Vitamin B2 enables regulation of fasting glucose availability
- Department of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, United States
- Department of Medicine, Baylor College of Medicine, United States
- Department of Molecular and Cellular Biology, Baylor College of Medicine, United States
- Department of Anesthesiology, University of Texas Health Sciences Center, United States
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, United States
- Department of Molecular and Human Genetics, Baylor College of Medicine, United States
- Department of Nutrition and Integrative Physiology, University of Utah, United States
- Department of Pediatrics, Baylor College of Medicine, United States
- Department of Nutritional Sciences and Toxicology, University of California, Berkeley, United States
Figures
Figure 1 with 3 supplements
Riboflavin deficiency alters body composition and energy expenditure.
(a) Ad libitum flavin adenine dinucleotide (FAD) concentrations measured from male WT mice (n = 3). (b) One-month-old male mice (n = 5 per group) were exposed to 99% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 1 mo, followed by metabolic phenotyping. (c) FAD concentrations in the fasted liver. (d) Body weight (g), (e) % body weight gain and (f) body composition (% of body mass). Mice were individually housed and monitored in CLAMS-HC. (g) Recorded traces of oxygen consumption (VO2), (h) respiratory exchange ratio (RER), (i) locomotor activity, and (j) cumulative food intake during dark (gray) and light (white) periods in the metabolic cages. One-way ANOVA with Sidak multiple-comparison tests was used for (a). Liver, heart, and kidney show statistically higher FAD levels versus other tissues. Other statistical analyses include two-way ANOVA with Sidak multiple-comparison tests for body weight (d, e) and body composition (fat and lean mass%) by Mann–Whitney for Ctrl v. B2D (f). Statistical analysis of CLAMS data was performed by ANCOVA with lean body mass as a co-variate (g–j). Data are represented as mean ± SEM. #p<0.10, *p<0.05, **p<0.02. Numerical data for individual panels are provided in Figure 1—source data 1.
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Figure 1—source data 1
Numerical data presented in Figure 1.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig1-data1-v1.zip
Figure 1—figure supplement 1
Effects of 99% riboflavin deficiency on brown adipose tissue (BAT) in male and female mice.
Analysis of BAT gene expression and tissue morphology (H/E) for (a) male and (b) female mice fed 99% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 4 wk. Scale bar is 50 μm for BAT morphology. Tbp served as the invariant control for gene expression measurements. Statistical analysis of gene expression was performed by Mann–Whitney test for (a) and (b). Data are represented as mean ± SEM. Numerical data for individual panels are provided in Figure 1—figure supplement 1—source data 1.
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Figure 1—figure supplement 1—source data 1
Numerical data presented in Figure 1—figure supplement 1.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Metabolic effects of 99% riboflavin deficiency in female mice.
One-month-old female mice (n = 5 per group) were exposed to 99% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 1 mo, followed by metabolic phenotyping. (a) Flavin adenine dinucleotide (FAD) concentrations in the fasted liver. (b) Body weight (g), (c) % body weight gain and (d) body composition (% of body mass). Mice were individually housed and monitored in CLAMS-HC. (e) Recorded traces of oxygen consumption (VO2), (f) Respiratory exchange ratio (RER), (g) locomotor activity, and (h) cumulative food intake during dark (gray) and light (white) periods in the metabolic cages. Statistical analyses are Mann–Whitney test (a), two-way ANOVA with Sidak multiple-comparison tests (b, c), body composition (fat and lean mass%) by Mann-Whitney for Ctrl vs. B2D (d), CLAMS by ANCOVA with lean body mass as a co-variate (e–h). Data are represented as mean ± SEM. #p<0.10. Numerical data for individual panels are provided in Figure 1—figure supplement 2—source data 1.
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Figure 1—figure supplement 2—source data 1
Numerical data presented in Figure 1—figure supplement 2.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig1-figsupp2-data1-v1.zip
Figure 1—figure supplement 3
Metabolic effects of 90% riboflavin deficiency in mice.
