Inhibition of Plasminogen Activator Inhibitor-1 Activation Suppresses High Fat Diet-Induced Weight Gain via Alleviation of Hypothalamic Leptin Resistance

Abstract

Leptin resistance is an important mechanism underlying the development and maintenance of obesity and is thus regarded as a promising target of obesity treatment. Plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue-type and urokinase-type plasminogen activators, is produced at high levels in adipose tissue, especially in states of obesity, and is considered to primarily be involved in thrombosis. PAI-1 may also have roles in inter-organ tissue communications regulating body weight, because PAI-1 knockout mice reportedly exhibit resistance to high fat diet (HFD)-induced obesity. However, the role of PAI-1 in body weight regulation and the underlying mechanisms have not been fully elucidated. We herein studied how PAI-1 affects systemic energy metabolism. We examined body weight and food intake of PAI-1 knockout mice fed normal chow or HFD. We also examined the effects of pharmacological inhibition of PAI-1 activity by a small molecular weight compound, TM5441, on body weight, leptin sensitivities, and expressions of thermogenesis-related genes in brown adipose tissue (BAT) of HFD-fed wild type (WT) mice. Neither body weight gain nor food intake was reduced in PAI-1 KO mice under chow fed conditions. On the other hand, under HFD feeding conditions, food intake was decreased in PAI-1 KO as compared with WT mice (HFD-WT mice 3.98 ± 0.08 g/day vs HFD-KO mice 3.73 ± 0.07 g/day, P = 0.021), leading to an eventual significant suppression of weight gain (HFD-WT mice 40.3 ± 1.68 g vs HFD-KO mice 34.6 ± 1.84 g, P = 0.039). Additionally, TM5441 treatment of WT mice pre-fed the HFD resulted in a marked suppression of body weight gain in a PAI-1-dependent manner (HFD-WT-Control mice 37.6 ± 1.07 g vs HFD-WT-TM5441 mice 33.8 ± 0.97 g, P = 0.017). TM5441 treatment alleviated HFD-induced systemic and hypothalamic leptin resistance, before suppression of weight gain was evident. Moreover, improved leptin sensitivity in response to TM5441 treatment was accompanied by increased expressions of thermogenesis-related genes such as uncoupling protein 1 in BAT (HFD-WT-Control mice 1.00 ± 0.07 vs HFD-WT-TM5441 mice 1.32 ± 0.05, P = 0.002). These results suggest that PAI-1 plays a causative role in body weight gain under HFD-fed conditions by inducing hypothalamic leptin resistance. Furthermore, they indicate that pharmacological inhibition of PAI-1 activity is a potential strategy for alleviating diet-induced leptin resistance in obese subjects.

Keywords: arcuate nucleus; food intake; leptin resistance; obesity; plasminogen activator inhibitor-1; thermogenesis.

Figure 1

Time courses of body weight changes, food intake, and oral glucose tolerance tests in PAI-1 knockout (KO) mice fed chow or the safflower oil-rich high fat diet (HFD). (A) Daily food intake of chow-KO and chow-WT mice from the 1st day through the 5th day of observation. Each bar represents the mean ± SE of n = 6 mice. (B) Body weight of chow fed PAI-1 KO mice (chow-KO) and wild-type (WT) control mice (chow-WT). Eight-week-old mice were weighed weekly. Each bar represents the mean ± SE of n = 6 mice. (C) Daily food intake of chow-KO and chow-WT mice from the 71st day through the 75th day of observation. Each bar represents the mean ± SE of n = 6 mice. (D) Body weight of safflower oil-rich high fat diet (HFD) fed PAI-1 KO mice (HFD-KO) and WT control mice (HFD-WT). Eight-week-old mice were fed safflower oil-rich HFD and then were weighed weekly. Each bar represents the mean ± SE of n = 8 mice. (E) Daily food intake of HFD-KO and HFD-WT mice from the 1st day through the 21st day of observation. Each bar represents the mean ± SE of n = 9 mice. (F) Average food intake of HFD-KO and HFD-WT mice from the 1st day through the 21st day of observation. Each bar represents the mean ± SE of n = 9 mice. (G) Blood glucose levels and (H) plasma insulin levels during oral glucose tolerance tests performed 21 days after the initiation of safflower oil-rich HFD feeding. Results are expressed as mean ± SE of n = 8 mice. ^*P < 0.05 and ^**P < 0.01.

