Natural Piperine Ameliorates Insulin Resistance And Obesity Related Type 2 Diabetes Through Anti-inflammation

Objective:Obesity,especially abdominal obesity?visceral obesity?has been warned as a high risk factor for type 2 diabetes?T2D?.This correlation exists because both the obesity and T2D are in a state of chronic low-grade inflammation.A huge number of immune cells aggregate in insulin target tissues?liver,muscle,fat,etc.?,especially M₁-like macrophages,secreting galectin-3?Gal-3?,IL-1?,TNF-?and other pro-inflammatory cytokines to block insulin signal transmission,hence mediating insulin resistance and T2DM.Piperine is a natural alkaloid isolated from the fruit of black pepper,which has a long edible history around the world.Studies have pointed out that piperine has a certain therapeutic effect on mice with type 2 diabetes induced by a high-fat diet,but its mechanism is not clear.In recent years,studies have revealed that piperine has an excellent therapeutic effect on a variety of chronic inflammatory disease.Based on the important role of chronic inflammation in the pathological process of obesity leading to type 2 diabetes,the study aims to explore whether piperine can play a certain therapeutic role on obese type 2 diabetes by inhibiting metabolic inflammation.Methods:?1?In vivo experiments.Newborn ICR mice on postnatal days 2 were injected subcutaneously?SC?with MSG?4 g/kg/d?to induce T2D mice model.After induction of T2D animal model,the 6-month-old MSG mice were divided into three groups,which were treated with metformin?150 mg/kg/day?,piperine?40 mg/kg/day?and vehicle for successive 10 weeks,respectively.Male ICR mice in the same age were used as the normal control group and given the same amount of 0.5%CMC-Na.At the end of the experiment,the obesity degree in each group were evaluated by measuring the body weight,Lee's index and abdominal fat index.The improvement effect of piperine on glycolipid metabolism disorder was evaluated by detecting related indicators of glycolipid metabolism in plasma.Oral glucose tolerance test?OGTT?and insulin tolerance test?ITT?were used to evaluate the improvement of piperine on glucose tolerance or insulin sensitivity.High glucose clamp experiment was conducted to investigate the effect of piperine on the islet function of MSG obese mice.The effect of piperine on blood inflammatory cells was evaluated using routine blood test.The serum pro-inflammatory cytokine concentration of each mouse was detected using Elisa and qPCR assay was used to detect the expression of pro-inflammatory cytokines in adipose tissue to evaluate the inhibitory effect of piperine on chronic inflammation.The effect of piperine on the protein expression of M₁-like polarization biomarker CD11c protein in adipose tissue macrophages was detected by immunohistochemical experiments.The effect of piperine on liver and adipose tissue pathology was examined by HE staining experiment.?2?In vitro experiments.In order to assess the inhibitory effect of piperine on pro-inflammatory macrophages,first,RAW264.7 macrophage cells were pretreated with different concentrations of piperine?20,40,80?M?for 12 h,and then stimulated with LPS solution?1?g/m L?for 24 h to inducing M₁-like polarization of macrophages and establishing inflammatory condition.The concentration of IL-1?in the cell supernatant was detected by Elisa.The expressions of CD11c,IL-1?,TNF-?at the mRNA level were detected in qPCR assay.Changes in protein levels of TLR-4,CD11c,and IL-1?were detected by WB experiments.Results:?1?In vivo experiments.By dynamically monitoring the changes of obesity degree and diabetes-related indicators in the process model establishment,we found that mice developed the obesity with type 2 diabetes by the age of 6 months.During the administration of piperine,we dynamically monitored the changes of body weight.The results showed that piperine could inhibit the body weight gain of MSG mice without affecting the food intake.In addition,the results showed that the 10-week administration of piperine?40 mg/kg/d?not only significantly decreased the elevated FBG,serum TC and TG levels,but also improved the oral glucose intolerance and abnormal insulin resistance and partly relieved the hyperinsulinemia in adult MSG obese mice.Moreover,piperine significantly decreased the total and differential white blood cell?WBC?count and the serum level of lipopolysaccharide?LPS?,pro-inflammatory cytokines such as galectin-3?Gal-3?,interleukin-1??IL-1??.Third,the results of HE staining experiments confirmed that piperine reduced white fat infiltration in liver tissues of obese mice,improved non-alcoholic fatty liver,and reduced hypertrophic fat cells in fat tissue.Furthermore,piperine clearly down-regulated the mRNA levels of pro-inflammatory cytokines and the protein levels of CD11c in adipose tissues.The above results validate the experimental hypothesis of this study at the animal level,that is,piperine can inhibit M1-like polarization of macrophages in visceral adipose tissue,reduce the degree of local and systemic chronic metabolic inflammation,improve hyperinsulinmia and disorder of glucose and lipid metabolism in obese MSG mice model,and thus produce the effect against obesity-related diabetes.?2?In vitro experiments.The results of qPCR experiments showed that piperine could significantly reduce the expression of pro-inflammatory factors at the mRNA level with a dose dependence.In addition,Elisa assay showed that found that the content of IL-1?in supernatant decreased with the concentration of piperine increased.Finally,WB experiment results showed that piperine significantly down-regulated the protein levels of IL-1?,CD11c and TLR-4.The above experimental results prove that piperine can inhibit M1-like polarization of macrophages induced by LPS at the cellular level,which is consistent with the whole animal experiment.Conclusions:Piperine can significantly delay the progression of obesity-related diabetes,and improve severe glycolipid metabolism disorders,oral glucose intolerance and insulin resistance.These effects are based on piperine on inhibition of M1 polarization in adipose tissue and probably interfere with the malignant induction between visceral adipocytes and macrophages.As a result,visceral fat and metabolic inflammation of the whole body are reduced to a great extent.Edible spice pepper is one of the important sources of piperine,both are safe and effective.Piperine is promising to be used as dietary regulators or drugs for the prevention and treatment of obesity-related diabetes.