Tissue Inhibitor of Metalloproteinase-3 Knockout Mice Exhibit Enhanced Energy Expenditure through Thermogenesis

<div><p>Tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme), its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO) mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT) mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.</p></div

Real-time PCR-based analysis of mitochondrial activity in soleus muscle of TIMP-3 KO and wild type mice.

<p>Expression of PGC-1α (A), PPAR-δ (B), PPAR-γ (C), UCP-2 (D), NRF-1 (E) and NRF-2 (F) in TIMP-3 knockout (KO) and wild type (WT) mice is presented as mean ± SD (n = 6–7/group). *p<0.05 ^†p<0.01.</p

Real-time PCR-based analysis of mitochondrial activity in gastrocnemius muscle of TIMP-3 KO and wild type mice.

<p>Expression of PGC-1α (A), PPAR-δ (B), PPAR-γ (C), UCP-2 (D), NRF-1 (E) and NRF-2 (F) in TIMP-3 knockout (KO) and wild type (WT) mice is presented as mean ± SD (n = 6–7/group). ^†p<0.01.</p

Metabolic parameters in TIMP-3 KO and wild type mice.

<p>Body temperature (A), body weight (B), food intake per body weight (C) and locomotor activity (D) in TIMP-3 knockout (KO) and wild type (WT) mice at 15 weeks or 8 months of age are presented as mean ± SD (n = 5–7/group). ^†p<0.01, ^‡p<0.001.</p

Cold exposure experiments in TIMP-3 KO and wild type mice.

<p>Mice at 15 weeks or 8 months of age were exposed to 4°C for 8 hours. Data are presented as mean ± SD (n = 5/group). *p<0.05 ^†p<0.01.</p

Western blot analysis of mitochondrial proteins in soleus muscle of TIMP-3 KO and wild type mice.

<p>Representative Western blot for each mitochondria-related gene (A), and average protein content of UCP-2 (B), NRF-1 (C), NRF-2 (D), VDAC (E) and COX-4 (F). Data are presented as mean ± SD (n = 3/group). *p<0.05.</p

Respiratory parameters in TIMP-3 KO and wild type mice.

<p>Oxygen consumption (VO₂) (A), carbon dioxide production (VCO₂) (B) and respiratory exchange ratio (RER) (C) in TIMP-3 knockout (KO) and wild type (WT) mice at 15 weeks or 8 months of age are presented as mean ± SD (n = 5–7/group). *p<0.05, ^†p<0.01.</p

Novel mutations in Patients 1 (P1) and 2 (P2).

<p>(A) Genomic DNA sequencing of exon 5 in the <i>BBS5</i> gene showed a C>T transition at the codon 89, resulting in arginine to stop (p.R89X). (B) RT-PCR revealed that an extra band with a shorter fragment in P1, P2, and their father (Fa), but not in normal control (C) or their mother (Mo). NC indicates no cDNA contained. (C) Sequencing of RT-PCR fragments showed that the shorter fragment lacked exon 8 with normal sequences in exon 5, while the normal-size fragment included exon 8, but had p.R89X mutation in exon 5. (D) Genomic DNA sequencing in exon 8 and surrounding introns in the <i>BBS5</i> gene showed IVS7-27 T>G mutation in the patients.</p

Clinical and genetic information on Japanese patients with Bardet-Biedl syndrome.

<p>*, left mild pyelectasis; BMI, body-mass index.</p><p>ST, strabismus; DC, dental crowding; CA, cardiac anomaly.</p

Protein analyses of BBS5.

<p>The immunoblot showed that Patients 1 (P1) and 2 (P2) had no full-length BBS5 protein, while control (C) had it. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.</p