Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , ( left ) Representative wheel-running behaviour and ( middle ) quantification of average daily onset of activity for TRF-Reg (n = 5) and TRF-CR (n = 5) mice throughout the duration of the 4-wk intervention (double plotted for clarity). ( right ) Background-corrected PER2::LUCIFERASE readings from excised suprachiasmatic nucleus (SCN) of TRF-Reg (n = 6) and TRF-CR (n = 6) mice. The grey shading indicates mean values ± SEM. Experiments were performed in 4–6 mo old male C57BL6/J mice. b , Body weight of TRF-Reg (n = 15) and TRF-CR (n = 15) mice over the duration of the 4-wk study. c , Blood glucose and serum insulin during oral glucose tolerance testing performed during the daytime (at ZT4) in TRF-Reg (n = 6) and TRF-CR (n = 6) mice. d , GAP/DHAP mass isotopomer distribution determined by mass spectrometry of liver from TRF-Reg (n = 3) and TRF-CR (n = 2) mice 30 min following i.p. administration of a 10:1 mix of U 13 C-lactate:U 13 C-pyruvate (1 g/kg). e-f , ( left ) Relative concentration of (e) NADH and (f) NAD + by HPLC in liver of TRF-CR compared to TRF-Reg mice during the day (ZT4) (n = 12 for TRF-Reg, n = 11 for TRF-CR,) and at night (ZT16) (n = 9 for each diet) for each time point. ( right ) Concentration of (e) NADH and (f) NAD + in ad lib fed wild-type mouse liver by HPLC every 6 hrs for 24 hrs (n = 7). g-h , Log 2 -FC in daytime liver acyl-carnitine levels in (g) TRF-CR compared to TRF-Reg mice and (h) Lb NOX- compared to null-overexpressing TRF-CR mice (n = 3). Data are presented as mean values ± SEM. Statistics were performed with unpaired, two-tailed student’s t test except as otherwise noted in the figure. *p < 0.5, ***p < 0.01.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Activity Assay, Luciferase, Mass Spectrometry, Concentration Assay, Two Tailed Test

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Model for the TRF-CR diet using an automated feeder system in 4–6-month-old male C57BL/6J mice. Control mice received a 300 mg pellet of regular chow every 1.2 h throughout the dark period (TRF-Reg), whereas TRF-CR mice received a 300 mg pellet of a carbohydrate-depleted, nutrient-controlled chow every 2 h throughout the dark period, resulting in a 40% reduction in calories. b , Relative concentration of NADH and NAD + by HPLC in liver of TRF-Reg ( n = 12) and TRF-CR ( n = 11) mice during the daytime (ZT4). c , log 2 (fold change) in daytime serum acyl-carnitine levels during TRF-CR compared with TRF-Reg mice ( n = 3). d , Body temperature rhythms monitored non-invasively using subcutaneous probes in TRF-Reg ( n = 12) and TRF-CR ( n = 9) mice over 24 h (double plotted for clarity). e , Model depicting NADH-consuming reaction of Lb NOX. Representative tissue-specific expression profile of cytonuclear Lb NOX in null- and Lb NOX-transduced mice relative to Lb NOX-transduced liver. f , Relative concentration of NADH and NAD + in liver of null- and Lb NOX-transduced TRF-Reg ( n = 6) or TRF-CR ( n = 5 for NADH, n = 6 for NAD + ) mice during the daytime. g , log 2 (fold change) in daytime serum acyl-carnitine levels in TRF-CR mice transduced with cytonuclear Lb NOX compared with TRF-CR mice transduced with null virus ( n = 3). h , Body temperature rhythms in Lb NOX-expressing TRF-Reg ( n = 12) or TRF-CR ( n = 9) mice over 24 h (double plotted for clarity). Data are presented as mean ± s.e.m. Statistics were performed with unpaired, two-tailed Student’s t test unless otherwise noted in the figure. * P < 0.05, *** P < 0.001. ANOVA, analysis of variance; Cereb, cerebellum; DC, dicarboxylate; Gastroc, gastrocnemius muscle; gWAT, gonadal white adipose tissue; Hypoth, hypothalamus; i, isomer; iWAT, inguinal white adipose tissue; OH, hydroxy; Quad, quadriceps muscle.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Control, Concentration Assay, Expressing, Transduction, Virus, Two Tailed Test