One-month-old male mice (n = 5 per group) were exposed to 90% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 1 mo, followed by metabolic phenotyping. (a) Flavin adenine dinucleotide (FAD) concentrations in the fasted liver. (b) Body weight (g), (c) % body weight gain and (d) body composition (% of body mass). (e) Blood glucose excursion in 90% B2D or Ctrl mice during pyruvate tolerance tests (n = 5). Mice were individually housed and monitored in CLAMS-HC. (f) Recorded traces of oxygen consumption (VO2), (g) Respiratory exchange ratio (RER), (h) locomotor activity, and (i) cumulative food intake during dark (gray) and light (white) periods in the metabolic cages. Statistical analyses are body weight by two-way ANOVA with Sidak multiple-comparison tests (b, c), body composition (fat and lean mass%) by Mann–Whitney for Ctrl vs. 90%B2D, pyruvate tolerance test (e) by two-way ANOVA with Sidak multiple-comparison tests and area under the curve (AUC) by Mann–Whitney test, CLAMS by ANCOVA with lean body mass as a co-variate (f–i). Data are represented as mean ± SEM. *p<0.05. Numerical data for individual panels are provided in Figure 1—figure supplement 3—source data 1.
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Figure 1—figure supplement 3—source data 1
Numerical data presented in Figure 1—figure supplement 3.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig1-figsupp3-data1-v1.zip
Figure 2
Liver glucose production requires bioavailable riboflavin.
(a) Liver flavin adenine dinucleotide (FAD) and glucose levels across light/dark cycles (Zeitgeber Time, ZT) in male mice. Overnight (16 hr) fasting and re-fed (6 hr) (b) blood glucose and (c) liver FAD levels from male WT mice (n = 4). (d) Blood glucose excursion during pyruvate tolerance tests (n = 5 per group). (e) Basal glucose production was measured in fasted (18 hr) conscious mice (n = 5 vitamin B2-deficient diet [B2D] or n = 7 Ctrl) using constant rate of intravenous infusion (0.1 uCi/min) of [3-3H]-glucose using a through a surgically implanted catheter in jugular vein. Whole-body basal glucose production rates (mg/kg/min) were calculated by dividing the [3-3H] glucose infusion rate by the plasma glucose-specific activity corrected to the body weight. (e’) Steady-state rates were reached within 1 hr of infusion. (e’’) Basal hepatic glucose production averaged across the three sampling times. (f) RNA-seq coupled with Panther Gene Ontology analysis identified pathways altered by B2D in the liver (n = 5 independent animals/diet). (g) Volcano plot depicting expression levels of flavoprotein genes (yellow) after B2D compared to control. (h) Lipidomic analysis of Ctrl and B2D (n = 5 per group). This analysis identified triglycerides significantly (p<0.05) increased in B2D versus Ctrl fed mice. (i) Representative Oil-Red-O stained liver sections from B2D or Ctrl. Scale bar 50 μm. (j) Enrichment of nodes encoded by genes that map to Mouse Genome Informatics (MGI) hypertriglyceridemia phenotypes among nodes with significant HCT intersections with B2D-induced genes. (k) Overlap of B2D-enriched nodes with nodes enriched in the human non-alcoholic fatty liver disease (NAFLD) gene expression consensome. The human NAFLD gene expression consensome ranks 18,162 genes based on their discovery across publicly archived clinical NAFLD case/control transcriptomic datasets. Statistical significance (*p<0.05) calculated by Mann–Whitney (b, c, e”) or two-way ANOVA with Sidak multiple-comparison test (d, e’). Statistical enrichment shown by hypergeometric test for (j, k). Data are represented as mean ± SEM. Numerical data for individual panels are provided in Figure 2—source data 1. Serum parameters in and liver lipid measurements are provided in Figure 2—source data 2 and Figure 2—source data 3, respectively.
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Figure 2—source data 1
Numerical data presented in Figure 2.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig2-data1-v1.zip
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Figure 2—source data 2
Serum parameters in vitamin B2-deficient diet (B2D) interventions.
Data are mean ± SEM. The number of animals in each group is indicated in parentheses. Statistical significance (p) was determined by Mann-Whitney tests.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig2-data2-v1.docx
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Figure 2—source data 3
Liver triglycerides and cholesterol in vitamin B2-deficient diet (B2D) interventions.
Data are mean ± SEM. The number of animals in each group is indicated in parentheses. Statistical significance (p) was determined by Mann–Whitney tests.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig2-data3-v1.docx
Figure 3 with 1 supplement
Liver PPARα governs glucogenic responses to dietary riboflavin.
