A disputed evidence on obesity: comparison of the effects of Rcan2(-/-) and Rps6kb1(-/-) mutations on growth and body weight in C57BL/6J mice

Abstract

It is widely accepted that body weight and adipose mass are tightly regulated by homeostatic mechanisms, in which leptin plays a critical role through hypothalamic pathways, and obesity is a result of homeostatic disorder. However, in C57BL/6J mice, we found that Rcan2 increases food intake and plays an important role in the development of age- and diet-induced obesity through a leptin-independent mechanism. RCAN2 was initially identified as a thyroid hormone (T3)-responsive gene in human fibroblasts. Expression of RCAN2 is regulated by T3 through the PI3K-Akt/PKB-mTOR-Rps6kb1 signaling pathway. Intriguingly, both Rcan2(-/-) and Rps6kb1(-/-) mutations were reported to result in lean phenotypes in mice. In this study we compared the effects of these two mutations on growth and body weight in C57BL/6J mice. We observed reduced body weight and lower fat mass in both Rcan2(-/-) and Rps6kb1(-/-) mice compared to the wild-type mice, and we reported other differences unique to either the Rcan2(-/-) or Rps6kb1(-/-) mice. Firstly, loss of Rcan2 does not directly alter body length; however, Rcan2(-/-) mice exhibit reduced food intake. In contrast, Rps6kb1(-/-) mice exhibit abnormal embryonic development, which leads to smaller body size and reduced food intake in adulthood. Secondly, when fed a normal chow diet, Rcan2(-/-) mice weigh significantly more than Rps6kb1(-/-) mice, but both Rcan2(-/-) and Rps6kb1(-/-) mice develop similar amounts of epididymal fat. On a high-fat diet, Rcan2(-/-) mice gain body weight and fat mass at slower rates than Rps6kb1(-/-) mice. Finally, using the double-knockout mice (Rcan2(-/-) Rps6kb1(-/-)), we demonstrate that concurrent loss of Rcan2 and Rps6kb1 has an additive effect on body weight reduction in C57BL/6J mice. Our data suggest that Rcan2 and Rps6kb1 mutations both affect growth and body weight of mice, though likely through different mechanisms.

Keywords: Body weight regulation; Growth; Obesity; Rcan2 gene; Rps6kb1 gene.

Fig. 1

Expression of Rps6kb1 protein in Rcan2 ^−/− mice and expression of Rcan2 mRNA in Rps6kb1 ^−/− mice (a) Expression of Rps6kb1 protein in Rcan2 ^−/− mice. Total protein was isolated from hypothalami of WT, Rcan2 ^+/−, and Rcan2 ^−/− mice and subjected to Western-blot probing for Rps6kb1 and β-actin (internal control). Genotyping of Rcan2 is shown for representative animals (top). (b) Expression of Rps6kb1 protein in Rps6kb1 ^−/− mice. (c) Expression levels of Rcan2-1 and Rcan2-3 mRNA in Rps6kb1 ^−/− mice. Total RNA was isolated from hypothalami of WT and Rps6kb1^−/−mice and subjected to real-time quantitative PCR analysis using primers specific for Rcan2-1, Rcan2-3, and β-actin (internal standard). mRNA expression levels are reported as relative to expression from WT mice. n=4 in each group. All values are given as mean±SEM

Fig. 2

Phenotypes of WT, Rcan2 ^−/−, and Rps6kb1 ^−/− mice fed NCD (a) Growth curves of WT, Rcan2 ^−/−, and Rps6kb1 ^−/− mice fed NCD. Body weights of 13 Rcan2 ^−/−, 11 Rps6kb1 ^−/−, and 21 WT male mice were analyzed from postnatal Week 4 to Week 20. ^* P<0.05, ^** P<0.001, ^*** P<0.0001 (Rcan2 ^−/− vs. WT);^### P<0.0001 (Rps6kb1 ^−/− vs. WT); ⁺ P<0.05, ⁺⁺ P<0.001, ⁺⁺⁺ P<0.0001 (Rps6kb1 ^−/− vs. Rcan2 ^−/−). (b) Representative examples of 20-week-old WT, Rcan2 ^−/−, and Rps6kb1 ^−/− males. (c) Mean tibia lengths of 13 20-week-old WT, 7 Rcan2 ^−/− and 11 Rps6kb1 ^−/− males. ^*** P<0.0001. (d) Mean weight of epididymal white adipose tissue in 13 WT, 7 Rcan2 ^−/−, and 11 Rps6kb1 ^−/− males at 20 weeks of age. ^* P<0.05, ^** P<0.001. All values are given as mean±SEM