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , RNA-Seq in liver in the morning (ZT4) demonstrating the effect of TRF-CR in null-transduced (left) ( n = 6) and Lb NOX-transduced (middle) ( n = 6 for TRF-Reg, n = 5 for TRF-CR) 4–6-month-old male C57BL/6J mice for genes differentially expressed (DESeq2 FDR-adjusted P < 0.05) by TRF-CR in null-transduced mice (930 genes). Venn diagram (right) displays overlap in differentially expressed genes by TRF-CR in null- and Lb NOX-transduced mice. b , Quadrant plot comparing transcriptional responses between TRF-CR ( x axis) and Lb NOX in TRF-CR ( y axis). Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced mice (930 genes). Colouring indicates genes within quadrant 2 (green) and quadrant 4 (red), and the percentages within each quadrant are shown. c , d , For the genes within quadrants 2 and 4 from b , the top 15 ( c ) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms enriched ( P < 0.05) following gene ontology analysis and ( d ) JASPAR motifs enriched ( P < 0.05) following HOMER DNA motif enrichment analysis are shown. e , Quadrant plots comparing the transcriptional response to TRF-CR in null-transduced mice ( x axis) with that of genetic ablation of either Pparα (top) or Bmal1 (bottom) ( y axis ) in animals fed ad libitum. Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced animals. Genes that have an absolute log (fold change) > 0.5 for both comparisons are coloured blue or black, and the percentage of genes was determined by quadrant ( n = 3). f , LC–MS metabolomics profiling of amino acids in liver during the daytime (ZT4). The log (fold change) from TRF-CR (blue) ( n = 5) and Lb NOX in TRF-CR (white) ( n = 6) is shown for select differential amino acids (two-tailed, unpaired Student’s t test with Benjamini and Hochberg adjustment for multiple measures FDR P < 0.05; see Supplementary Table for full list of amino acids). Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles. g , Model depicting the interrelationship of NADH during TRF-CR to the activity of PPARα and BMAL1 and the transcription of downstream oxidative gene networks.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Whisker Assay, Activity Assay

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , ( left ) RNA-seq reads per 10 million sequenced reads (RPM) that align to Lb NOX in Lb NOX- vs null-transduced TRF-Reg (n = 6 per genotype) and TRF-CR (n = 6 for null; n = 5 for Lb NOX) liver. ( right ) Representative in situ immunohistochemistry against FLAG- Lb NOX in liver of null- and Lb NOX-transduced mice. Experiments were performed in 4–6 mo old male C57BL6/J mice unless otherwise noted. b , Heatmap depicting log 2 -FC in gene expression from TRF-CR and Lb NOX in TRF-CR for genes within gluconeogenic and glycolytic gene ontology groups. c , Unbiased principal components analysis during the day (ZT4) or night (ZT16) of null- and Lb NOX-expressing TRF-Reg or TRF-CR mice for genes that are differentially-expressed by TRF-CR in null mice at respective time points (DESeq2 p-adj<0.05) (n as in panel a). d , Log 2 -transformed FC in expression of select fatty acid metabolism genes during TRF-CR (blue) (n = 6), following Lb NOX-overexpression during TRF-CR (white) (n = 5), and in Bmal1 knockout liver (purple) (n = 3). e , 24-hour fasted body temperatures in 4–6 mo old female liver-specific Bmal1 fx/fx mice before and after retro-orbital administration of AAV8–TBG-iCre (n = 6) ( left : ANOVA, p < 0.01; right: *p < 0.05 in paired, two-way student’s t test). f , Log 2 -transformed FC in expression of genes within the methionine pathway for conditions (n as in panel d). g , ( top ) Summary of methionine pathway and ( bottom ) metabolomics of effect of TRF-CR and Lb NOX in TRF-CR on end-products of methionine metabolism in liver and serum performed during the daytime (ZT4) (n = 3). Data are presented as mean values ± SEM.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: RNA Sequencing, In Situ, Immunohistochemistry, Gene Expression, Expressing, Transformation Assay, Over Expression, Knock-Out

