Cathepsin K null mice show reduced adiposity during the rapid accumulation of fat stores

Abstract

Growing evidences indicate that proteases are implicated in adipogenesis and in the onset of obesity. We previously reported that the cysteine protease cathepsin K (ctsk) is overexpressed in the white adipose tissue (WAT) of obese individuals. We herein characterized the WAT and the metabolic phenotype of ctsk deficient animals (ctsk-/-). When the growth rate of ctsk-/- was compared to that of the wild type animals (WT), we could establish a time window (5-8 weeks of age) within which ctsk-/-display significantly lower body weight and WAT size as compared to WT. Such a difference was not observable in older mice. Upon treatment with high fat diet (HFD) for 12 weeks ctsk-/- gained significantly less weight than WT and showed reduced brown adipose tissue, liver mass and a lower percentage of body fat. Plasma triglycerides, cholesterol and leptin were significantly lower in HFD-fed-ctsk-/- as compared to HFD-fed WT animals. Adipocyte lipolysis rates were increased in both young and HFD-fed-ctsk-/-, as compared to WT. Carnitine palmitoyl transferase-1 activity, was higher in mitochondria isolated from the WAT of HFD treated ctsk-/- as compared to WT. Together, these data indicate that ctsk ablation in mice results in reduced body fat content under conditions requiring a rapid accumulation of fat stores. This observation could be partly explained by an increased release and/or utilization of FFA and by an augmented ratio of lipolysis/lipogenesis. These results also demonstrate that under a HFD, ctsk deficiency confers a partial resistance to the development of dyslipidemia.

Figure 1. Young C57Bl6 ctsk −/− mice display reduced body weight.

Growth curves from 10 to 120 days for male (A) and female (B) mice (n=12 for males and n=9 for females for each genotype. Statistical details for Bonferroni post‐tests: males df=22, t=2.15 for day 32, t=3.54 for day 42, t=2.08 for day 56; females df=16, t=2.63 for day 21, t=2.81 for day 32, t=2.14 for day 39, t=3.22 for day 56. * P<0.05 . (C) Scattergram showing the positive and significant correlation (Spearman regression analysis) (P<0.05) between MRI data, expressed as (fat volume)/(total body volume), and the ratio fat pad/body weight of 10 animals.

Figure 2. WT and ctsk −/− mice belonging to 2 independent colonies of mice (respectively bred on the mixed sv129/C57Bl6 and pure C57Bl6 genetic background) were fed a high‐fat diet (HFD) starting at 8–9 weeks of age.

(A) Representative graph showing food intake in C57Bl6 male mice (n=10). Food intake was monitored weekly in the two groups studied (see text for further explanations) and no significant differences between genotypes were observed. (B) Graphs showing the increase in body weight with time for the sv129/C57Bl6 females and for the C57Bl6 males (n=10 for both groups). The increases in body weight were calculated based on the initial body weight at day 0 of HFD feeding. (C) Weights of epididymal WAT, perirenal WAT and BAT, taken from 5 month C67Bl6 old male mice (n=9 for ctsk−/− and for WT) after 12 weeks of HFD. Results are mean ± SEM * p<0.05, ** p<0.01, ***p<0.001. (D) Representative histological sections of epididymal WAT from C57Bl6 ctsk+/+ (left panel) and ctsk−/− (right panel) mice maintained on a high fat diet for 12 weeks.

Figure 3. FFA utilization and release in ctsk +/+ and ctsk −/− mice.

(A) CPT activity of WAT mitochondria from HFD‐fed C67Bl6 WT and ctsk−/− mice. Results are the mean ± SEM for 4 WT and 3 male ctsk−/− mice (40 days‐old) p<0.03 compared to WT mice. (B) Glycerol release was measured in adipocytes isolated from the visceral adipose tissue of C57Bl6 WT and ctsk−/− mice maintained in basal medium (ctr), or stimulated for 1 h with 1 µM isoproterenol (iso). Top and bottom panels show respectively the results obtained with young male animals (44 days‐old) and with male animals maintained on HFD for 12 weeks. Results are the mean ± SEM for 4 independent experiments. * p<0.05, ** p<0.01.

Figure 4. Ctsk deficiency interferes with adipogenesis.

(A) Adipocyte differentiation from mouse embryo fibroblasts of C57Bl6 wild type (WT) and ctsk−/− mice (KO). Mouse embryo fibroblasts were prepared 12 days post-coitum and were induced to differentiate in adipocytes. At 10 days after the addition of adipocyte differentiation media we performed oil red O staining (10 X magnification). (B) Quantification of aP2, leptin and LPL mRNA expression after real time PCR in WT (black bars) and ctsk−/− (white bars) MEF. Results are the mean ± SEM for 4 independent experiments. Statistical details for aP2, leptin and LPL are respectively: t = 9.15, t = 2.96 and t = 4.26, df = 5 in all cases. * p<0.05, **p<0.01, ***p<0.001 versus WT.

Figure 5. Hypothetical scheme representing how ctsk ablation in mice may result in partial resistance to increase adipose mass in conditions requiring rapid accumulation of fat stores.

In wt mice (intact genome, left side of the panel) ctsk participates to WAT extra-cellular matrix remodelling, thus facilitating the enlargement of the adipose mass, which implies adipocyte size increase and de novo differentiation of pre-adipocytes. Mechanisms involving an intracellular action of ctsk are also hypothesized. In ctsk deficient animals (right side of the panel) stimuli directed to increase the adipose mass are partially blunted and the extra caloric intake is partly utilized in a futile cycle, consisting of increased synthesis of triglycerides, increased triglycerides hydrolysis and increased β-oxidation of FFA.