(a) Top enriched, vitamin B2-deficient diet (B2D) repressed gene sets using the EnrichR transcription factor collection (left). Scatterplot showing enrichment of known BioGRID-curated PPARα interacting nodes (yellow) among nodes with the most significant intersections with B2D-repressed genes (right). (b) Volcano plot depicting expression levels of PPARα-regulated flavoprotein genes (yellow) after Ctrl or B2D. (c) One-month-old Pparaflox/flox male mice (n = 4–5 per group) received either AAV-TBG-GFP or AAV-TBG-Cre. Two weeks later, mice were provided 99% B2D or isocaloric diet (Ctrl) for 1 mo. (d) Body weight (g), (e) % body weight gain and (f) body composition (% of body mass). (g) Blood glucose levels and (h) area under the curve (AUC) during pyruvate tolerance tests. (i) Representative Oil-Red-O stained liver sections after B2D, scale bar 100 μm. (j) Flavin adenine dinucleotide (FAD) concentrations in the fasted liver after 4 wk of B2D. (k) Relative mRNA expression of Ppara and Acox1. Tbp served as the invariant control. (l) HepG2 cells were transiently transfected with a PPARα expression plasmid. PPRE luciferase reporter activity was measured in response to PPARα agonist (WY-14643) ± lumiflavin and normalized to β-galactosidase (n = 4). Statistics: *p<0.05 vs. control GFP (a, B2D GFP; b, B2D Cre; c, Ctrl Cre) by two-way ANOVA with Fisher LSD (d, e, g), *p<0.05 for body composition (fat and lean mass%) by Mann–Whitney vs. control GFP, *p<0.05, #p<0.10 vs. control GFP by one-way ANOVA with Dunnet’s test and Fisher LSD (h, j, k). *p<0.05 by two-way ANOVA with Tukey’s multiple-comparison test for (l), a and b are statistical differences vs. vehicle and WY-14643, respectively. Statistical enrichment shown by hypergeometric test (a, right, b). Data are mean ± SEM. Numerical data for individual panels are provided in Figure 3—source data 1.
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Figure 3—source data 1
Numerical data presented in Figure 3.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig3-data1-v1.zip
Figure 3—figure supplement 1
Ppara whole-body knockout responses to dietary riboflavin depletion.
(a) One-month-old Ppara-/- male mice (n = 4 per group) were exposed to 99% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 1 mo. (b) Body weight (g), (c) % body weight gain and (d) blood glucose levels during pyruvate tolerance tests. Statistical analysis of phenotypes was analyzed by two-way ANOVA with Sidak multiple-comparison tests (b–d). Data are represented as mean ± SEM. *p<0.05. Numerical data for individual panels are provided in Figure 3—figure supplement 1—source data 1.
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Figure 3—figure supplement 1—source data 1
Numerical data presented in Figure 3—figure supplement 1.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig3-figsupp1-data1-v1.zip
Figure 4
Remnant PPARα activation rescues fasting responses impaired by vitamin B2-deficient diet (B2D).
(a) One-month-old male mice (n = 10 per group) were exposed to 99% vitamin B2-deficient diet (B2D) or isocaloric control diet (Ctrl) for 9 wk. For weeks 7–9, mice were gavaged daily with fenofibrate (FF). (b) Body weight (g) and (c) body weight gain during treatments. For post-gavage (week 9), mice were fasted overnight and administered FF 2 hr before measurements. (d) Overnight fasting blood glucose levels for pre-gavage (gray) and after 2 wk of FF treatment (orange). (e) Representative H&E-stained liver sections from Ctrl or B2D mice following FF treatment. Scale, 100 μm. (f) Expression level of flavoprotein genes in B2D-fed mice following FF. (g) Enrichment of B2D+FF with gene footprints repressed in human NAFLD. Statistical analyses include two-way ANOVA with Sidak multiple-comparison test (b–d) and *p<0.05 for (b, c). *p<0.05 pre-gavage Ctrl vs. B2D or **p<0.05 post-gavage vs. pre-gavage were p-value cutoffs for (d). Statistical enrichment shown by hypergeometric test (f, g). Data are represented as mean ± SEM. Numerical data for individual panels are provided in Figure 4—source data 1.
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Figure 4—source data 1
Numerical data presented in Figure 4.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig4-data1-v1.zip
Figure 5 with 1 supplement
Fenofibrate impacts mitochondrial respiratory chain efficiency but does not rescue flavin adenine dinucleotide (FAD) levels in vitamin B2-deficient diet (B2D).
