type 2 diabetes mellitus ( t2 dm ) is a chronic metabolic syndrome with an increasing prevalence throughout the world . the most recent data report 37.1 million t2 dm patients worldwide ; the percentage of these t2 dm patients in china is 11.6% and 11.3% in the united states [ 1 , 2 ] . ir is a physiological condition in which cells fail to respond adequately to the normal actions of the hormone insulin . the body may continue to produce insulin , but the cells in the body become resistant to its actions and , therefore , are unable to use it as effectively , leading to hyperglycemia . to reduce ir , there are several insulin sensitizers that are commonly used , including thiazolidinediones ( e.g. , rosiglitazone and pioglitazone ) and biguanides ( e.g. , metformin ) . these agents can improve ir and increase peripheral utilization of insulin , resulting in a decrease in blood glucose . thiazolidinediones carry a risk of bone fractures , bladder cancer , and , of special concern , cardiovascular side effects . although some studies have shown that thiazolidinediones have no additional risks for myocardial infarction or cardiac death [ 5 , 6 ] , caution in its use has been recommended by the u.s . food and drug administration . the biguanides remain for use in the treatment of ir , and these agents may have gastrointestinal side effects . however , in the clinical setting , we often face the difficulty of having no effective insulin sensitizers to use in certain t2 dm patients , especially those who can not tolerate biguanides . tianmai xiaoke pian or tmxkp ( hebei fuge pharmacy co. , ltd ) is a traditional chinese medicine tablet , comprised of chromium picolinate ( 1.6  mg per tablet , 200  g elemental chromium ) , tianhuafen ( radix trichosanthis , snake gourd root ) , maidong ( radix ophiopogonis , dwarf lily turf tuber ) , and wuweizi ( fructus schisandrae chinensis , chinese magnoliavine fruit ) . , tmxkp was shown to decrease serum glucose and glycated hemoglobin ( hba1c ) in t2 dm patients and also in rats [ 79 ] . therefore , in this present study , we examined the effects of tmxkp on insulin downstream signaling pi3-k / akt pathway , an important insulin pathway , in rats with t2 dm . spf grade male wistar rats ( weighing 250 to 300  g ) were obtained from the laboratory animal investigation center of china medical university ( liaoning , china ) . the rats were housed in spf facilities at the animal center of shengjing hospital of china medical university , under a 12-hour dark - light cycle and fed ad libitum during the experiment . the animal protocols used were approved by the institutional animal care and use committee at shengjing hospital of china medical university . all animal experiments were carried out in accordance with the national research council guide for the care and use of laboratory animals . based on previous articles about the t2 dm model [ 1012 ] and our preliminary experiment , a t2 dm model was induced by high - fat diet and streptozotocin ( stz ) injections . rats were fed with a high - fat diet ( 22% fat , 48% carbohydrates , and 20% protein , 20.08  kj / g ) [ 13 , 14 ] for 4 weeks prior to intraperitoneal injection of a single 25  mg / kg dose of stz ( dissolved in 0.05  m citrate buffer at ph 4.5 , prepared immediately before use ) . two weeks after the stz injection , rats were assessed by fasting blood glucose with blood collected from the tail vein . t2 dm was diagnosed as a fasting blood glucose 7.8  mmol / l on two occasions or a nonfasting blood glucose 11.1  mmol / l ; rats induced with t2 dm were considered as diabetic rats and were selected for subsequent experiments . diabetic rats were randomly divided into two groups : a diabetic group ( n = 10 ) and a tmxkp - treated group ( n = 10 , given 50  mg / kg tmxkp intragastrically daily ) . weight - matched normal rats were selected as the control group ( n = 10 ) . based on the recommended dose of tmxkp for humans ( 0.48  g / d ) , a corresponding dose for rats was estimated at 50  mg / kg . the tmxkp - treated group received 0.48  g tmxkp dissolved in 2 ml 0.9% saline ; the control group received an equal volume 0.9% saline without tmxkp . all diabetic rats received a high - fat diet during the experiment , while nondiabetic rats in the control group were fed a normal diet ( 5% fat , 53% carbohydrate , and 23% protein , 14.6  kj / g ) . blood , left gastrocnemius muscle , greater omentum adipose tissue , and liver were removed immediately for further analysis . fasting blood glucose and oral glucose tolerance tests ( ogtt ) were performed every 2 weeks after diagnosis of diabetes using blood samples taken from the tail vein . generally