LCoRL Regulates Growth and Metabolism

Abstract

Genome-wide association studies (GWAS) in humans and livestock have identified genes associated with metabolic traits. However, the causality of many of these genes on metabolic homeostasis is largely unclear due to a lack of detailed functional analyses. Here we report ligand-dependent corepressor-like (LCoRL) as a metabolic regulator for body weight and glucose homeostasis. Although GWAS data show that LCoRL is strongly associated with body size, glucose homeostasis, and other metabolic traits in humans and livestock, functional investigations had not been performed. We generated Lcorl knockout mice (Lcorl-/-) and characterized the metabolic traits. We found that Lcorl-/- pups are born smaller than the wild-type (WT) littermates before reaching normal weight by 7 to 9 weeks of age. While aging, Lcorl-/- mice remain lean compared to WT mice, which is associated with a decrease in daily food intake. Glucose tolerance and insulin sensitivity are improved in Lcorl-/- mice. Mechanistically, this stunted growth is linked to a reduction of circulating levels of IGF-1. The expression of the genes downstream of GH signaling and the genes involved in glucose and lipid metabolism are altered in the liver of Lcorl-/- mice. Furthermore, Lcorl-/- mice are protected against a high-fat diet challenge and show reduced exercise capacity in an exercise stress test. Collectively, our results are congruent with many of the metabolic parameters linked to the Lcorl locus as reported in GWAS in humans and livestock.

Keywords: Lcorl; adiposity; body weight; exercise; glucose homeostasis.

Figure 1.

Expression of lcorl and validation of Lcorl^−/− mouse. (A) LCoRL is expressed ubiquitously when we perform quantitative PCR. (B) Schematic diagram and the gel image show the targeted axons, the position of loxP site, and the deletion. (C-E) mRNA expression of Lcorl, Ncapg, and Lcor in hypothalamus (Hypoth), liver, gastrocnemius (Gastroc), and testis in wild-type and Lcorl^−/− males. n = 7-12 per group. Error is SEM. **** P < .0001 by unpaired t-test.

Figure 2.

The early growth is delayed in Lcorl^−/− mice with low circulating IGF-1 level. (A-B) Body weight curves of males and females show Lcorl^−/− mice were born lighter compared to the WT. The males reach to the normal weight by 8 weeks, but the females stay lean until 16 weeks even when their size becomes normal (Fig. 2C). (C-D) The growth curves of WT and Lcorl^−/− mice between postnatal day 0 and postnatal day 21. (E-F) Tibia lengths of 3-week, 8-week, and 18-week-old WT and Lcorl^−/− mice. (G) Circulating GH levels of 3-week and 8-week-old mice. (H) Circulating IGF-1 levels of 3-week and 8-week-old female mice. (I-K) Expression level of each component of the hypothalamus-pituitary-somatostatin axis in 3- and 8-week-old female WT and Lcorl^−/− mice. (F) Hypothalamic Ghrh and Sst, G: pituitary Gh, Ghrhr, Sstr1-3, 5 (Sstr4 is not expressed in pituitary) or H: liver Ghr, Igf1, Igfals, Igfbp3. n = 8-18. Error is SEM. Statical tests are a 2-way repeated measures ANOVA, a 2-way ANOVA, or a mixed effect analysis with a Sidak's multiple comparison post hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Figure 3.

LCoRL regulates expression of the genes involved in the hypothalamus-pituitary-somatostatin axis and liver metabolism. (A) Volcano plot of gene expression in 3-week-old female mouse livers compared to WT. (B) Normalized counts of liver genes in the hypothalamus-pituitary-somatostatin axis. (C) Heat map of differently expressed genes. (D-E) KEGG pathways enrichment of downregulated (D) and upregulated genes (E). (F) ChEA analysis of transcription factors regulating the downregulated and upregulated genes. (G) Serum insulin (G) levels of 3- and 8-week-old WT and Lcorl^−/− mice. Adjusted P-values for RNA sequencing are calculated with a Wald test with a Benjamini-Hochberg correction (A-B). A 2-way ANOVA with a Sidak's multiple comparison post hoc test (G) was used. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Figure 4.

Lcorl^−/− mice have reduced fat mass and food intake. (A-D) Fat mass (A-B) and lean mass (C-D) of male and female WT and Lcorl^−/− mice measured by nuclear magnetic resonance. (E-F): Weight of liver and adipose tissue in male and female 18-week WT and Lcorl^−/− mice. (G-H) Food intake of male and female WT and Lcorl^−/− mice on chow. Error is SEM. Statical tests: 2-way repeated measures ANOVA, 2-way ANOVA with a Sidak's multiple comparison post hoc test, or unpaired t-test. n = 8-13. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Figure 5.

Lcorl^−/− mice have alterations respiratory exchange ratio. (A-B) Locomotor activity of male and female WT and Lcorl^−/− mice. (C-D) vO₂ of male and female WT and Lcorl^−/− mice. (E-F) Average RER of male and female WT and Lcorl^−/− mice. (G-H) Daily traces of RER of male and female WT and Lcorl^−/− mice. The values are mean ± SEM. n = 6. Statical tests: unpaired t-test. * P < .05, ** P < .01.

Figure 6.

Lcorl^−/− mice have improved glucose tolerance independent of weight. (A-D) Glucose tolerance test in male and female WT and Lcorl^−/− mice (A-B) and area under the curve (C-D). (E-F) ITT in male and female WT and Lcorl^−/− mice and (G-H) area under the curve for ITT. Statical tests: 2-way repeated measures ANOVA, 2-way ANOVA with a Sidak's multiple comparison post hoc test, or unpaired t-test. The value is mean ± SEM. n = 7-10. * P < .05, ** P < .01.

Figure 7.

Lcorl^−/− mice are protected against HFD with improved glucose homeostasis. (A-J) Body weight (A-B), fat and lean mass measured by nuclear magnetic resonance (C-F), fat and liver weight in HFD-fed mice (G-H) and food intake (I-J) indicates that Lcorl^−/− mice are protected against HFD-induced weight and fat gains. (K-R) GTT in male and female WT and Lcorl^−/− mice (K-L) and AUC for GTT (M-N). (O-R) ITT in male and female WT and Lcorl^−/− mice (O-P) and AUC for ITT (Q-R). Statical tests: 2-way repeated measures ANOVA, 2-way ANOVA with a Sidak's multiple comparison post hoc test, or unpaired t-test. The value is mean ± SEM, n= 8-16. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Figure 8.

Lcorl^−/− mice have reduced exercise capacity. Running endurance curve (A), time run (B), maximum speed (C), work performed (D), maximal vO₂ (E) of male mice during an exercise stress test. Values are the mean ± SEM. Statical tests are log-rank (Mantel-Cox) test (A) or unpaired t-test (B-E). n = 15-16. * P < .05, ** P < .01, *** P < .001, **** P < .0001.