Vitamin B2 enables regulation of fasting glucose availability
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Masschelin et al. investigate the role of Vitamin B2 (riboflavin), an essential cofactor for FAD and FMN coenzymes involved in the electron transport chain and TCA cycle, in fasting glucose metabolism. This study phenotypes B2-deficient mice liver and provides valuable data on genes and metabolites that are changed with B2 depletion +/- Fenofibrate administration. The work employs solid methodology and will be of interest to liver physiologists interested in fasting in the context of PPAR.
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Abstract
Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Masschelin et al. describes how Vitamin B2 deficiency affects body composition, energy expenditure, and glucose metabolism. B2 deficient mice have lower O2 consumption, and locomotor activity, with no difference in food intake. These mice also have lower liver FAD levels, which is expected given that B2 is a necessary cofactor for this coenzyme. Additionally, these mice have lower blood glucose levels following pyruvate injection, implying a lower capacity for gluconeogenesis. Using PPAR KO mice, they show that this effect on pyruvate tolerance is due to PPARα activation, though there is still a minor difference between wildtype and KO mice. Importantly, they show that fenofibrate PPAR agonism can improve glucose output following pyruvate injection in the absence of B2. …
Author Response
Reviewer #1 (Public Review):
The manuscript by Masschelin et al. describes how Vitamin B2 deficiency affects body composition, energy expenditure, and glucose metabolism. B2 deficient mice have lower O2 consumption, and locomotor activity, with no difference in food intake. These mice also have lower liver FAD levels, which is expected given that B2 is a necessary cofactor for this coenzyme. Additionally, these mice have lower blood glucose levels following pyruvate injection, implying a lower capacity for gluconeogenesis. Using PPAR KO mice, they show that this effect on pyruvate tolerance is due to PPARα activation, though there is still a minor difference between wildtype and KO mice. Importantly, they show that fenofibrate PPAR agonism can improve glucose output following pyruvate injection in the absence of B2. The authors also perform robust metabolomics in each experimental condition and phenotype of the mouse well.
Thank you for the positive input.
- The authors have yet to explore other explanations of differences in glucose metabolism under B2D +/Fenofibrate. The canonical targets of PPARα are involved in fatty acid oxidation, ketogenesis, and VLDL/HDL metabolism, in addition to gluconeogenesis (Bougarne et al. 2018). Gluconeogenesis is more of a fasting response due to CREB, FOXO1/PGC1a activation rather than PPAR. In response to B2D, the PPARα KO mice have increased plasma TGs, which may suggest a difference in VLDL TG secretion (Suppl. S3). Perhaps lipid metabolism is more directly affected, and changes in glucose metabolism are secondary to that of triglyceride metabolism. Regarding ketogenesis, the fenofibrate+ B2D fed mice have decreased plasma betahydroxybutyrate, suggesting decreased ketogenesis, which is a more canonical PPARα pathway (Suppl. S3). Testing each of these processes would help control that this mechanism is specific to gluconeogenesis and not secondary to something else.
We value this reviewer’s comment. To address this point, we considered other mechanisms in our revised Discussion. In future studies, we plan to further explore these metabolic effects and to use ATAC-Seq to understand the transcription factors responsive to B2D. We anticipate these studies will take additional years to complete. Nonetheless, the present studies set the foundation for future work to investigate how FAD influences transcriptional regulation of metabolism.
- Is the effect on ISR dependent on PPARα? Is the mechanism of Fenofibrate on the liver, or on another cell type? In Figure 1, the authors state that Riboflavin deficiency alters body composition and energy expenditure, and then focuses on the liver. However, FAD levels are also increased in the heart and kidneys in addition to the liver. These tissues also respond to PPARα agonism, in addition to the muscle which plays a role in regulating glucose metabolism (B2D mice also have a higher lean mass (Fig 1e)). Additionally, the authors haven't shown specifically if the effects of Fenofibrate on electron transport and the ISR are dependent on the presence of PPARα (Figure 5, 6).
We agree that knowing whether the effects of Fenofibrate on the ISR require liver PPARA is a critical issue, which will require dedicated studies for a thorough and meaningful conclusion. In new experiments, we knocked down Ppara in the liver using AAV8-Cre administration to Pparaflox/flox mice. Our data show liver-specific Ppara knockdown recapitulates whole-body B2D effects on pyruvate tolerance and hepatic steatosis (Figure 3I). These results agree with findings in whole-body Ppara knockout mice (Supplemental Figure 4), reinforcing the idea that the direct impact of B2D mainly occurs via PPARA activity in the liver. We acknowledge in the discussion ATF4 and ISR activation may contribute to PPARA-independent responses to B2D (Biochem J 443:165–71, 2012; Gut 65:1202-1214, 2016).
