Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate

Abstract

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods¹, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease^2-4. Fructose intake triggers de novo lipogenesis in the liver^4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates⁷. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases⁸. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota⁹, and this supplies lipogenic acetyl-CoA independently of ACLY¹⁰. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

ED Figure 1.. Hepatic ACLY deficiency minimally impacts the response to dietary fructose.

a, Schematic of fructolysis and glycolysis feeding into de novo lipogenesis. F1P = fructose-1-phosphate, F-1,6-BP = fructose-1,6-bisphosphate, GA = glyceraldehyde, DHAP = dihydroxyacetone phosphate, G3P = glyceraldehyde-3-phosphate. b, Body weights of WT and LAKO mice on CD or HFrD for 18 weeks (n = WT-CD:13, LAKO-CD:5, WT-HFrD:14, LAKO-HFrD:5), data are mean ± SD. c, Weights of liver, posterior subcutaneous (sWAT) and perigonadal (pgWAT) adipose tissues in WT and LAKO mice on CD or HFrD for 18 weeks (n Liver/sWAT/pgWAT = WT – CD: 7/7/7, LAKO – CD: 2/5/5, WT – HFrD: 6/12/12, LAKO – HFrD: 3/5/5). d, Representative images of Periodic Acid Schiff (PAS) stain for glycogen and Trichrome (TC) histological stain for fibrosis in livers from WT or LAKO mice on HFrD. Scale bars = 100 μm. e, Triglyceride content in WT or LAKO mice on CD or HFrD for 18 weeks, n = (WT-CD: 4, LAKO-CD: 3, WT-HFrD: 4, LAKO-HFrD: 3), statistical significance determined using Welch’s t test. For panels c and e, bars represent mean.

Extended Data Figure 2.. Hepatic ACLY deficiency results in modest metabolic alterations on high fructose diet.

a, Volcano plot of hepatic metabolites in WT and LAKO mice on CD or HFrD for 4 weeks, pink dots indicate significant hits as determined by a fold-change threshold of 2 and P-value threshold of 0.1, assuming equal variance. b, Principle component analysis of log-transformed data in Supplementary Table 1, each dot represents a unique sample, 95% CI shown in corresponding color. c, Relative metabolite abundance, normalized to WT-CD group, p values determined using Welch’s t test, n = (WT-CD:5, LAKO-CD: 3, WT-HFrD: 5, LAKO-HFrD: 4). Bars represent mean.

Extended Data Figure 3.. High fructose diet alters hepatic lipid metabolism.

a, Hierarchical clustering of relative hepatic triglyceride abundance in WT or LAKO mice on CD or HFrD for 4 weeks, clustering performed using one minus pearson correlation and average linkage. b, Relative abundance of hepatic triglycerides composed of 16:0 to 18:1 fatty acids, subset of data in panel a. c, Principle component analysis of log-transformed data in Supplementary Table 2, each dot represents a unique sample, 95% CI shown in corresponding color.

Extended Data Figure 4.. Fructose induces steatosis and contributes substantially to newly synthesized fatty acids in the liver independently of ACLY.

a, Schematic of experimental design of drinking water study. b, Daily consumption of unsweetened (H₂O) or 15% fructose + 15% glucose sweetened (Fruc:Gluc) water per mouse, each dot represents a repeat measurement (n = H₂O: 6, Fruc:Gluc: 7), statistical significance determined using Welch’s t test. c, Weight gain of WT or LAKO mice given H₂O or Fruc:Gluc water for 4 weeks (n = WT – H₂O: 4, LAKO – H₂O: 4, WT – Fruc:Gluc: 8, LAKO Fruc:Gluc: 6), p value indicated comparing all H₂O vs. Fruc:Gluc mice determined by Welch’s t test. d, Representative H&E and Oil Red O histological stains of livers from mice in panel c. Scale bars = 100 μm. e, Experimental design for data shown in Figure 1c. f, Isotopologue distribution of labeled serum saponified fatty acids shown in Figure 1c. For all panels, data are mean ± SD.

Extended Figure Data 5.. Fructose signals the use of acetate for de novo lipogenesis.

