Effect of ADAMTS5 deficiency on diet-induced obesity.

<p>(A-C) Expression of <i>Adamts5</i> (A disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5), <i>Ucp-1</i> (Uncoupling protein-1) and <i>Cidea</i> in subcutaneous (s) white adipose tissue (WAT) of ADAMTS5-J mice (n = 5–6). Data are corrected for the housekeeping gene <i>β-actin</i> and normalized to wild-type (WT) mice (+/+). Data are means ± SEM of n determinations. (D) Western blot analysis of UCP-1 protein levels in sWAT of <i>Adamts5</i>^+/+-P (n = 2), <i>Adamts5</i>^-/--P (-/-; n = 1), <i>Adamts5</i>^+/+-J (n = 5) and <i>Adamts5</i>^-/--J (-/-; n = 5) mice. The expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.</p

Effect of ADAMTS5 deficiency on diet-induced obesity.

<p>(A-C) Expression of <i>Adamts5</i> (A disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5), <i>Ucp-1</i> (Uncoupling protein-1) and <i>Cidea</i> in subcutaneous (s) white adipose tissue (WAT) of ADAMTS5-J mice (n = 5–6). Data are corrected for the housekeeping gene <i>β-actin</i> and normalized to wild-type (WT) mice (+/+). Data are means ± SEM of n determinations. (D) Western blot analysis of UCP-1 protein levels in sWAT of <i>Adamts5</i>^+/+-P (n = 2), <i>Adamts5</i>^-/--P (-/-; n = 1), <i>Adamts5</i>^+/+-J (n = 5) and <i>Adamts5</i>^-/--J (-/-; n = 5) mice. The expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.</p

Functional role of ADAMTS5 in adiposity and metabolic health

<div><p>Previous studies with gene-deficient mice (ADAMTS5-P) revealed that ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs, member 5) plays a functional role in adiposity and metabolic health. To confirm these observations, we have performed similar studies with an independently generated strain of ADAMTS5 deficient mice (ADAMTS5-J). Upon cold exposure as well as after high-fat diet feeding (diet-induced obesity or DIO model), these knockout (KO) mice developed less subcutaneous and gonadal white adipose tissue (WAT) as compared to their wild-type (WT) littermates (reduction was more pronounced in ADAMTS5-P mice). Enhanced browning of WAT, as monitored by expression of UCP-1 was seen in the ADAMTS5-J KO mice upon cold exposure but not in the DIO model (seen in both conditions with the ADAMTS5-P mice). Brown adipose tissue (BAT) mass was not different between KO and WT ADAMTS5-J mice, either upon cold exposure or in the DIO model (in contrast to the enhanced BAT mass with the ADAMTS5-P mice). Energy expenditure and thermogenesis were not significantly different between KO and WT ADAMTS5-J mice (in contrast to somewhat enhanced levels in ADAMTS5-P mice). Insulin sensitivity was improved in the ADAMTS5-J KO mice, and they were protected against non-alcoholic steatohepatitis in the DIO model (as the ADAMTS5-P mice). These data are thus similar for both strains of KO mice, confirming specificity of the phenotype, but some quantitative and qualitative differences are also observed.</p></div

Effect of ADAMTS5 genotype on AT after cold exposure for 2 weeks.

<p>(A) Body weight of ADAMTS5-J WT (+/+; n = 7) or homozygous deficient (-/-; n = 9) mice at start and end of the experiment. (B) Mass of sWAT, GON AT and BAT for WT (white bars; n = 8) and <i>Adamts5</i>^-/--J mice (black bars; n = 9). (C) Gene expression levels of markers of browning of sWAT, normalized to the housekeeping gene <i>Tbp</i> and shown relative to WT (n = 7–8). (D) sWAT western blots for UCP-1 (n = 7–8) and PGC1α (n = 7–9), and quantitation by densitometry, normalized to GAPDH and shown relative to WT. (E) sWAT sections of WT (+/+) and <i>Adamts5</i>^-/- -J mice were stained with haematoxylin & eosin (upper panel) or with antibodies directed against UCP-1 (lower panel; brown color) to illustrate browning of sWAT. Magnification: 400x. Scale bar = 10 μm. Data are means ± SEM of n determinations. * p<0.05, ** p<0.01 <i>versus</i> WT mice according to the non-parametric Mann-Whitney U test. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; WAT, white adipose tissue; UCP-1, uncoupling protein-1; s, subcutaneous; WT, wild-type; GON, gonadal; BAT, brown adipose tissue; <i>Tbp</i>, TATA-box binding protein; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-α and GAPDH, glyceraldehyde 3-phosphate.</p

Characterization of ADAMTS5-P and ADAMTS5-J mice.