An assessment of genetic requirements will require a large, rigorous set of experiments to identify the ratelimiting responses for fenofibrate activities during B2D, which we plan to do in the future. For this report, we decided to focus exclusively on tissue-specific knockout of Ppara. We will establish evidence for ISR responses to B2D in a separate study based on the feedback received here.
Reviewer #2 (Public Review):
The objective of this work by Masschelin et al. is to investigate the physiological relevance of flavin adenine dinucleotide (FAD). In particular, FAD supports the activity of flavoproteins involved in the production of cellular energy. Mutations in genes encoding flavoproteins often are associated with inborn errors of metabolism (IEMs), thus the clinical interest in investigating in more depth the physiological role of FAD. In this study, the authors first subjected male mice to a vitamin B12 deficient diet (B2D), demonstrating that loss of B12 replicates the phenotypes often observed with IEMs, including loss of body weight, hypoglycemia, and fatty liver. Using a combination of metabolomic phenotyping, transcriptomic analyses, and pharmacology (treatment with Fenofibrate, a PPARa agonist), the authors then reach the general conclusion that activation of the nuclear receptor PPARa can rescue the B2D phenotypes, thus revealing that PPARa directly controls the metabolic responses to FAD availability. Although the phenotypic analysis of the mice subjected to B2D increases our knowledge of the physiological impact of depleting the FAD pools on global energy metabolism, not all conclusions and statements made by the authors are totally supported by the data. In particular, the study is overall too descriptive and lacks mechanistic insights. While PPARa is likely an important player in the metabolic response to FAD availability, the molecular details on how FAD controls the activity of PPARa either directly or indirectly are entirely missing. Therefore, the authors are encouraged to directly assess whether B2D directly influences PPARa activity on the genes identified in the study, perform rescue experiments in the liver of PPARa KO mice and explore the possibility that other factors (including nuclear receptors) also participate in the response to B2 deficiency and diminished FAD pools.
We appreciate the input from Reviewer 2. The direct and indirect effects of B2D on PPARA activity are likely not trivial. However, we performed experiments to determine how FAD depletion affects PPARA transcriptional activity using the riboflavin analog and competitive inhibitor lumiflavin (Figure 3L). We found lumiflavin reduced PPRE-luciferase activity in the presence of PPARA agonist. Although the assay is a synthetic reporter expressed in vitro, the experiment provides evidence of how B2D influences PPARA transcriptional activity. And, yes, we agree that our manuscript does not completely reconcile the factor(s) explaining the effects of B2D on gene expression, and expanded the discussion to comment on this point. In future studies, we intend to identify which transcription factor(s) regulate the liver responses to B2D, and further elucidation of the molecular mechanisms will be a central objective of future work.
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eLife assessment
Masschelin et al. investigate the role of Vitamin B2 (riboflavin), an essential cofactor for FAD and FMN coenzymes involved in the electron transport chain and TCA cycle, in fasting glucose metabolism. This study phenotypes B2-deficient mice liver and provides valuable data on genes and metabolites that are changed with B2 depletion +/- Fenofibrate administration. The work employs solid methodology and will be of interest to liver physiologists interested in fasting in the context of PPAR.
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Reviewer #1 (Public Review):
The manuscript by Masschelin et al. describes how Vitamin B2 deficiency affects body composition, energy expenditure, and glucose metabolism. B2 deficient mice have lower O2 consumption, and locomotor activity, with no difference in food intake. These mice also have lower liver FAD levels, which is expected given that B2 is a necessary cofactor for this coenzyme. Additionally, these mice have lower blood glucose levels following pyruvate injection, implying a lower capacity for gluconeogenesis. Using PPAR KO mice, they show that this effect on pyruvate tolerance is due to PPARα activation, though there is still a minor difference between wild-type and KO mice. Importantly, they show that fenofibrate PPARagonism can improve glucose output following pyruvate injection in the absence of B2. The authors also …
Reviewer #1 (Public Review):
The manuscript by Masschelin et al. describes how Vitamin B2 deficiency affects body composition, energy expenditure, and glucose metabolism. B2 deficient mice have lower O2 consumption, and locomotor activity, with no difference in food intake. These mice also have lower liver FAD levels, which is expected given that B2 is a necessary cofactor for this coenzyme. Additionally, these mice have lower blood glucose levels following pyruvate injection, implying a lower capacity for gluconeogenesis. Using PPAR KO mice, they show that this effect on pyruvate tolerance is due to PPARα activation, though there is still a minor difference between wild-type and KO mice. Importantly, they show that fenofibrate PPARagonism can improve glucose output following pyruvate injection in the absence of B2. The authors also perform robust metabolomics in each experimental condition and phenotype of the mouse well.
