| Background and Objective:Soluble leptin receptor (sOB-R) is the product of proteolysis of membrane leptinreceptors at its extracellular region. As the major leptin binding protein in circulation,sOB-R is important in regulation of the free/bound leptin index, thus plays a crucial role inregulating the bioavailability and physiological function of leptin. Accumulatingepidemiological studies show that level of sOB-R in plasma is negatively correlated todisease states associated with abnormal leptin levels, such as Obese, T2DM and colorectalcancer. In a prospective investigation into the association of sOB-R with risk of metabolicdisorders in a healthy population, it has been showed that plasma sOB-R level is inverselyassociated with several important metabolic factors known to be involved in etiology ofmetabolic syndromes, such as fasting plasma glucose(FPG), LDL-lipoprotein, cholesteroland blood pressure, and is positively correlated to HDL-lipoprotein. Studies show thatsOB-R levels increased in obeses subjects with low calorie diet.In a prospective studyobserving effects of weight loss on18morbidly obese women one year after adjustablegastric banding surgery, it has been demonstrated that leptin levels decreased, whereassOB-R levels and the receptor bound fraction of leptin increased. Thus, the increased levelof sOB-R may negatively regulate free leptin and serve as a biomarker for an improvedmetabolic status in obese and T2DM patients.Though diet management and metabolic surgery are effective glucose-loweringtherapies, especially, for diabesity patients, the majority still need pharmaceutical therapy.As the first choice in oral diabetes treatment, the anti-diabetic drug metformin has beenreported to decrease plasma glucose by multiple mechanisms, such as promoting weightloss, decreasing hepatic glucose production, activating peripheral glucose utilization andalleviating insulin resistance. However, the relation of metformin treatment to plasmasOB-R levels in diabetic patients has so far not been determined. Therefore, this study explored the association of metformin treatment with plasma sOB-R levels in animalmodels as well as clinical patients. We have conducted the following works:(1) Firstly, we set up an animal model of impaired glucose tolerance (IGT) via feedinggenetically normal C57BL/6mice to a HF diet for5months with chow diet-fed mice as thecontrol. After measuring the FPG and performing the intraperitoneal glucose tolerance test(IPGTT) to confirm the establishment of IGT model, we started administration ofmetformin with a low (50mg/kg/d) and high dose (200mg/kg/d) for a continuous15days.The effects of metformin on FPG, glucose tolerance, and plasma sOB-R were thendetermined.(2) Next, we examined the effects of metformin on sOB-R levels on clinical patients.T2DM patients who have been freshly diagnosed as diabetes and accepted metformin(1.5g/d) treatment for a month. A follow-up visit were arranged and their plasma sampleswere collected. The influence of metformin on sOB-R level were then determined andcompared to its plasma level before using this drug in each individual patient.(3) In order to understand the underlying mechanism leading metformin treatment toan enhanced sOB-R production, we detected the influence of metformin on leptin receptorgene expression, particularly in mice liver, as well as in cultured hepatocytes (L02).Materials and methods：1. To set up an animal model with impaired glucose metabolism, C57BL/6mice, allmale, aged4–6weeks were randomized into two groups; one set of animals(n=21)receivedstandard laboratory chow and the other set (n=21)a high-fat (HF) diet. The mice were fedthe chow or HF diets for five months. All animals had free access to water. Body weight,FPG and glucose tolerance test were then measured.2. Afterwards, each group of mice was divided into3subgroups and gavaged withmetformin at50mg/kg/d or200mg/kg/d once a day for a continuous15days, with the salinegavaged mice as control. At the end of treatment, mice were fasted overnight beforesacrifice, and body weight, FPG、fasting serum insulin(Fins), HOMA-IR and sOB-R levelswere determined.3. Next, we examined the effects of metformin on sOB-R levels on clinical patients.T2DM patients who have been freshly diagnosed as diabetes and accepted metformin(1.5g/d) treatment for a month. Plasma samples were then collected during their follow-up visit. Leptin and sOB-R levels were determined.4. In order to understand the underlying mechanism leading metformin treatment to anenhanced sOB-R production, we detected leptin receptor gene expression, particularly inmice liver, as well as in cultured hepatocytes (L02) with real-time PCR.Results：1. The FPG levels were significantly increased in HF-fed mice after5months feedingcompared to chow-diet fed group (CD)(9.27±1.36vs6.87±0.15mmol/L).IPGTT showedsignificantly higher blood glucose concentrations at30,60and120minutes in HF micecompared to CD mice, indicating that impaired glucose tolerance (IGT) is induced by HFfeeding.2. In HF group, compared to none-drug-treated mice, administration of metformin(50mg/kg/d、200mg/kg/d) significantly decreased FPG(9.70±0.75vs5.85±1.33vs5.11±1.04mmol/L)、Fins(30.62±5.34vs26.52±9.37vs23.28±8.25mU/L) and the index ofinsulin resistance(11.46±1.84vs6.40±2.15vs6.01±1.81) and improved insulin sensitivity,confirming a therapeutic effect on improved glucose metabolism in IGT mice model.3. Importantly, serum sOB-R levels significantly increased in both CD and HF micewith metformin treatment, and in a dose-dependent manner (CD group：13.49±0.00vs27.37±0.01vs54.08±0.04ng/ml；HF group：23.92±0.00vs37.16±0.01vs57.94±0.01ng/ml). Moreover, enhanced sOB-R levels were associated with improved glucosemetabolism in IGT disease mice, no harmful effects were seen in CD mice.4. The effect of metformin treatment on sOB-R levels was also determined in T2DMpatients. And result showed that plasma sOB-R levels with metformin (1.5g/d) treatmentsignificantly increased (30.03±0.09ng/ml) compared to each individual patient before usingthis medicine (12.07±0.13ng/ml). Conversely, leptin levels decreased with metformintreatment vs. none-treatment (22.32±0.14ng/ml vs31.29±0.08ng/ml).5. As leptin receptors are abundantly expressed in liver, and ectodomain shedding ofthese receptors constituents the major source of sOB-R, we measure all isoforms for OB-R(OB-Rt) mRNA expression in animal livers from both CD and HF mice receivingmetformin treatment and compared to saline treated mice. Quantitative results fromreal-time PCR showed that the OB-Rt mRNA amount significantly increased withmetformin (50mg/kg/d、200mg/kg/d) by fold increase as1:2.49:7.36in CD group, and 1:6.77:10.70in HF group..6. We further tested the effect of metformin on OB-Rt mRNA expression in culturedhuman L02cells. Results from real-time PCR showed that OB-Rt mRNA levelsincreased2.5fold with metformin treatment.Conclusion：1. Metformin upregulates plasma sOB-R levels in mice and clinical patients. Theeffect is dose-dependent and is not altered by dietary types, indicating a drug specificinfluence.2. The mRNA of leptin recpetor significantly increased in livers from metformin-treatmice as well as in cultured L02hepatocytes compared to none treatment control.Together, our study showed that metformin dose-dependently upregulates serumsOB-R levels in mice likely due to increased liver leptin receptor gene expression whichmay contribute to improved blood glucose control. For the best of our knowledge, this isthe first study to show that metformin treatment is associated with increased sOB-R levelswhich is accompanied with improved glucose metabolism. It is thus tempting to speculatethat sOB-R may serve as a beneficial biomarker for monitoring metformin therapeuticeffect in diabetic patients. |
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