a, mRNA expression of ChREBP and its target genes in livers of WT or LAKO mice fed CD or HFrD (n = 4 mice/group), p values for WT-CD vs. WT-HFrD (blue text) and LAKO-CD vs. LAKO-HFrD (purple text) determined using two-sided t tests with Holm-Sidak method for multiple comparisons. b, mRNA expression of lipogenic genes in livers of WT or LAKO mice given H₂O or Fruc:Gluc water for 4 weeks (n = 4/group), statistical comparisons WT-H₂O vs. WT-Fruc:Gluc, p values for WT – H₂O vs. WT – Fruc:Gluc (blue font) and LAKO – H₂O vs. LAKO Fruc:Gluc (purple font) determined using two-sided t test with Holm-Sidak method for multiple comparisons. c, Immunoblots of lipogenic enzymes in livers of WT or LAKO mice given H₂O or Fruc:Gluc water for 4 weeks, each lane represents an individual mouse. d, Immunohistochemistry staining against ACLY in WT or LAKO livers on H₂O or Fruc:Gluc water for 4 weeks. Yellow boxes approximate location of 20X panels. Scale bars = 100 μm for 10X, 50 μm for 20X. e, H3K27ac ChIP-qPCR of livers from WT mice provided either water for 24 hours followed by an oral gavage of saline, or Fruc:Gluc water for 24 hours, followed by an oral gavage of 2.0 g/kg glucose and 2.0 g/kg fructose (Mlxipl: n = 3, Acss2: n = 3, Pklr: n = 4), livers harvested 90 minutes after gavage. p1 and p2 are two different primer sets. f, ChIP-seq tracks of Mlxipl, Pklr, Acss2 genomic loci, red bars indicate genomic regions used to design ChIP-qPCR primers. For panels a-b and e-f, bars represent mean.

Extended Data Figure 6.. Depletion of microbiome blocks substrate contribution, but not signaling component, of de novo lipogenesis following fructose consumption.

a, Experimental set-up for antibiotic depletion of the microbiome followed by ¹³C-fructose tracing into DNL. b, Representative images of cecums from a saline and antibiotic treated mouse. c, Relative abundance of bacterial abundance in cecal contents from mice treated with saline (n = 9) or antibiotics (n = 9) as determined by 16s RT-qPCR to a reference standard of E. coli DNA. P value determined using Welch’s t test. d, Heat map of microbial metabolite abundance in the portal blood, collected 1 hour after gavage. e, Relative abundance of portal blood ¹³C-fructose in WT – saline (n = 7) vs. WT – Antibiotics (n = 7) and LAKO – saline (n = 4) vs. LAKO – antibiotics (n = 4) following gavage and f, % total labeled carbons in portal blood glucose, p values determined using Welch’s t test. g, mRNA expression of ChREBPβ, Acss2, and Fasn in liver collected 1 hour after gavage (n = 4/group), p values determined using two-sided t tests with Holm-Sidak method for multiple comparisons. For c, e-g, bars represent mean.

Extended Data Figure 7.. Bolus fructose is converted into acetate in a microbiome-dependent manner.

a, TIC of labeled liver F1P, pyruvate, and acetyl-CoA, concentrations (μM) of portal blood labeled acetate, and % labeling of liver saponified 16:0 and 18:0 in saline- or antibiotic-treated WT mice gavaged with 2.0 g/kg ¹³C-fructose + 2.0 g/kg unlabeled glucose, n = 3 mice/time point. Data for saline-treated mice is also shown in Figure 2d. b, Isotopologue distribution of serum saponified fatty acids, collected 6 hours after gavage, data are mean ± SD, n = (WT-Saline: 8, LAKO-Saline: 4, WT-Antibiotics: 8, LAKO-Antibiotics: 4).

Extended Data Figure 8.. Bolus fructose-dependent DNL requires microbial acetate and hepatic ACSS2.

a, Concentrations (μM) of portal blood labeled acetate, propionate, and butyrate, n = (WT-Saline: 8, LAKO-Saline: 4, WT-Antibiotics: 8, LAKO-Antibiotics: 4). b, Abundance of cecal labeled acetate, propionate, and butyrate in WT mice, n = 3 mice/timepoint, except for saline-180 n = 2 mice. c, Heat map of hepatic triglyceride abundance in livers of saline- or antibiotic-treated mice following an oral gavage of 2.0 g/kg fructose + 2.0 g/kg glucose. d, Concentrations of portal and systemic blood acetate following gavage, each data point represents an individual mouse sacrificed at indicated time, p value determined using two-sided t tests with Holm-Sidak method for multiple comparisons. e, Weight gain in WT and LAKO mice 1 week following tail-vein injection with AAV8-GFP or AAV8-shAcss2, p value determined using Welch’s t test. f, Liver weight as % of body weight of WT and LAKO mice 1 week following tail-vein injection with AAV8-GFP or AAV8-shAcss2. g, Western blot of liver lysates from WT and LAKO mice 1 week following tail-vein injection with AAV8-GFP or AAV8-shAcss2.

Extended Data Figure 9.. Gradual fructose consumption promotes greater acetate usage in LAKO mice.

a, Experimental set-up for ¹³C-acetate tracing into DNL prior to and after gradual fructose administration. b, Western blot of ACLY, ACSS2, and S6 in liver lysates from WT and LAKO mice after 1 day or 14 days of Fruc:Gluc water. c, Representative H&E stains of livers from WT and LAKO mice provided Fruc:Gluc water for 2 weeks. Scale bars = 100 μm d, Relative abundance of acetate, propionate, and butyrate in the cecal contents of WT and LAKO mice treated with saline or antibiotics for 1 week (n = 4 mice/group). p values determined using Welch’s t test.