1. The authors have yet to explore other explanations of differences in glucose metabolism under B2D +/-Fenofibrate. The canonical targets of PPARα are involved in fatty acid oxidation, ketogenesis, and VLDL/HDL metabolism, in addition to gluconeogenesis (Bougarne et al. 2018). Gluconeogenesis is more of a fasting response due to CREB, FOXO1/PGC1activation rather than PPAR. In response to B2D, the PPARα KO mice have increased plasma TGs, which may suggest a difference in VLDL TG secretion (Suppl. S3). Perhaps lipid metabolism is more directly affected, and changes in glucose metabolism are secondary to that of triglyceride metabolism. Regarding ketogenesis, the fenofibrate+ B2D fed mice have decreased plasma beta-hydroxybutyrate, suggesting decreased ketogenesis, which is a more canonical PPARα pathway (Suppl. S3). Testing each of these processes would help control that this mechanism is specific to gluconeogenesis and not secondary to something else.
2. Is the effect on ISR dependent on PPARα? Is the mechanism of Fenofibrate on the liver, or on another cell type? In Figure 1, the authors state that Riboflavin deficiency alters body composition and energy expenditure, and then focuses on the liver. However, FAD levels are also increased in the heart and kidneys in addition to the liver. These tissues also respond to PPARα agonism, in addition to the muscle which plays a role in regulating glucose metabolism (B2D mice also have a higher lean mass (Fig 1e)). Additionally, the authors haven't shown specifically if the effects of fenofibrate on electron transport and the ISR are dependent on the presence of PPARα (Figure 5, 6).
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Reviewer #2 (Public Review):
The objective of this work by Masschelin et al. is to investigate the physiological relevance of flavin adenine dinucleotide (FAD). In particular, FAD supports the activity of flavoproteins involved in the production of cellular energy. Mutations in genes encoding flavoproteins often are associated with inborn errors of metabolism (IEMs), thus the clinical interest in investigating in more depth the physiological role of FAD. In this study, the authors first subjected male mice to a vitamin B12 deficient diet (B2D), demonstrating that loss of B12 replicates the phenotypes often observed with IEMs, including loss of body weight, hypoglycemia, and fatty liver. Using a combination of metabolomic phenotyping, transcriptomic analyses, and pharmacology (treatment with fenofibrate, a PPARa agonist), the authors …
Reviewer #2 (Public Review):
The objective of this work by Masschelin et al. is to investigate the physiological relevance of flavin adenine dinucleotide (FAD). In particular, FAD supports the activity of flavoproteins involved in the production of cellular energy. Mutations in genes encoding flavoproteins often are associated with inborn errors of metabolism (IEMs), thus the clinical interest in investigating in more depth the physiological role of FAD. In this study, the authors first subjected male mice to a vitamin B12 deficient diet (B2D), demonstrating that loss of B12 replicates the phenotypes often observed with IEMs, including loss of body weight, hypoglycemia, and fatty liver. Using a combination of metabolomic phenotyping, transcriptomic analyses, and pharmacology (treatment with fenofibrate, a PPARa agonist), the authors then reach the general conclusion that activation of the nuclear receptor PPARa can rescue the B2D phenotypes, thus revealing that PPARa directly controls the metabolic responses to FAD availability. Although the phenotypic analysis of the mice subjected to B2D increases our knowledge of the physiological impact of depleting the FAD pools on global energy metabolism, not all conclusions and statements made by the authors are totally supported by the data. In particular, the study is overall too descriptive and lacks mechanistic insights. While PPARa is likely an important player in the metabolic response to FAD availability, the molecular details on how FAD controls the activity of PPARa either directly or indirectly are entirely missing. Therefore, the authors are encouraged to directly assess whether B2D directly influences PPARa activity on the genes identified in the study, perform rescue experiments in the liver of PPARa KO mice and explore the possibility that other factors (including nuclear receptors) also participate in the response to B12 deficiency and diminished FAD pools.
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