Bonferroni's test for multiple comparisons. All the data represent mean +/− SEM. Pyruvate kinase, Pklr; solute carrier family 2-facilitated glucose transporter member 5, Glut5; triose kinase, Tk; acetyl-CoA carboxylase 1, Acc1; ATP-citrate synthase, Acly; aldolase B, AldoB; fatty acid synthase, Fasn.

Figure 5

Reversal of hypertriglyceridemia, hyperinsulinemia, and steatosis in the rats fed an “American diet”. (A) Final body weights of rats fed an American diet (AD) for 9 weeks and administered vehicle or the indicated dose of PF-06835919 in the final week of the study. (B) Plasma insulin, (C) plasma triglycerides (D), hepatic triglycerides, (E) plasma-free fatty acids, (F) plasma ApoC3, (G) plasma cholesterol, and (H) plasma adiponectin (n = 8/group). Statistical significance determined by ANOVA and Bonferroni's multiple comparison test. All the data represent mean +/− SEM.

Figure 6

KHK inhibition in humans increased plasma fructose. (A) Plasma fructose levels measured in healthy subjects administered a single dose of PF-06835919. At the indicated time points, the subjects were provided a fructose-sweetened beverage with breakfast, lunch, and dinner. (B) Plasma fructose AUC_0–10hr measured in the subjects administered the indicated dose of PF-06835919. (C) Predictive mathematical modeling demonstrating the predicted % KHK inhibition based on the change in AUC_0–10hr. (D) Model representing fructose metabolism and how KHK inhibition improves metabolic dysfunction. Ingested fructose is primarily metabolized by enterocytes. Some fructose escapes intestinal metabolism and is metabolized by the liver, promoting ChREBP activation, insulin resistance, DNL, and steatosis. With excessive fructose consumption, hepatic metabolism of fructose metabolism increases due to saturation of intestinal fructose metabolism and/or increased gut permeability, further augmenting ChREBP activation, DNL, insulin resistance, and steatosis. KHK inhibition prevents the metabolism of fructose in enterocytes and hepatocytes, thus reducing ChREBP activation, insulin resistance, DNL, and steatosis (n = 6/group in A and B). All the data represent mean +/− SEM.