Loss of CTRP10 results in female obesity with preserved metabolic health

Figures

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  Figure 1.. Nutritional regulation of Ctrp10 expression. (A) Sequence alignment of full-length human (GenBank # NP_872334), mouse (NP_997116), chicken (XP_046777733), Xenopus frog (XP_031749381), and zebrafish (XP_001920705) CTRP10/C1ql2 using Clustal-omega (Sievers and Higgins, 2018). Identical amino acids are shaded black and similar amino acids are shaded grey. Gaps are indicated by dash lines. Signal peptide, collagen domain with characteristic Gly-X-Y repeats, and the C-terminal globular C1q domain are indicated. (B) Ctrp10 expression across different mouse tissues (n=10). (C) Expression of Ctrp10 across mouse tissues in response to an overnight (16 hr) fast or fasting followed by 2 hr refeeding. (D) Expression of Ctrp10 across mouse tissues in response to a high-fat diet (HFD) for 12 weeks or a control low-fat diet (LFD). (E) Generation of Ctrp10 knockout (KO) mice. The entire protein coding region in exon 1 and 2 of Ctrp10 was deleted using the CRISPR/Cas9 method and confirmed with DNA sequencing. (F) Wild-type (WT) and KO alleles were confirmed by PCR genotyping. (G) The complete loss of Ctrp10 transcript in KO mice was confirmed in mouse cortex, one of the tissues with high Ctrp10…

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  Figure 2.. Ctrp10-KO mice fed a low-fat diet have normal body weight and energy balance. (A-B) Body weight (A) and body composition analysis (B) of fat mass, % fat mass (relative to body weight), lean mass, and % lean mass of WT (n=17) and KO (n=14) male mice at 18 weeks of age. (C-D) Body weight (C) and body composition analysis (D) of fat mass, % fat mass (relative to body weight), lean mass, and % lean mass of WT (n=9) and KO (n=6) female mice at 13 weeks of age. (E-G) Food intake, physical activity, and energy expenditure (EE) in male mice at 18 weeks of age across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fasted, refed; WT, n=11–12; KO, n=10–12). (H-J) Food intake, physical activity, and energy expenditure in female mice at 13 weeks of age (WT, n=9; KO, n=6). All data are presented as mean ± S.E.M.

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  Figure 3.. Ctrp10-KO mice fed a low-fat diet have normal fasting-refeeding response and glucose homeostasis. (A-B) Overnight fasted and refed blood glucose, serum insulin, triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate levels in male (A) and female (B) mice. (C-D) Blood glucose levels during glucose tolerance tests (GTT; C) and insulin tolerance tests (ITT; D) in WT (n=17) and KO (n=14) male mice at 12 weeks of age. (E-F) Blood glucose levels during glucose tolerance tests (GTT; E) and insulin tolerance tests (ITT; F) in WT (n=9) and KO (n=6) female mice at 20 and 21 weeks of age, respectively. All data are presented as mean ± S.E.M. * p<0.05 (two-way ANOVA with Sidak’s post hoc tests).

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  Figure 4.. Ctrp10-KO female mice on a low-fat diet develop obesity with age. (A) Body weights over time of WT and KO female mice fed a low-fat diet (LFD). (B) Representative image of WT and KO female on LFD for 40 weeks. (C) Body composition analysis of WT (n=9) and KO (n=6) female mice fed a LFD. (D) Representative H&E stained histology of gonadal white adipose tissue (gWAT) and the quantification of adipocyte cell size (n=6 per genotype). Scale bar = 100 µM. (E) Representative H&E stained histology of inguinal white adipose tissue (iWAT) and the quantification of adipocyte cell size (n=6 per genotype). Scale bar = 100 µM. (F) 24 hr food intake data measured manually. (G) Fecal frequency, fecal weight, and fecal energy over a 24 hr period. (H) Deep colon temperature measured at the light and dark cycle. (I-K) Food intake, physical activity, and energy expenditure in female mice across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fasted, refed; WT, n=9; KO, n=6). Indirect calorimetry analysis was performed after female mice were on LFD for 30 weeks. (L) Overnight (16 hr) fasted blood glucose, serum insulin, triglyceride, cholesterol, non-esterified free fatty…

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  Figure 4—figure supplement 1.. No significant differences in serum estradiol levels between WT (n=9) and Ctrp10 KO (n=6) female mice fed a low-fat diet (LFD). We included WT male sera (n=5) as additional controls as chow-fed male mice are expected to have much lower levels of serum estradiol.

