| Background and Objective:Type 2 diabetes mellitus(T2DM) is a very common endocrine and metabolic disease, which accounts for 90% ~ 95% of all cases of diabetes mellitus(DM). It is caused by the interaction of genetic and environmental factors. In recent years, lifestyle changes further stimulate an explosive growth of T2 DM incidence, especially in China. Currently, China has the largest diabetes population in the world[1]. Insulin resistance is both the important characteristic and the pathogenesis for the onset of T2 DM. It is a condition that is often associated with central obesity which is a well established independent risk factor for the development of T2 DM, especially the visceral fat[2,3,4,5,6,7,8]. With the increase in fat mass, obese patients typically display an increasing amount of circulating leptin[9] and decreasing soluble leptin receptor(s LR) [10,11].Leptin, a hormone that is secreted from adipose tissue, shows multiple functions ranging from regulating food intake, glucose and lipid metabolism to immune inflammation[12]. Being the major leptin binding protein in plasma, s LR is the key regulator that modulates leptin’s biological functions[13]. Serum s LR levels are decreased in obese T2 DM patients compared to healthy individuals, and are inversely associated with plasma glucose, low-density lipoprotein(LDL) cholesterol and blood pressure[14]. Whereas, s LR are increased after weigh loss surgery, diet therapy or physical training[15,16,17], indicating that s LR may play a beneficial role in alleviating metabolic disturbance in obese T2 DM patients. In agreement, the previous studies in our lab showed that s LR increased in both high-fat diet(HFD)-induced insulin resistant mice and T2 DM patients that received metformin treatment[18]. Understanding the mechanisms for metformin in upregulating s LR may provide new approach for prevention obesity and its related metabolic disorders.We now know that s LR is generated by shedding of membrane-bound leptin receptor at its ectodomain region[19]. In human, there are four LR isoforms, namely LR-a, LR-b, LR-c and LR-d, all of which are encoded by the same leptin receptor gene that are generated via differential splicing after being transcribed into precursor m RNA during their maturation[20]. Among them, LRb is the only full-length isoform capable for mediating leptin signaling[21]. Other leptin receptor isoforms may also exist in rodents, such as membrane-bound LR-f and secretion isoform LR-e[22]. Liver shows the highest expression in leptin receptor, thus plays an important role in modulating plasma s LR levels[23,24]. In addition, kidney also shows abundant expression in leptin receptor[23]. So far, much less is known about the shedding enzymes for this receptor. A recent in vitro study showed that ADAM10(A Disintegrin And Metalloproteinase 10) and ADAM17 are involved in proteocleavage of leptin receptor[25]. In this study, we tested the effects of metformin on gene expression of major isoforms of membrane-bound leptin receptor(LRa,LRb,LRc,LRd)and its proteolytic enzymes(ADAM10 and ADAM17) in HFD-induced insulin resistanct mice. We also tested a dose-dependent effect of metformin on these gene expression at both m RNA and proteins levels. Materials and methods:Forty-two C57BL/6 mice with age of 6~8 weeks and body weight of 17.02±1.91 grams were randomly divided into 2 groups. One group were fed with standard chow diet(CD)(n=21) while the other with high-fat diet(HFD)(n=21) for up to 5 months.Then