speaking , following a 12-hour overnight fast , blood from the tail vein was collected at 0 , 30 , 60 and 120 minutes after glucose lavage ( at 1  g / kg of body weight ) . the area under the curve ( auc ) was calculated for blood glucose ( bg ) from the ogtt as follows : auc = 1/4  ( bg0 ) + 1/2  ( bg30 ) + 3/4  ( bg60 ) + 1/2  ( bg120 ) . blood glucose was measured using an accu - check performa nano glucose meter ( roche , germany ) , and insulin was detected by enzyme - linked immunoassay ( elisa ; rat insulin elisa kit , thermo scientific ) in accordance with the method of kekow et al . . homeostatic model assessment of insulin resistance ( homa - ir ) was calculated as : ( 1)homa - ir = fbg  mmol / lfins  u / ml22.5,where fbg is fasting blood glucose and fins is fasting insulin concentration . the total rna of skeletal muscle , liver , and adipose tissues from rats was extracted using the trizol method , according to manufacturer 's instructions . irs-1 , irs-2 , pi3-kp85 , akt2 , and -actin gene expression was detected by reverse transcription - polymerase chain reaction ( rt - pcr ) analysis . liver , adipose tissue , and skeletal muscle lysates were homogenized by centrifuging at 15,000  r / min for 30 minutes at 4c . nitrocellulose membranes were incubated at 4c overnight with primary antibodies against irs-1 , irs-2 , pi3-k , akt2 , p - akt2 , and -actin , respectively . immunoreactive proteins were visualized using enhanced chemiluminescence western blotting detection reagents and detected by hmias-2000 imaging system . band densities were determined by biorad quantity one software and quantified as the ratio to -actin . statistical significance was determined by one - way analysis of variance ( anova ) followed by dunnett c test for unequal variances data . for data with equal variances assumed , at the beginning and end of the experiment , weight , total cholesterol , and triglycerides for all rats were recorded . as shown in table 2 , we could observe an obvious decrease in body weight in diabetic rats compared with normal ( control group ) rats ( p < 0.05 ) . and there was no difference in body weight between the tmxkp - treated and untreated diabetic groups . total cholesterol and triglycerides levels were elevated in the diabetic group compared with the control group , but tmxkp was expected to decrease these values in the treated diabetic group ( p < 0.05 ) . the fasting blood glucose of diabetic rats was significantly higher than that in the control group at all time points . fasting blood glucose in the tmxkp - treated group decreased significantly compared with the untreated diabetic group ( p < 0.05 , figure 1(a ) ) . for the ogtt test , blood glucose levels in the untreated diabetic group were higher than those of the control group at all time points after oral glucose administration ( p < 0.05 ) . however , blood glucose after ogtt in the tmxkp - treated group was significantly lower compared with the untreated diabetic group ( p < 0.05 , figure 1(b ) ) . from the auc of the three groups , we could observe an obvious decrease in glucose in the tmxkp - treated group compared with the untreated diabetic group ( p < 0.05 , figure 1(c ) ) . after comparisons , the homa - ir value in tmxkp - treated group was significantly decreased compared with that of the untreated diabetic group ( p < 0.05 , figure 1(d ) ) . tmxkp is tianmai xiaoke pian ; auc is area under curve , mmol / lh ; ogtt is oral glucose tolerance test ; homa - ir is homeostatic model assessment of insulin resistance ; p < 0.05 , versus control group . there were significant differences when compared with control group ; p < 0.05 , versus diabetic group . pi3-k / akt signal pathway is thought to be an important signal pathway in ir . to detect the effects of tmxkp on ir , liver , left gastrocnemius muscle , and greater omentum adipose tissues pi3-k / akt signal pathway - related gene expression levels were detected by rt - pcr analysis , including irs-1 , irs-2 , pi3-k p85 , and akt2 genes . the primers used are listed in table 1 . in the untreated diabetic group , all gene expression levels after tmxkp treatment , gene expression levels were increased compared with the untreated diabetic group ( p < 0.05 ) . irs-1 , irs-2 , pi3-k , and akt2 protein expression levels were detected by western blot method in liver , skeletal muscle , and adipose tissues . after comparison , we observed an obvious decrease in protein concentrations in all tissues in the untreated diabetic group compared with the control group ( p < 0.05 , figure 3 ) . after tmxkp treatment for 8 weeks , pi3-k / akt signal pathway - related proteins were also determined in tmxkp - treated