CTRP11 contributes modestly to systemic metabolism and energy balance

Figures

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  FIGURE 1. Ctrp11 expression is regulated by nutritional and metabolic states. (A) Expression of Ctrp11 in different mouse tissues in response to overnight (~16 h) food withdrawal and 3‐h refeeding (after an overnight food deprivation). (B) Expression of Ctrp11 in different mouse tissues in response to a 12‐week period of control low‐fat diet (LFD) or a high‐fat diet (HFD). All data are presented as mean ± SEM. *p < .05; **p < .01; ***p < .001; ****p < .0001

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  FIGURE 2. CTRP11‐null mice have normal testosterone level and locomotor function. (A) Gene targeting strategy used to generate Ctrp11 knockout (KO) mice. Exon 1 that encodes 179 amino acids (75% of the entire coding region) was replaced by the bGEO (a fusion of the β‐galactosidase and neomycin resistance genes) and Puro (Puromycin resistance gene) cassette. The strategy ensures a complete null allele. The location of the forward (P36) and reverse (P37 and GT‐IRES) primers used to genotype the wild‐type (WT) and KO mice are indicated by the red arrows. (B) Representative PCR genotyping results indicating the successful generation of WT (+/+), heterozygous (+/–), and homozygous KO (–/–) mice. (C) Quantitative real‐time PCR analysis confirming the complete absence of Ctrp11 transcript in inguinal white adipose tissue of male mice. (D) Serum testosterone levels in WT (n = 10) and KO (n = 7) male mice at ~24 weeks of age. (E) Grip strength analysis of male (WT, n = 11; KO, n = 10) and female (WT, n = 10; KO, n = 6) mice. (F) Rotarod analysis of locomotor function in male and female WT and Ctrp11 KO mice. (G) Representative gastrocnemius histology and the quantification of muscle fiber…

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  FIGURE 3. Basal metabolic profiles of WT and Ctrp11‐KO mice fed a standard chow. (A) Body weights of WT littermates and Ctrp11 KO male mice fed a standard chow over time (WT, n = 12; KO, n = 7). (B) Body composition analysis of % fat mass (relative to body weight) and % lean mass (relative to body weight) of WT and KO male mice. (C) Body weights of WT littermates and Ctrp11 KO female mice fed a standard chow over time (WT, n = 10; KO, n = 11). (D) Body composition analysis of fat mass and lean mass of WT and KO female mice. (E) Overnight fasted serum glucose, triglyceride, cholesterol, non‐esterified free fatty acids (NEFA), and β‐hydroxybutyrate in WT and KO male mice. (F) Overnight fasted serum glucose, triglyceride, cholesterol, non‐esterified free fatty acids (NEFA), and β‐hydroxybutyrate in WT and KO female mice. (G) Fasted (6 h) serum insulin levels in WT and KO male mice. (H) Blood glucose levels during glucose tolerance tests in WT and KO male mice. (I) Fasted (6 h) serum insulin levels in WT and KO female mice. (J) Blood glucose levels during glucose tolerance tests in WT and KO female mice. (K) Blood glucose levels during insulin tolerance tests in male and female WT and KO mice.…

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  FIGURE 4. Food intake, oxygen consumption, energy expenditure, and physical activity of chow‐fed WT and Ctrp11‐KO female mice under ad libitum fed, fasted, and refed states. Indirect calorimetry analyses of WT littermates and KO female mice. Dark and light cycle food intake during ad libitum (A), food withdrawal (B), and refeeding phase (C). Dark and light cycle oxygen consumption rate (VO2) during ad libitum (D), food withdrawal (E), and refeeding phase (F). Dark and light cycle energy expenditure (EE) during ad libitum (G), food withdrawal (H), and refeeding phase (I). Dark and light cycle ambulatory activity levels during ad libitum (J), food withdrawal (K), and refeeding phase (L). Oxygen consumption rate and energy expenditure data were normalized to lean mass. All data are presented as mean ± SEM. *p < .05. WT (n = 10); KO (n = 11)

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  FIGURE 5. Meal pattern analysis of chow‐fed Ctrp11 KO female mice in the refed period following food deprivation. (A) Food intake in the first 6 h when food was reintroduced after a 24‐h food withdrawal. (B) Total food intake in the light and dark cycle, as well as, over the 24‐h period. (C) Meal size, (D) meal number, (E) meal duration, (F) ingestive rate, (G) intermeal interval, and (H) satiety ratio in WT (n = 10) and KO (n = 11) female mice. All data are presented as mean ± SEM. *p < .05; ***p < .001

