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 (133). 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 h) fast or fasting followed by 2 h 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 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 expression (WT, n = 5; KO, n = 5). All expression levels were normalized to β-actin. All data are presented as mean ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001.

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.

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).

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 acids, and β-hydroxybutyrate levels. (M) Very-low density lipoprotein-triglyceride (VLDL-TG) and high-density lipoprotein-cholesterol (HDL-cholesterol) analysis by FPLC of pooled (n = 6-7 per genotype) mouse sera. (N) Blood glucose levels during glucose tolerance tests (GTT). (O) Blood glucose levels during insulin tolerance tests (ITT). GTT and ITT were performed when the female mice reached 28 and 29 weeks of age, respectively. WT, n = 9; KO, n = 6.

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 = 17) and KO (n = 13) 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 ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Ctrp10-KO mice on a high-fat diet 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).

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) female mice. (I) Representative H&E stained histological sections of liver and the quantification of hepatic lipid content (% lipid area; n = 7 per genotype). Scale bar = 100 μM. (J) Hepatic expression of genes associated with inflammation, fibrosis, ER and oxidative stress, lipid synthesis, and lipid catabolism in WT and KO female mice. Gene expression data were obtained from RNA-seq. (K-L) Quantification of hydroxyproline (marker of fibrosis) and malondialdehyde (MDA; marker of oxidative stress) in liver. WT, n = 15; KO, n = 11. All data are presented as mean ± S.E.M. * P < 0.05; ** P < 0.01.

Transcriptomic analysis of liver, adipose tissue, and skeletal muscle of female Ctrp10 KO mice fed a high-fat diet.

(A-D) Cropped volcano plot views of all differentially expressed genes (DEGs, Log2(Fold Change) >1 or <-1 with a p-value <0.05) of the liver, gonadal white adipose tissue (gWAT), inguinal WAT (iWAT), or skeletal muscle (gastrocnemius). (E) Overlap analysis of tissue DEGs showing (top panel) expression unique to gonadal white adipose tissue (gW), inguinal white adipose tissue (iW), liver (L), or skeletal muscle (M). Percent (%) represents percent DEGs unique to each tissue. Bottom panel show DEGs shared across multiple tissues, with all the shared DEGs listed. (F) Enrichr analysis (129) of biological pathways and processes significantly (p<0.01) affected across the CTRP10 deficient female mice. Top pathways and processes derived from Gene Ontology (GO), Reactome (R-HAS), WikiPathway human (WP), and mammalian phenotype (MP). All up- or down-regulated DEGs across all tissues were used for analysis. The tissues contributing to the highest ranked pathways and processes are specified. n = 6 KO and 6 WT for RNA-seq experiments.

Loss of CTRP10 induces significant and wide-spread alterations in the expression of key transcription factors, secreted protein, membrane receptors, and metabolism-associated genes.

(A-D) Selected genes from the DEG list of each tissue organized based on gene type (genes encoding transcription factors, secreted proteins, receptors, and proteins involved in metabolism) and ranked from highest to lowest row z-score. N = 6 per genotype

GTEx genetic co-correlation of mouse differentially expressed gene (DEG) orthologues.

(A-B) Heatmaps showing biweight midcorrelation (bicor) coefficient among human tissue DEG orthologues in females (A) and males (B) in GTEx. Y-axis color indicates tissue of origin, P-value based on students’ regression P-value. (C) T-tests between correlation coefficient in males and females among all DEG orthologue gene pairs for subcutaneous (SubQ) adipose tissue, visceral (visc) adipose tissue, liver, and skeletal muscle. (D) the same as in C, except comparisons are shown for all gene-gene pairs between tissues. For example, the top left graph compared the connectivity of males (blue color) vs females (green color) for correlation between subcutaneous (SubQ) and visceral (Visc) adipose tissue DEG orthologues.