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  Figure 4—figure supplement 2.. Mitochondrial respiration through complex I (CI), CII, and CIV in liver and skeletal muscle (gastrocnemius) of WT (n=9) and Ctrp10 KO (n=6) female mice fed a low-fat diet (LFD). No significant differences in liver and skeletal muscle (gastrocnemius) mitochondrial respiration through complex I (CI), CIII, and CIV between WT (n=9) and Ctrp10 KO (n=6) female mice. (A and D) Average oxygen consumption rate (OCR) traces per group, normalized to mitochondrial content. Each group tracing represents the average trace of nine WT and six KO samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: (i) basal reads, (ii) addition of NADH (activation of respiration through complex I), (iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), (iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally (v) addition of azide (inhibitor of complex IV). (B and E) The same information as presented in (A and D) conducted on the same samples, but the NADH injection step is…

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  Figure 5.. Sexually dimorphic response of Ctrp10-KO mice to an obesogenic diet. (A) Body weights over time of WT and KO male mice fed a high-fat diet (HFD). (B) Body composition analysis of WT (n=17) and KO (n=14) male mice fed a HFD for 9 weeks. (C-E) Food intake, physical activity, and energy expenditure in male mice across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fasted, refed; WT, n=11; KO, n=11). Indirect calorimetry analysis was performed after male mice were on HFD for 10 weeks. (F) Body weights over time of WT and KO female mice fed a high-fat diet. (G) Representative image of WT and KO female mice after 13 weeks of high-fat feeding. (H) Body composition analysis of WT (n=15) and KO (n=12) female mice on HFD for 6 weeks. (I-K) Food intake, physical activity, and energy expenditure in female mice (WT, n=11–12; KO, n=12) across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fasted, refed). Indirect calorimetry analysis was performed after female mice were on HFD for 6 weeks. (L) ANCOVA analysis of energy expenditure using body weight as a covariate. (M) Respiratory exchange ratio (RER). All data are presented as mean ±…

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  Figure 5—figure supplement 1.. No differences in food intake, body temperature, or changes in fecal output, frequency, and energy content between WT and Ctrp10 KO female mice fed a high-fat diet. Sample size for food intake, fecal frequency, and fecal weight: WT = 6 and KO = 6; sample size for fecal energy and total fecal energy content: WT = 9 and KO = 6; sample size for deep colon temperature: WT = 9 and KO = 10.

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  Figure 5—figure supplement 2.. No significant differences in serum estradiol levels between WT (n=6) and Ctrp10 KO (n=11) female mice fed a high-fat diet (HFD). We included WT male sera (n=5) as additional controls as chow-fed male mice are expected to have much lower levels of serum estradiol.

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  Figure 5—figure supplement 3.. Mitochondrial respiration through complex I (CI), CII, and CIV in liver and skeletal muscle (gastrocnemius) of WT (n=7) and Ctrp10 KO (n=8) female mice fed a high-fat diet. (A and D) Average oxygen consumption rate (OCR) traces per group, normalized to mitochondrial content. Each group tracing represents the average trace of seven WT and eight KO samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: (i) basal reads, (ii) addition of NADH (activation of respiration through complex I), (iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), (iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally (v) addition of azide (inhibitor of complex IV). (B and E) The same information as presented in (A and D) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). (C and F) Average values of all data presented for liver (A-B)…

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  Figure 6.. Ctrp10-KO mice on a high-fat diet (HFD) have normal glucose and insulin tolerance. (A-B) Overnight fasted and refed blood glucose, serum insulin, triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate levels in male (A) and female (B) mice fed a HFD for 10 weeks. (C-D) Blood glucose levels during glucose tolerance tests (GTT; C) and insulin tolerance tests (ITT; D) in WT (n=17) and KO (n=14) male mice fed a HFD for 10 weeks. (E-F) Blood glucose levels during glucose tolerance tests (GTT; E) and insulin tolerance tests (ITT; F) in WT (n=16) and KO (n=12) female mice fed a HFD for 8 weeks. (G) VLDL-TG and HDL-cholesterol analysis by FPLC of pooled female mouse sera. All data are presented as mean ± S.E.M. ** p<0.01; *** p<0.001; **** p<0.0001 (two-way ANOVA with Sidak’s post hoc tests for fasted/refed data).

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  Figure 7.. Ctrp10-KO female mice fed a HFD do not develop adipose tissue dysfunction and fatty liver. (A) Representative images of dissected gonadal white adipose tissue (gWAT) and the quantification of gWAT weight in WT (n=15) and KO (n=12) female mice fed a HFD for 14 weeks. (B) Representative H&E stained histological sections of gWAT and the quantification of adipocyte cell size (n=7 per genotype). Scale bar = 100 µM. (C) Representative images of dissected inguinal white adipose tissue (iWAT) and the quantification of iWAT weight in WT (n=15) and KO (n=12) female mice. (D) Representative H&E stained histological sections of iWAT and the quantification of adipocyte cell size (n=7 per genotype). Scale bar = 100 µM. (E) Expression of genes associated with inflammation, fibrosis, ER and oxidative stress in gWAT and iWAT of WT (n=6) and KO (n=6) female mice fed a HFD for 14 weeks. Gene expression data were obtained from RNA-seq. (F-G) Quantification of hydroxyproline (marker of fibrosis) and malondialdehyde (MDA; marker of oxidative stress) in gWAT and iWAT. WT, n=15; KO, n=11. (H) Representative images of dissected liver and the quantification of liver weight in WT (n=15) and KO (n=12)…