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  FIGURE 6. Metabolic profiles of WT and Ctrp11‐KO mice fed a high‐fat diet. (A) Body weights of WT littermates and Ctrp11 KO male mice fed a high‐fat diet (HFD) over time (WT, n = 22; KO, n = 18). (B) Body composition analysis of % fat mass (relative to body weight) and % lean mass (relative to body weight) of WT and KO male mice. (C) Body weights of WT littermates and Ctrp11 KO female mice fed HFD over time (WT, n = 21; KO, n = 18). (D) Body composition analysis of % fat mass and % lean mass of WT and KO female mice. (E) Blood glucose levels during glucose tolerance tests in WT and KO male mice. (F) Area under curve for data shown in E. (G) Serum insulin levels at time 0 and 15 post glucose injection in male mice. (H) Blood glucose levels during glucose tolerance tests in WT and KO female mice. (I) Area under curve for data shown in H. (J) Serum insulin levels at time 0 and 15 post glucose injection in female mice. (K) Blood glucose levels during insulin tolerance tests in WT and KO male mice. (L) Area under curve for data shown in K. (M) Blood glucose levels during insulin tolerance tests in WT and KO female mice. (N) Area under curve for data shown in M. (O) Serum inflammatory cytokine…

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  FIGURE 7. Tissue histology and gene expression in WT and Ctrp11‐KO male mice fed a high‐fat diet. (A) Representative histology of visceral (gonadal) white adipose tissues and the quantification of adipocyte cell size (cross‐sectional area, CSA) in WT and KO male mice. (B) Representative histology of subcutaneous (inguinal) white adipose tissues and the quantification of adipocyte cell size (cross‐sectional area, CSA) in WT and KO male mice. (C and D) Expression of anti‐inflammatory M2 macrophage markers, pro‐inflammatory M1 macrophage markers, fibrotic and oxidative stress genes in the gonadal white adipose tissue (C) and inguinal white adipose tissue (D) of WT and KO male mice. (E) Representative histology of liver tissues and the quantification of lipid area (% of total area) in WT and KO male mice. (F) Hepatic TG levels in WT and KO male mice. (G) Expression of anti‐inflammatory M2 macrophage markers, pro‐inflammatory M1 macrophage markers, fibrotic and oxidative stress genes in the liver of WT and KO male mice. All data are presented as mean ± SEM. *p < .05

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  FIGURE 8. Fasting–refeeding lipid profiles and lipid tolerance tests of WT and Ctrp11‐KO mice fed a high‐fat diet. Blood glucose (A), serum insulin (B), triglyceride (C), cholesterol (D), non‐esterified free fatty acids (NEFA; E), and β‐hydroxybutyrate (F) levels in overnight fasted and 2 h refed WT (n = 21) and KO (n = 18) male mice on HFD. Blood glucose (G), serum insulin (H), triglyceride (I), cholesterol (J), non‐esterified free fatty acids (NEFA; K), and β‐hydroxybutyrate (L) levels in overnight fasted and 2 h refed WT (n = 19) and KO (n = 18) female mice on HFD. (M and N) Serum triglyceride levels during lipid tolerance tests in male (M) and female (N) WT and KO mice on HFD. All data are presented as mean ± SEM

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  FIGURE 9. Metabolic profiles of obese WT and Ctrp11‐KO mice subjected to obesity reversal. (A–F) Body weight and body composition (% fat mass and lean mass normalized to body weight) of HFD‐induced obese male and female mice before (time 0) and after 4 weeks of diet reversal (switching from HFD to LFD). (G and H) Blood glucose levels during glucose tolerance tests in WT and KO male (G) and female (H) mice subjected to obesity reversal. (I) Blood glucose levels during insulin tolerance tests in male WT and KO mice subjected to obesity reversal. (J) Area under curve for data shown in I. (K) Insulin tolerance tests in female WT and KO mice subjected to obesity reversal. (L‐M) Fasting (~16 h) blood glucose, serum insulin, triglyceride, cholesterol, non‐esterified free fatty acids (NEFA), and β‐hydroxybutyrate levels in male (L) and female (M) mice subjected to obesity reversal. (N) Serum IL‐1β, IL‐6, TNF‐α, and MCP‐1/CCL2 levels in WT and KO male mice. All data are presented as mean ± SEM. *p < .05. Male (WT, n = 10; KO, n = 11); Female (WT, n = 9; KO, n = 11)