Extended Data Figure 10.. Fructose provides signal and substrate to promote hepatic de novo lipogenesis.

a, Proposed model of bolus fructose-induced hepatic DNL. Fructose catabolism in hepatocytes acts as a signal to induce DNL genes including ACSS2, while fructose metabolism by the gut microbiome provides acetate as a substrate to feed DNL, mediated by ACSS2. b, Proposed model of gradual fructose-induced hepatic DNL. Like the bolus model, fructose catabolism in hepatocytes acts as a signal to induce DNL genes. Hepatic fructose and glucose (made from fructose by the small intestine) catabolism provide citrate as a substrate to feed DNL, mediated by ACLY. Metabolism of fibers and other dietary components by the gut microbiome provides acetate as a substrate to feed DNL, following its conversion to acetyl-CoA by hepatic ACSS2. Created with BioRender.com.

Figure 1.. Fructose-dependent fatty acid synthesis is ACLY-independent.

a, Representative H&E and Oil Red O histological stains of livers of WT or LAKO mice on chow (CD) or high fructose diet (HFrD) from two independent experiments of n = 4 WT and LAKO mice/diet (4 weeks) and n = 13 WT mice/diet and n = 6 LAKO mice/diet (18 weeks). Scale bars = 100 μm. b, Relative deuterium incorporation in palmitic acid (16:0) and stearic acid (18:0) after 24 hour D₂O labeling of mice, D₂O set to 1 and compared to D₂O Fruc:Gluc within each genotype, significance determined by two-sided t tests. c, % total labeled carbons in serum saponified fatty acids from mice gavaged with 1:1 fructose:glucose, ¹³C-labeled substrate indicated, bars represent mean. d, Immunoblots of lipogenic enzymes in livers of WT or LAKO mice fed CD or HFrD for 4 weeks.

Figure 2.. Lipogenic acetyl-CoA is preferentially produced from acetate in hepatocytes.

a, Pathways for lipogenic acetyl-CoA production from fructose, glucose, or acetate. b-c, % total labeled carbons in fructolytic intermediates (b) and acetyl-CoA, malonyl-CoA, or HMG-CoA (c) in primary hepatocytes incubated for 6 hours with 25mM fructose + 1mM acetate, ¹³C-labeled substrate indicated, bars represent mean, n = 3. d, Total ion counts (TIC) of labeled liver F1P, pyruvate, and acetyl-CoA, concentrations (μM) of portal blood labeled acetate, and % labeling of liver 16:0 and 18:0 in saline-treated WT mice gavaged with 1:1 ¹³C-fructose:glucose, data are mean ± SEM, n = 3 mice/time point.

Figure 3.. Metabolism of bolus fructose by the microbiome feeds hepatic lipogenesis.

a, Area under curve (AUC_0–240 min) of labeled hepatic F1P, pyruvate, acetyl-CoA, palmitate and portal blood acetate, data are mean ± SEM. See Extended Data Figure 8a for curves. b, % total labeled carbons in serum saponified fatty acids from saline- or antibiotic-treated LAKO mice gavaged with ¹³C-fructose:glucose, significance determined using two-sided t tests. c, % total labeled carbons in serum saponified fatty acids from saline- or antibiotic-treated LAKO mice gavaged with 1:1 fructose:glucose + 0.5 g/kg acetate, ¹³C-labeled substrate indicated, significance determined by two-sided t test. d, % total labeled carbons in serum saponified fatty acids from WT and LAKO mice 1 week after injection with AAV-GFP or AAV-shAcss2, significance determined by two-sided t test. For b-c, bars represent mean.

Figure 4.. Gradual fructose consumption promotes hepatic lipogenesis from ACLY- and ACSS2-derived acetyl-CoA.

a, Experimental design for gradual fructose consumption. b, % total labeled carbons from ¹³C-fructose or glucose in hepatic saponified fatty acids, WT vs. LAKO. c, % total labeled hydrogens from D₂O in hepatic saponified fatty acids. d, % total labeled carbons from ¹³C-acetate in serum saponified fatty acids, see Extended Data Fig. 10a for experimental design. e, total % labeled hydrogens from D₂O in hepatic saponified fatty acids in WT and LAKO mice following 1 week treatment with saline or antibiotics (abx). f, mRNA expression of ChREBP and lipogenic genes in livers of mice in (e). g, total % hydrogens labeled in hepatic saponified fatty acids in WT and LAKO mice 1 week after injection with AAV-GFP or AAV-shAcss2. For b-g, bars represent mean, b-e,g, significance determined using 2-way ANOVA with Tukey’s test for multiple comparisons.