(a) Fasting liver sphingolipid species (pmol/mg protein) and pool sizes by mass spectrometry (n = 4 per group). (b) Hepatic NAD/NADH ratio. (c) Fasting liver FAD and flavin mononucleotide (FMN) relative abundance by mass spectrometry (n = 4 per group). (d) Western blot analysis of oxidative phosphorylation complexes. HSP90 served as the invariant control. (e) Ratio of oxidized and reduced liver coenzyme Q10 and Q9 by mass spectrometry (n = 4 per group). Statistics are Mann–Whitney test for Ctrl vs. B2D (-FF) or Ctrl vs. B2D (+FF) for (a, b, c, e). *p<0.05, #p<0.10. Data are represented as mean ± SEM. Numerical data for individual panels are provided in Figure 5—source data 1. Full gel images are provided in Figure 5—source data 2.
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Figure 5—source data 1
Numerical data presented in Figure 5.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig5-data1-v1.zip
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Figure 5—source data 2
Full gel images and original image files for western blots in Figure 5.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig5-data2-v1.zip
Figure 5—figure supplement 1
Vitamin B2-deficient diet (B2D) effects on sphingolipids in the liver.
Fasting liver sphingolipid and ceramide species (pmol/mg protein) by mass spectrometry (n = 4 per group). Statistics are Mann–Whitney test for Ctrl vs. B2D (-FF) or Ctrl vs. B2D (+FF). *p<0.05. Data are represented as mean ± SEM. Numerical data for individual panels are provided in Figure 5—figure supplement 1—source data 1.
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Figure 5—figure supplement 1—source data 1
Numerical data presented in Figure 5—figure supplement 1.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig5-figsupp1-data1-v1.zip
Figure 6
Integrated stress response (ISR) activity forms the basis to reconcile flavin adenine dinucleotide (FAD) disruption.
(a) Glycolysis, tricarboxylic acid (TCA), amino acid, fatty acid oxidation (carnitines) metabolites measured by mass spectrometry across vitamin B2-deficient diet (B2D) and FF treatments (shown as Z-score). (b) Metaboanalyst integration demonstrates B2D causes organic acidemias. (c) RNA-seq coupled with Gene Set Enrichment Analysis (GSEA) identified gene signatures altered by B2D in the liver (n = 5 independent animals/diet). (d) Change in glucose levels 30 min after pyruvate injection pre- and post-fenofibrate gavage (n = 5). (e) Amino acid, glucose metabolism, and ISR genes in B2D or B2D + FF, shown as Z-score from RNA-seq data. (f) Western blot analysis for ISR proteins in liver lysates from mice treated with B2D (4 wk total) or B2D+FF (9 wk total) and fasted overnight. (g) B2D + FF activates the ISR, increasing amino acids that restore glucose availability. Statistical significance was assessed by Mann–Whitney (a, e) and two-way ANOVA and Fisher LSD (d). Data are represented as mean ± SEM. *p<0.05, #p<0.10. Numerical data for individual panels are provided in Figure 6—source data 1. Full gel images are provided in Figure 6—source data 2.
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Figure 6—source data 1
Numerical data presented in Figure 6.
Excel spreadsheet containing raw data for panels presented in the figure.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig6-data1-v1.zip
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Figure 6—source data 2
Full gel images and original image files for western blots in Figure 6.
- https://cdn.elifesciences.org/articles/84077/elife-84077-fig6-data2-v1.zip
Author response image 1
One-week of B2D does not affect food intake or ad libitum blood glucose.
Individually housed one-month old male mice (n=3 per group) were exposed to 99% B2D or isocaloric control diet (Ctrl) for one week. (a) Cumulative food intake. (b) ad libitum blood glucose. Data are represented as mean +/- SEM. Mann-Whitney test for a and b.
Additional files
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Supplementary file 1
Macronutrient compositions of 90% B2D used in the study.
- https://cdn.elifesciences.org/articles/84077/elife-84077-supp1-v1.zip
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Supplementary file 2
Macronutrient compositions of 99% B2D used in the study.
- https://cdn.elifesciences.org/articles/84077/elife-84077-supp2-v1.zip
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Supplementary file 3
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/84077/elife-84077-supp3-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/84077/elife-84077-mdarchecklist1-v1.pdf
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Vitamin B2 enables regulation of fasting glucose availability
eLife 12:e84077.
https://doi.org/10.7554/eLife.84077