the mice were divided into 3 subgroups(n=7) in each group, and administrated with water, low dose of metformin as 50 mg/(kg.d) and high dose of metformin as 200 mg/(kg.d) via gastric lavage daily for 15 consecutive days, respectively [18]. Animals were then sacrificed, and liver and kidney tissues were stored in- 80 ℃ refrigerator for later use.Expressions of total leptin receptor(LRt), its major isoforms(LRa,LRb,LRc,LRd) and its shedding enzymes(ADAM10、ADAM17) in liver and kidney were then determined for m RNA by real-time PCR, and proteins by Western blot. Results:1. Metformin upregulates the expression of leptin receptor gene in liver tissue, but shows no effect in kidney tissue. In liver, the relative fold expression of LRt m RNA, compared to none-drug treated mice, dose-dependently inceased in both CD group [CD-W by promoting the expressions of its shedding enzymes(ADAM10 and ADAM17) gene or proteins in liver tissue. 3. Metformin up-regulates membrane-bound short isoforms of leptin receptors(LRa, LRc, LRd) in liver which may contribute to increased serum s LR. LRb is not important for s LR, but its up-regulation by high dose of metfomin could modulate leptin’s biological functions. vs CD-LM vs CD-HM, 1.04±0.34 vs 1.63±0.18 vs 5.26 ±0.89(P<0.01)] and HF group[HF-W vs HF-LM vs HF-HM, 2.25 ±0.10 vs 4.96±0.88(P<0.05) vs 9.11 ±1.33(P<0.01)].2. In liver, metformin-induced major isoforms of membrane-bound leptin receptor were LRa {CD group[1.02±0.24 vs 1.42±0.47 vs 2.92 ±0.68(P<0.05)];HF group[1.13 ±0.62 vs 2.80±0.18(P<0.05) vs 6.04 ±1.00(P<0.01)]}、LRc {CD group[1.00±0.06 vs 1.02±0.13 vs 1.97 ±0.25(P<0.01)];HF group[0.97±0.12 vs 2.60±0.18(P<0.01) vs 4.58 ±0.11(P<0.01)]}、LRd {[1.02±0.25 vs 0.69±0.27 vs 2.48 ±0.15(P<0.01)]; HF group[1.22±0.23 vs 2.55±0.16(P<0.01) vs 4.82±0.03(P<0.01)]}。3. Interestingly, liver expression of LRb m RNA demonstrated no obvious changes in CD group [1.02 ± 0.30 vs 0.69 ± 0.16 vs 0.32 ± 0.09], but increased significantly in high dose of metformin treatment in HF group [0.69 ± 0.26 vs 0.77 ± 0.15 vs 2.39 ± 0.59(P<0.01)].4. In kidney, metformin shows no significant effect on leptin receptor expressions: LRt [CD group(1.06±0.43 vs 1.03±0.32 vs 0.85 ±0.08);HF group(0.37 ±0.13 vs 0.36±0.17 vs 0.74 ±0.27)], LRa[CD group(1.05±0.45 vs 0.87±0.22 vs 0.36 ±0.07);HF group(0.56 ±0.21 vs 0.57±0.23 vs 0.57±0.08)], LRb {CD group[1.00±0.15 vs 0.58±0.16(P<0.01) vs 0.12±0.02(P<0.01)]; HF group(0.14±0.04 vs 0.08±0.05 vs 0.08±0.03)}, LRc[CD group(1.03±0.32 vs 1.05±0.16 vs 0.40 ±0.06);HF group(0.69±0.25 vs 0.67±0.21 vs 0.48 ±0.14)], LRd{CD group[1.11±0.52 vs 0.79±0.18 vs 0.44±0.14(P<0.05)]; HF group(0.28±0.07 vs 0.21±0.04 vs 0.67±0.13)}regardless of the food types.5. Metformin also shows no significant impact on ADAM10 m RNA [ liver:CD group(1.02±0.22 vs 1.06±0.14 vs 0.76 ±0.10);HF group(0.79 ±0.14 vs 0.82±0.07 vs 0.88 ±0.05)]{kidney:CD group[1.01±0.17 vs 0.95±0.16 vs 0.56 ±0.16(P<0.05)]; HF group(0.38 ±0.19 vs 0.23±0.04 vs 0.33 ±0.09)} and ADAM17 m RNA [liver:CD group(1.00±0.01 vs 1.07±0.07 vs 0.85±0.09); HF group(0.81 ±0.02 vs 0.82±0.06 vs 0.84 ±0.09)] {kidney: CD group[1.02±0.29 vs 1.00±0.08 vs 0.42±0.07(P<0.05)];HF group(0.40±0.18 vs 0.21±0.06 vs 0.40 ±0.16)}or proteins levels no matter in liver or kidney tissue. Conclusion:1. Metformin up-regulates the expression of LR gene in liver but not in kidney tissue.2. Metformin up-regulates serum s LR levels by promoting LR gene expression but not... |
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