Fig. 6: Fructose metabolism drives ISC proliferation by engaging glycolysis.

a Representative immunofluorescent images of Olfm4 (Red) in intestinal organoids treated with or without fructose. Nuclei are stained blue. Scale bar, 50 μm. Images are representative of n = 3 biologically independent samples. b Heatmap showing a differential metabolic profile of fructose (10 mM)-treated intestinal organoids for 24 h. c Pathway enrichment analysis of differential metabolites from intestinal organoids treated with fructose (10 mM) as compared to PBS-treated controls by using Ingenuity Pathway Analysis (IPA). Red circles denote pathways that have significant changes. d Relative mRNA expression of key enzymes involved in glycolysis. hk, hexokinase; fbp, fructose-bisphosphatase; eno, enolase; pfk, phosphofructokinase; pgk, phosphoglycerate kinase; pk, pyruvate kinase; ldh, lactate dehydrogenase; pdh: pyruvate dehydrogenase complex. Data are expressed as mean fold change ± SEM for n = 3, unpaired two-tailed Student’s t-test (p = 0.05 for fbp1; p = 0.04 for fbp2; p = 0.01 for eno1; p = 0.01 for eno2; p = 0.08 for eno3; p = 0.03 for pfk; p = 0.02 for pkl; p = 0.001 for pkm; p = 0.04 for idh2; p = 0.01 for pdha1; p = 0.05 for pdhb). e Percent labeling of lactate following treatment of intestinal organoids with 10 mM U-[¹³C]-Glucose (¹³C Glu) after 24 h treatment with 10 mM fructose (Fru). The # in the (M + #) designation indicated how many [¹²C] were replaced with [¹³C] in the isotopic labeled metabolites. The abundance is expressed as percent labeling among all isotopomers of the metabolites. Data are expressed as mean ± SEM for n = 3, unpaired two-tailed Student’s t-test, **p < 0.01 for (M + 3) lactate. f Relative mRNA expression of aldonase isoforms involved in fructose utilization. aldo, aldolase. Data are expressed as mean fold change ± SEM for n = 3, unpaired two-tailed Student’s t-test (p = 0.03 for aldoa: p = 0.02 for aldob; p = 0.01 for aldoc). g Percent labeling of the indicated metabolites following treatment of mature intestinal organoids with 10 mM U-[¹³C]-Fructose (¹³C Fru) for 6 h. Data are represented as mean ± SEM for n = 3. h Schematic summary of the conversion of [¹³C]-fructose into key metabolites in glycolysis, tricarboxylic acid (TCA) cycle, and glutamine metabolism pathway as found in intestinal organoids.