<p>Genotyping of wild-type (+/+), heterozygous (+/-) and homozygous (-/-) deficient ADAMTS5-P and ADAMTS5-J mice. L represents the basepair (bp) ladder. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5.</p

Genotype distribution of littermates obtained by interbreeding heterozygous deficient ADAMTS5 mice.

<p>Genotype distribution of littermates obtained by interbreeding heterozygous deficient ADAMTS5 mice.</p

Histological analysis of liver sections of WT (+/+) and <i>Adamts5</i>^-/--J (-/-) mice kept on HFD for 15 weeks.

<p>(A) Representative Haematoxylin & Eosin (H&E) (left) and Sirius Red (right) stainings. Magnification: 200x. The white arrows indicate foci of inflammation in the H&E stainings and sinusoidal fibrosis in the Sirius Red stainings. (B) Histological scoring of steatosis, hepatocyte ballooning, inflammation, fibrosis and non-alcoholic fatty liver (NALF) activity score (NAS). (C) Disease severity distribution, as number of mice without or with NALF/NASH. Data are means ± SEM of 6 (WT) or 7 (<i>Adamts5</i>^-/--J) experiments for panels B and C. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; WT, wild-type; HFD, high-fat diet and NASH, non-alcoholic steatohepatitis.</p

Effect of ADAMTS5 genotype on AT during cold exposure for 72 h.

<p>(A, B) Gene expression levels of <i>Ucp-1</i> (A) and <i>Cidea</i> (B) in sWAT of WT (+/+) and homozygous deficient (-/-) mice from the ADAMTS5-P (+/+: n = 10; -/-: n = 8) and ADAMTS5-J strains (+/+: n = 11; -/-: n = 7). Gene expression levels are normalized to the housekeeping gene <i>Tbp</i> and shown relative to WT mice at +4°C. (C,D) UCP-1 protein levels in sWAT. The Western blots (C) are quantitated by densitometry, normalized to β-actin and expressed relative to WT (D). (E-G) Relative ADAMTS5 gene expression in sWAT (E), GON AT (F) and BAT (G) of WT mice kept at 24°C or at 4°C. Gene expression levels are normalized to the housekeeping gene <i>Tbp</i> and shown relative to WT mice at 24°C. Data are means ± SEM of 4–8 and 5–11 determinations for panel D and panel E-G, respectively. ** p<0.01, *** p<0.001 versus 24°C according to the non-parametric Mann-Whitney U test. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; WAT, white adipose tissue; UCP-1, uncoupling protein-1; s, subcutaneous; WT, wild-type; GON, gonadal; BAT, brown adipose tissue and <i>Tbp</i>, TATA-box binding protein.</p

Effect of ADAMTS5 deficiency on metabolic parameters of mice kept on a high-fat diet (HFD) for 15 weeks.

<p>Effect of ADAMTS5 deficiency on metabolic parameters of mice kept on a high-fat diet (HFD) for 15 weeks.</p

Indirect calorimetry with ADAMTS5-J mice after 10 weeks of HFD feeding.

<p>(A) Body weight change of wild-type (white bars; +/+) and <i>Adamts5</i>^-/--J mice (black bars; -/-) over 72 h. (B,C) Food and water intake. (D,E) Oxygen consumption (VO2) and carbon dioxide production (VCO2). (F) Calculated respiratory exchange rate (RER). (G) Locomotive activity during dark and light phase. (H,I) Energy expenditure and heat production over 24 h. Data are means ± SEM of 3 determinations for all panels. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; HFD, high-fat diet.</p