Figure 2

Body weight and oral glucose tolerance tests in lard-rich HFD fed mice treated with TM5441. (A) Body weight of WT mice fed safflower oil-rich HFD or lard-rich HFD. Each bar represents the mean ± SE of n = 5 mice. (B) Body weight of lard-rich HFD-fed mice treated with TM5441. Ten-week-old WT mice that had been pre-fed the lard-rich HFD for 2 weeks were given either lard-rich HFD mixed with TM5441 (HFD-WT-TM5441 mice) or lard-rich HFD (HFD-WT-Control mice) and then were weighed weekly. Each bar represents the mean ± SE of n = 10 mice. (C) Liver weight of HFD-WT-TM5441 and HFD-WT-Control mice. Each bar represents the mean ± SE of n = 9 mice. (D) Representative hematoxylin eosin (HE) staining of liver sections of HFD-WT-TM5441 and HFD-WT-Control mice (original magnification: 20×, scale bars: 100 μm). (E) Epididymal white adipose tissue (WAT) weight of HFD-WT-TM5441 and HFD-WT-Control mice. Each bar represents the mean ± SE of n = 9 mice. (F) Representative HE staining of WAT sections of HFD-WT-TM5441 and HFD-WT-Control mice (original magnification: 20×, scale bars: 100 μm). (G) Adipocyte size of HFD-WT-TM5441 and HFD-WT-Control mice. Each bar represents the mean ± SE of n = 9 mice. (H) Locomotor activities of HFD-WT-TM5441 and HFD-WT-Control mice during light hours and dark hours. Each bar represents the mean ± SE of n = 7 mice. (I) Blood glucose levels and (J) plasma insulin levels during oral glucose tolerance tests performed 35 days after the initiation of lard-rich HFD feeding. Results are expressed as mean ± SE of n = 9 mice. ^*P < 0.05 and ^**P < 0.01.

Figure 3

Body weight of lard-rich HFD fed PAI-1 knockout (KO) mice treated with TM5441. (A) Body weight of lard-rich HFD-fed PAI-1 KO mice treated with TM5441. Ten-week-old PAI-1 KO mice that had been pre-fed the lard-rich HFD for 2 weeks were given either lard-rich HFD mixed with TM5441 (HFD-KO-TM5441 mice) or lard-rich HFD (HFD-KO-Control mice) and then were weighed weekly. Each bar represents the mean ± SE of n = 9 mice. (B) Liver weight of HFD-KO-TM5441 and HFD-KO-Control mice. Each bar represents the mean ± SE of n = 9 mice. (C) WAT weight of HFD-KO-TM5441 and HFD-KO-Control mice. Each bar represents the mean ± SE of n = 9 mice.

Figure 4

Evaluation of leptin sensitivity in lard-rich HFD fed mice treated with TM5441. (A) Plasma leptin levels of HFD-WT-TM5441 mice and HFD-WT-Control mice. Results are expressed as mean ± SE of n = 9 mice. (B) Body weight of HFD-WT-TM5441-Leptin mice, HFD-WT-Control-Leptin mice, HFD-WT-TM5441-Saline mice, and HFD-WT-Control-Saline mice. Ten-week-old WT mice that had been pre-fed the lard-rich HFD for 2 weeks were given either lard-rich HFD mixed with TM5441 or lard-rich HFD for 1 week. Results are expressed as mean ± SE of n = 4 mice. (C) Body weight changes during leptin treatment. Mice were given a daily intraperitoneal injection of either leptin (HFD-WT-TM5441-Leptin mice or HFD-WT-Control-Leptin mice) or saline (HFD-WT-TM5441-Saline mice or HFD-WT-Control-Saline mice) for 5 consecutive days. Body weight immediately before the first leptin injection was subtracted from body weight 24 h after the last injection. Results are expressed as mean ± SE of n = 4 mice. (D) Body weight of HFD-WT-TM5441-Leptin mice, HFD-WT-Control-Leptin mice, HFD-WT-TM5441-Saline mice, and HFD-WT-Control-Saline mice. Ten-week-old WT mice that had been pre-fed the lard-rich HFD for 2 weeks were given either lard-rich HFD mixed with TM5441 or lard-rich HFD for 1 week. Results are expressed as mean ± SE of n = 4 mice. (E) Relative mRNA expressions of agouti-related peptide (agrp), neuropeptide y (npy), and proopiomelanocortin (Pomc) in the hypothalamic arcuate nucleus (ARC). Mice were fasted for 24 h and then given a single intraperitoneal injection of either leptin (HFD-WT-TM5441-Leptin mice or HFD-WT-Control-Leptin mice) or saline (HFD-WT-TM5441-Saline mice or HFD-WT-Control-Saline mice). Brain samples were harvested 4 h after the injection. Results are expressed as mean ± SE of n = 4 mice. (F) Relative mRNA expressions of uncoupling protein 1 (ucp1), peroxisome proliferative activated receptor gamma coactivator 1 alpha (pgc-1a), type II iodothyronine deiodinase (dio2), cell death-inducing DNA fragmentation factor alpha subunit-like effector A (cidea), and PR domain containing 16 (prdm16) in brown adipose tissue (BAT). Ten-week-old WT mice that had been pre-fed the lard-rich HFD for 2 weeks were given either lard-rich HFD mixed with TM5441 or lard-rich HFD for 1 week. Results are expressed as mean ± SE of n = 9 mice. ^*P < 0.05 and ^**P < 0.01.