diabetic rats . we observed a significant increase in protein concentrations in all tissues ( p < 0.05 , figure 3 ) . these findings indicate that ir was reduced by tmxkp through the pi3-k / akt signal pathway . nearly 1 in 10 of the world 's population is affected by t2 dm . and so , the question of how to best fight against t2 dm is an important issue for investigators all over the world . they both play a crucial role in the treatment of t2 dm . however , with the widespread use of these agents , new problems have gradually arisen . thiazolidinediones have many adverse effects , including the risk of cardiovascular adverse events , bone fractures , bladder cancer , and hepatotoxicity . the major side effects of metformin , a biguanide , are gastrointestinal symptoms ( e.g. , nausea , vomiting , and diarrhea ) and a metallic taste in the mouth . so there are limitations to the use of biguanides in some patients . as a result , in actual clinical practice , if a patient is intolerant to metformin , there are no other insulin sensitizers available as an alternative . in this study , to further understand the effects of tmxkp on ir , we tested this agent in rats with stz - induced t2 dm . and the results were in accordance with a previous study by zhang et al . [ 7 , 9 ] . after treatment with tmxkp for 8 weeks , characteristic variables of ir including fasting blood glucose , blood glucose following ogtt , auc of ogtt blood glucose , and homa - ir were decreased significantly in tmxkp - treated diabetic rats compared with diabetic rats . therefore , tmxkp can decrease both blood glucose and ir in diabetes . as we know , the pi3-k / akt signal pathway is a classical pathway of insulin in glucose metabolism , having a role in glucose uptake by the liver , skeletal muscles , and adipose tissues [ 19 , 20 ] . decreasing or blocking this pathway will reduce the insulin physiological effects that lead to ir . to investigate the further mechanisms of the effects of tmxkp on ir , we used an rt - pcr analysis method to test the pi3-k / akt signal pathway - related gene , including irs-1 , irs-2 , pi3-k p85 , and akt2 genes in insulin affected tissues : liver , skeletal muscle , and adipose tissues . after tmxkp treatment , gene expression levels were increased , which indicates alleviators of ir in all insulin - affected tissues . similar to the regulation of gene expression , we observed increased irs-1 , irs-2 , pi3-k p85 , akt2 , and p - akt2 expression after tmxkp treatment . many researchers suggest a beneficial effect on glucose intolerance [ 21 , 22 ] ; however , other research has shown it to be ineffective [ 23 , 24 ] . these findings suggest that although chromium is the main component of tmxkp , perhaps tmxkp does not act exactly in the same manner as chromium . in this experiment because of the issues with the clinical use of insulin sensitizers , we are often faced with a dilemma of choice of treatment . in our investigation , we have shown the anti - ir effects of tmxkp through the pi3k / akt pathway in detail and have provided evidence for its use . tmxkp may be a promising direction in clinical medicine for the treatment of t2 dm .
in the clinical setting , given the potential adverse effects of thiazolidinediones and biguanides , we often have difficulty in treatment that no other insulin sensitizers are available for use in type 2 diabetic mellitus ( t2 dm ) patients . tianmai xiaoke pian ( tmxkp ) is a traditional chinese medicine tablet , which is comprised of chromium picolinate , tianhuafen , maidong , and wuweizi . to understand its mechanism of action on insulin resistance , tmxkp ( 50  mg / kg orally ) was tested in t2 dm rats ( induced by a high - fat diet and streptozotocin ) . 
 eight weeks later , fasting blood glucose ( fbg ) and oral glucose tolerance tests ( ogtt ) were performed . 
 area under the curve ( auc ) and homeostatic model assessment of insulin resistance ( homa - ir ) were calculated , and pi3-k / akt signal pathway - related genes and proteins were tested by reverse transcription - polymerase chain reaction ( rt - pcr ) and western blot analysis in muscle , adipose , and liver tissues , respectively . 
 tmxkp significantly reduced fbg , ogtt , auc , and homa - ir in diabetic rats ( p < 0.05 ) . 
 furthermore , we also observed that tmxkp could significantly decrease irs-1 , irs-2 , pi3-k p85 , and akt2 gene expression and also irs-1 , irs-2 , pi3-k , akt2 , and p - akt2 protein expression levels ( p < 0.05 ) in diabetic rats . 
 these findings confirm that tmxkp can alleviate insulin resistance in t2 dm rats through the pi3k / akt pathway . 
 thus tmxkp appears to be a promising insulin sensitizer .