EMBO/SourceData huggingface dataset:
Figure 1(A-C). Mice body weight gain (A), the lean mass and fat mass (B) and gWAT index (C). At 8 weeks of age, male C57BL/6 mice were switched to HFD. After 12 weeks of HFD feeding, mice were divided into three groups receiving non-exercise, endurance exercise or resistance exercise for 14 days. (n = 8 per group).(D). Relative changes in metabolites in response to resistance exercise. Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non-exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group). Shades of yellow and blue represent fold increase and fold decrease of a metabolite, respectively (see color scale).(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).(H). Serum AKG concentration-time profile obtained before and after 40-min resistant exercise. At 8 weeks of age, male C57BL/6 mice were switched to HFD and continuously fed with HFD for 12 weeks. At 20 weeks of age, mice received resistance exercise for 40 min. Another group of chow fed male C57BL/6 mice (10 weeks) received resistance exercise for 40 min. The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after exercise (n = 8-10 per group).(I). Serum AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1 day or resistance exercise for 40 min.(n = 8-10 per group).(J). Muscles AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group).Muscle ICDHm (K), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).GDH (L), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).α-KGDH (M) enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).
Figure 2(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group).(B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).(G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group).(J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group).(K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group).(M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).(P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group).(R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group).(U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).
Figure 3(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).
Figure 4(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).
Figure 5(A). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).(B). The mRNA expression of OXGR1 in different tissues of 12-weeks male C57BL/6 mice (n = 3 per group).(C). OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 (green).(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).(E). Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control (NC) siRNA or siOXGR1 (n = 3 per group).(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).Oxygen consumption (P-Q) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).
Figure 6Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).
Figure 7Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group).Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group).Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group).Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).The mRNA expression in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).protein expression of UCP1 in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).Oxygen consumption in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).RER in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).
Figure 8Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).Immunoblots and quantification of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.Immunoblots and quantification of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).
Figure 1Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.
Figure 2(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.
Figure 3(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).
Figure 4WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.
Figure 5Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.
Figure 6in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates.The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively.
Figure 7Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.
f1(b) in N2 wild type (black diamonds) and ung-1 mutants (red squares), (c) following the depletion of EXO-3 (red triangles), APN-1 (blue circles) or control RNAi (L4440, black diamonds) in the N2 wild-type background, and (d) in msh-2(ok2410) (orange triangles), msh-6(pk2504) (green circles) and mlh-1(ok1917) (grey squares) mutants.(e) Western blottings showing expression of MSH-6 protein in the wild type strain grown on E. coli expressing control (N2) or the indicated RNAi, as well as in msh-6(pk2504) and msh-2(ok2410) mutants (top panel), and expression of MSH-2::GFP fusion protein in transgenic worms grown on E. coli expressing RNAi for MSH-2, MSH-6 or empty vector control as indicated (lower panel). Actin was used as the loading control.(f) F1 survival after depletion of MSH-2 and MSH-6 in the mlh-1(ok1917) mutant. (g,h) F1 survival after depletion of the AP endonucleases EXO-3 and APN-1 in (g) the msh-6(pk2504) or (h) msh-2(ok2410) mutant background. Kruskal-Wallis one-way analysis on ranks (P=0.963 and P=0.985 in h and g, respectively). Mann-Whitney rank-sum test failed to identify a difference in median survival after depleting EXO-3 in the msh-6 mutant (g, P=0.929). (i) F1 survival following depletion of the indicated genes in exo-3(tm4374) mutants. (j) F1 survival in msh-6(pk2504), exo-3(tm4374) and exo-3;msh-6 double mutants. (k) F1 survival in exo-3(tm4374), mlh-1(ok1917) and exo-3;mlh-1 double mutants. All survival curves (b-d,f-k) show the mean±s.d. for each data point from three independent experiments.
f2(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm).b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci.(c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1).(d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci.(f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.
f3a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).(b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm).(c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product.(d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments.(e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes.(f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm).(g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment.(h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.