Theophylline-Based KMUP-1 Improves Steatohepatitis via MMP-9/IL-10 and Lipolysis via HSL/p-HSL in Obese Mice

Abstract

KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) has been reported to cause hepatic fat loss. However, the action mechanisms of KMUP-1 in obesity-induced steatohepatitis remains unclear. This study elucidated the steatohepatitis via matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNFα), and related lipolysis via hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) by KMUP-1. KMUP-1 on steatohepatitis-associated HSL/p-HSL/ATGL/MMP-9/TNFα/interleukin-10 (IL-10) and infiltration of M1/M2 macrophages in obese mice were examined. KMUP-1 was administered by oral gavage from weeks 1-14 in high-fat diet (HFD)-supplemented C57BL/6J male mice (protection group) and from weeks 8-14, for 6 weeks, in HFD-induced obese mice (treatment group). Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of tissues, oil globules number and size, infiltration and switching of M1/M2 macrophages were measured to determine the effects on livers. IL-10 and MMP-9 proteins were explored to determine the effects of KMUP-1 on M1/M2 macrophage polarization in HFD-induced steatohepatitis. Long-term administration of KMUP-1 reversed HFD-fed mice increased in body weight, sGOT/sGPT, triglyceride (TG) and glucose. Additionally, KMUP-1 decreased MMP-9 and reactive oxygen species (ROS), and increased HSL/p-HSL and IL-10 in HFD mice livers. In conclusion, KMUP-1, a phosphodiesterase inhibitor (PDEI), was shown to reduce lipid accumulation in liver tissues, suggesting that it could be able to prevent or treat steatohepatitis induced by HFD.

Keywords: M1/M2 macrophage; adipose triglyceride lipase; fatty liver; hormone sensitive lipase; matrix metallopeptidase 9; tumor necrosis factor α.

Figure 1

Effects of KMUP-1 on high-fat diet (HFD)-induced body weight, serum glutamic-oxaloacetic transaminase (sGOT), serum glutamic-pyruvic transaminase (sGPT), triglyceride (TG), and glucose in serum of mice treated with KMUP-1. (A) Protection/Treatment protocol in the mice model; (B) Oral administration of KMUP-1 (2.5 mg/kg/day for 14 weeks) prevented HFD-induced body weight increase; (C) Treatment with KMUP-1 (1, 2.5, 5 mg/kg/day for last 6 weeks) attenuated HFD-induced body weight gain from week 8 to week 14; (D) HFD-induced sGOT, sGPT, TG and glucose levels were reduced by KMUP-1. Data are means ± Standard error (S.E.), n = 6–8. * p < 0.05; ** p < 0.01; *** p < 0.001 versus HFD group.

Figure 2

Morphology of livers protected or treated with KMUP-1 in HFD mice shown by H&E staining and the diameter of oil globules measured. (A) Normal morphology of livers in mice fed with normal chow diet (ND) for 14 weeks; (B) Mice fed with HFD for 14 weeks induced relevant fatty liver; (C,D) represent the Treatment and Protection of liver changes by oral gavage of KMUP-1 (2.5 mg/kg/day) for 14 weeks in HFD mice; (E) Excessive oil globules in HFD liver at 14 weeks; (F) Treatment and (G) protection of fatty livers by KMUP-1 for 6 weeks and 14 weeks, respectively; (H) Large amount of Mallory’s hyaline bodies (purple dots indicated by black arrow) were observed in the HFD group; (I) Obese animal treated with KMUP-1 (2.5 mg/kg/day) for 6 weeks decreased the worst pathologic changes at 14 weeks, i.e., the oil globules and Mallory’s hyaline bodies were attenuated at 14 weeks; and this response was more prominent in the (J) protection group; (K) shows H&E staining of the liver from normal chow diet (ND) mice as a control; (L) A representative example for measuring of hepatic oil globules from Figure 2F. The standardized diameters of 10, 25, 50 and 100 µm are depicted.

Figure 3

Immunohistochemistry (IHC) staining of tumor necrosis factor α (TNFα)/matrix metallopeptidase 9 (MMP-9), hormone sensitive lipase (HSL)/phosphorylated HSL (p-HSL) and adipose triglyceride lipase (ATGL) in HFD-induced liver steatosis and oil globules protected/treated with KMUP-1 for 14 weeks/6 weeks. HFD-induced fatty liver at 14 weeks implied that oil globules were rich in liver tissues. The expression of HFD-induced TNFα (A) and MMP-9 (B) in the treatment and protection groups; Treatment and/or protection with KMUP-1 sharply reduced the number and diameter of oil globules (G,H); HFD-induced the expression of HSL/p-HSL showed that KMUP-1 could affect the HSL protein (brown, C); and significantly enhanced the active form of HSL (p-HSL) in the protection group (deep brown, D) and matched data regarding the number and diameter of oil globules depicted in (I,J); ATGL expression is not affected by KMUP-1 in both treatment and protection groups (E); but markedly attenuated the number and diameter of oil globules (K); (F,L) negative control of protein expression, and the number and diameter of oil globules. Data are means ± S.E. of three independent experiments. *** p < 0.001 versus HFD group.

Figure 4

IHC staining of infiltrated M1/M2 macrophages and accumulated oil globules treated/protected with KMUP-1. KMUP-1 (2.5 mg/kg/day) administration for 6 weeks/14 weeks modulates the balance of infiltrated macrophages 1 (M1; A,B) and macrophages 2 (M2; C,D). KMUP-1 significantly affected the M1 (CD11c, dark brown)/M2 (CD209a, dark brown) macrophages polarization, but little affected the balance of M1 (F4/80, brown) and M2 (CD206, brown), in treatment and protection groups. All the accompanied oil globules were reduced by KMUP-1. The number and diameter changes of oil globules are in the average from M1/M2-positive cells (E–H). Data are means ± S.E. of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus HFD group. Scale bar: 100 µm.

Figure 5

Expression of IL-10 and MMP-9 in mice livers treated with KMUP-1. Treatment/protection with KMUP-1 (2.5 mg/kg/day) for 6 weeks/14 weeks increased the expression of IL-10 (A) and decreased the expression of MMP-9 (B) in mice livers. The protein expression of IL-10 and MMP-9 was described in Materials and Methods. Data are means ± S.E. of six independent experiments. ** p < 0.01 versus HFD group.

Figure 6

The levels of hepatic ROS was reduced by treating KMUP-1 (1, 2.5 mg/kg/day). HFD induced accumulation of ROS in livers. Protection/treatment with KMUP-1 for 6 weeks/14 weeks decreased the hepatic ROS. ROS was determined as described in Materials and Methods. Data are means ± S.E. of six independent experiments. * p < 0.05; ** p < 0.01 versus HFD group. ND: normal chow diet.