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Lineweaver-Burk transformation of data from SAMDI-MS. b , Relative NADH quantified by HPLC following supplementation with pyruvate or lactate compared to controls (n = 3). c , Densitometric quantification of western blots from liver of 4–6 mo old male mice in indicated genotypes at ZT4 for p53-(K379)Ac ( left ) and FOXO1-(K242, K245, K262)Ac ( right ) relative to control TRF-Reg (from Fig. ) (n = 3). d , FOXO1 ChIP-seq from liver collected during the daytime comparing effect of FOXO1 binding in TRF-CR (x-axis) with the effect of hepatic SIRT1 ablation ( L-Sirt1 − / − ) (y-axis). Each point indicates a FOXO1 peak in control liver. Peaks that have absolute log 2 (fold change) >0.5 for both comparisons are colored blue or black and counted by quadrant (n = 2). e , Densitometric quantification of western blots as in (c) relative to null-transduced TRF-Reg mice (from Fig. ) (Ac-p53: n = 4 for TRF-CR, Lb NOX. n = 6 for all other conditions; Ac-FOXO1: n = 2 for TRF-CR, Lb NOX. n = 3 for all other conditions). f , FOXO1 ChIP-seq from liver during the daytime comparing effect of TRF-CR (x-axis) (n = 3) with effect of (f) Lb NOX in TRF-CR mice (y-axis) (n = 2) with coloring and counting as in panel d. (n = 2–3). g , Western blotting for Ac-FOXO1 during the nighttime (ZT16) in null- and Lb NOX-transduced mice on TRF-Reg and TRF-CR (n.s. non-specific) and densitometric quantification (n = 3). h , BMAL1 ChIP-seq from liver during the daytime comparing the effect of TRF-CR and L-Sirt1 − / − as in panel d (n = 3). Uncropped Western blot scans labelled with molecular weight markers are presented in the Source Data Files. i , RNA-seq reads mapping to the exon of Sirt1 that is flanked by LoxP sites (Ex4) relative to exon 9 (Ex9) of Sirt1 , and RNA-seq reads per 10 million sequenced reads (RPM) that align to iCre and Lb NOX in null- or Lb NOX-transduced TRF-CR mice co-transduced with iCre (n = 6). Null-expressing mice on TRF-CR (blue) (n = 5) are shown as reference. j , Quadrant plot comparing transcriptional responses to TRF-CR in null-transduced mice (x-axis) (n = 6) and Lb NOX-expression in TRF-CR, L-Sirt1 − / − mice (y-axis) (n = 6). Each point indicates a gene that is DE by TRF-CR in null-transduced mice (930 genes). Percentages of genes within each quadrant are shown. k , Average 48-hour fasting body temperature in 4–6 mo old female liver-specific Sirt1 − / − mice (n = 5). Data are presented as mean values ± SEM. Statistics were performed with unpaired, two-tailed student’s t test except as otherwise noted in the figure. *p < 0.05.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Transformation Assay, Western Blot, Control, ChIP-sequencing, Binding Assay, Molecular Weight, RNA Sequencing, Transduction, Expressing, Two Tailed Test

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: a , Model to examine the role of SIRT1 in the NADH-dependent effects on RNAs, metabolites and body temperature during TRF-CR. b , Deacetylation rate for SIRT1 with increasing concentrations of NAD + and NADH assayed by SAMDI-MS ( n = 4). c , Model of pyruvate/lactate equilibrium shows that supplementation with pyruvate and lactate reduces and elevates NADH, respectively. Western blotting for SIRT1 targets, Ac-H3K9 and Ac-H4K16, in immortalized mouse embryonic fibroblasts treated with pyruvate or lactate to modulate NADH ( n = 3). d , e , Western blotting for SIRT1 targets, Ac-p53 and Ac-FOXO1, in TRF-Reg or TRF-CR liver of 4–6-month-old male ( d ) control or liver-specific Sirt1 − / − mice or ( e ) null- or Lb NOX-transduced mice. n.s., non-specific. Uncropped western blot scans labelled with molecular weight markers are presented in the Source Data files. f , BMAL1 ChIP-Seq in liver of TRF-Reg or TRF-CR mice. Peaks demonstrating an absolute log 2 (fold change) > 0.5 are coloured black. Box–whisker plots of BMAL1 ChIP-Seq demonstrating the effect of TRF-CR in null- ( n = 6) and Lb NOX-transduced ( n = 3) liver on BMAL1 peaks identified in controls and with an absolute log 2 (fold change) > 0.5 in controls. Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles. g , Heatmap depicting log 2 (fold change) in gene expression (left), liver metabolite concentrations (middle) and serum acyl-carnitine levels (right) in indicated conditions/genotypes at ZT4. Heatmaps are subdivided into the genes, metabolites and acyl-carnitines that are regulated by Lb NOX during TRF-CR through mechanisms requiring SIRT1 and sorted by effect of TRF-CR (RNA-Seq, n = 6; metabolomes, n = 5–6; acyl-carnitines n = 3). h , Body temperature rhythms over 24 h from subcutaneous probes implanted in null- ( n = 6 for control, n = 5 for L-Sirt1 − / − ) or Lb NOX-expressing ( n = 5) control and liver-specific Sirt1 − / − mice on TRF-CR. Data are presented as mean values ± s.e.m.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Western Blot, Control, Molecular Weight, ChIP-sequencing, Whisker Assay, Gene Expression, RNA Sequencing, Expressing

Journal: Nature Metabolism

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

doi: 10.1038/s42255-021-00498-1

Figure Lengend Snippet: TRF-CR generates rhythmic bouts of daytime torpor through increased levels of NADH in liver, inhibition of SIRT1 and downregulation of oxidative gene networks controlling acyl-carnitines and core body temperature. Reducing levels of NADH in the morning through the transduction of Lb NOX in TRF-CR mice increases lipid oxidation through the activation of SIRT1, resulting in elevated daytime body temperature rhythms. These findings identify NAD(H) redox state as a link between TRF-CR and whole-body metabolism.

Article Snippet: Briefly, Lb NOX and control plasmids (Addgene 75285) were cloned into AAV expression vectors under the thyroid-binding globulin (TBG) promoter (to avoid inflammation associated with adenoviral-based methods), packaged into AAVs of serotype 8, purified, and concentrated by Vector Biolabs.

Techniques: Inhibition, Transduction, Activation Assay