Fig. 3

Phenotypes of WT, Rcan2 ^−/−, and Rps6kb1 ^−/− mice fed HFD (a) Growth curves of WT, Rcan2 ^−/−, and Rps6kb1 ^−/− mice fed HFD. Body weights of 11 Rcan2 ^−/−, 9 Rps6kb1 ^−/−, and 12 WT males were analyzed from postnatal Week 4 to Week 20. ^* P<0.05, ^** P<0.001, ^*** P<0.0001 (Rcan2 ^−/− vs. WT); ^## P<0.001, ^### P<0.0001 (Rps6kb1 ^−/− vs. WT); ⁺ P<0.05, ⁺⁺ P<0.001, ⁺⁺⁺ P<0.0001 (Rps6kb1 ^−/− vs. Rcan2 ^−/−). (b) Representative examples of 20-week-old WT, Rcan2 ^−/− and Rps6kb1 ^−/− males. (c) Mean tibia lengths of 12 WT, 9 Rcan2 ^−/− and 8 Rps6kb1 ^−/− males at 20 weeks of age. ^*** P<0.0001. (d) Mean weight of epididymal and retroperitoneal white adipose tissue (EWAT and RWAT, respectively) and liver in 12 20-week-old WT, 9 Rcan2 ^−/−, and 8 Rps6kb1 ^−/− males. ^* P<0.05, ^** P<0.001, ^*** P<0.0001. (e) Biweekly food intake measured from postnatal Week 5 to Week 20 in 4 WT, 4 Rcan2 ^−/−, and 6 Rps6kb1 ^−/− mice. ^* P<0.05, ^** P<0.001 (Rcan2 ^−/− vs. WT); ^# P<0.05, ^## P<0.001 (Rps6kb1 ^−/− vs. WT). (f) Cumulative food intake measured from postnatal Week 4 to Week 20 in 4 WT, 4 Rcan2 ^−/−, and 6 Rps6kb1 ^−/− mice. ^*** P<0.0001. All values are given as mean±SEM

Fig. 4

Histological analysis of epididymal white adipose tissue (EWAT) and liver (a) EWAT morphology. Rcan2 ^−/−, Rps6kb1 ^−/−, and WT mice were fed either NCD or HFD from postnatal Week 4 to Week 20; animals were humanely sacrificed and histological analysis was performed on EWAT samples. Representative H & E stains of EWAT from each of the six treatment groups are presented. Arrows indicate the presence of crown-like structures in the HFD-fed WT and Rps6kb1 ^−/− mice. Scale bar: 100 μm. (b) Average size of adipocytes. Adipocyte size was determined using ImageJ software. Statistics were performed in each diet group using one-way ANOVA, and individual group differences were measured using Bonferroni correction. ^*** P<0.0001. All values are given as mean±SEM. (c) Liver histology. Rcan2 ^−/−, Rps6kb1 ^−/−, and WT mice were fed either NCD or HFD from postnatal Week 4 to Week 20; animals were humanely sacrificed and histological analysis was performed on liver samples. Representative micrographs of H & E-stained liver sections demonstrate much more severe steatosis in livers of HFD-fed WT mice than Rps6kb1 ^−/− mice, while Rcan2 ^−/− mice did not suffer hepatic steatosis, even on HFD. Scale bar: 100 μm

Fig. 5

A pair-feeding study between WT and Rcan2 ^−/− mice on HFD (a) Growth curves of the WT and Rcan2 ^−/− mice after a 16-week pair-feeding study. (b) Mean weight gain measured from postnatal Week 4 to Week 20 in WT and Rcan2 ^−/− mice. (c, d) Mean weights of epididymal and retroperitoneal white adipose tissue (EWAT and RWAT, respectively; c) and of liver (d) in WT and Rcan2 ^−/− mice after pair-feeding for 16 weeks. n=4 animals for each groups. All values are given as mean±SEM

Fig. 6

Phenotypes of double-mutant (Rcan2 ^−/− Rps6kb1 ^−/−) mice (a) Growth curves of Rps6kb1 ^−/− and double-mutant mice fed NCD. The body weights of 8 Rps6kb1 ^−/− and 8 double-mutant males were measured from postnatal Week 4 to Week 20. ^* P<0.05, ^** P<0.001. (b) Mean tibia lengths from 8 Rps6kb1 ^−/− and 8 double-mutant males. (c, d) Weights of epididymal and retroperitoneal white adipose tissue (EWAT and RWAT, respectively; c) and of liver (d) from 8 Rps6kb1 ^−/− and 8 double-mutant males. ^* P<0.05, ^** P<0.001. All values are given as mean±SEM