NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Data and Materials Availability: Data generated in this study are publicly available in the GEO repository (GSE151281). We also utilized publicly-accessible RNA-seq data from GEO repositories GSE133989 and GSE118787. JASPAR databases are found at http://jaspar.genereg.net/search? nature research | reporting summary April 2020 q=&collection=CORE&tax_group=vertebrates . Source data are provided with this paper. Correspondence and requests for materials should be addressed to Joseph Bass (j-bass@northwestern.edu). Reprints and permissions information is available at www.nature.com/reprints.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Power calculations were performed to identify the minimum number of mice for each assay that would achieve 90% power given measured differences in means and standard deviations for each assay in previous work in our lab as in (Levine DC et al, Molecular Cell, 2020).
Data exclusions We used pre-determined criteria to exclude outliers including IQR*1.5 rule or PCA clustering at a substantial distance away from other replicates. One replicate from the AAV8-LbNOX-transduced, TRF-CR group was excluded as a result of being identified as an outlier by the above criteria in assays measuring protein and RNA levels of LbNOX, NADH, and also in clustering of RNA-seq data.

Replication
All findings in mice were successfully replicated at each attempt and were replicated in at least one independent cohort containing multiple mice. In cell-based studies, results were repeated in at least 3 independent experiments collected and assayed on different days.
Randomization Littermates were distributed evenly into each group.

Blinding
Code-names were applied to samples to blind researchers during sample processing at the bench and during data analysis.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Validation
Antibodies chosen have been widely published, contain validation statements in the vendor websites, and/or were validated by the Antibody Validation Database (Park Lab). For the FOXO1 Abcam ChIP antibody, ChIP-seq datasets were validated by confirming expected phenotypes (i.e. increased binding to known targets following prolonged fasting and in insulin receptor knockouts), by performing unbiased DNA motif analysis with HOMER, and by comparing results to a Santa Cruz FOXO1 antibody (PMID: 30532187) using peaks from our antibody as reference.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) Immortal wild type mouse embryonic fibroblasts were generated in lab as previously described (Levine DC et al, Molecular Cell, 2020). HEK293T cells were obtained from Takara (632180).

nature research | reporting summary
April 2020

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals 4-6 mo old male C57B6/J mice were used in TRF-Reg and TRF-CR mice +/-LbNox. We also utilized liver-specific Sirt1 KO mice (generated either by crossing Sirt1 fx/fx mice with Alb-Cre mice or by injecting Sirt1 fx/fx mice with AAV8-TBG-iCre) and liver-specific Bmal1 KO mice (generated by injecting Bmal1 fx/fx mice with AAV8-TBG-iCre). Male 4-6 month old male mice were used in all experiments, except for studies monitoring fasted body temperature which were performed in 4-6 month old female mice.

Wild animals
No wild animals were used in this study.
Field-collected samples No samples were collected in the field in this study.

Ethics oversight
All animal procedures were in accordance with guidelines of the Institutional Animal Care and Use Committee at Northwestern University. Mouse protocols approved in this study include: IS00003543, IS00007712, IS00001143, and IS00000601.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.
Data in this study is publicly available in the GEO repository (GSE151281). Correspondence and requests for materials should be addressed to Joseph Bass (j-bass@northwestern.edu).