Jiao-Tai-Wan Ameliorates the Insulin Resistance and Lipid Metabolism in T2DM Rats by Regulating Neurotransmitters


 Background: Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance and β-cell dysfunction, and accompanied by neuroendocrine disorders. Recently, Jiao-Tai-Wan (JTW) has been reported to exert hypoglycemic effects against diabetes. However, its mechanism has not been clarified. Therefore, we attempted to explore the effect of JTW on alleviating insulin resistance and lipid metabolism disorder in T2DM rats by regulating the level of neurotransmitters. Methods: Sprague-Dawley (SD) rats were treated with a high-fat diet/streptozotocin to induce T2DM and then gavaged with JTW for 4 weeks. Afterwards, endpoints including body weight, fasting blood glucose, glucose tolerance, serum insulin, and lipid index were determined, and we analyzed pathological changes in the liver and kidney. Meanwhile, the level of neurotransmitter neurotransmitters in the central nervous system and peripheral tissues was measured by UPLC-MS/MS. Furthermore, the expression of neurotransmitter transporter mRNA and protein levels in the brain and kidney of T2DM rats was analyzed by qRT-PCR and WB. Results: The results showed that JTW ameliorated glucose homeostasis, insulin resistance, and lipid metabolism in T2DM rats by regulating the disorder of neurotransmitter distribution in the brain, kidney, intestine, adrenal gland, blood, and urine of T2DM rats. Mechanically, JTW may improve neurotransmitter disturbance by reducing mRNA and protein expression of SERT, DAT, and GAT-1 and increasing mRNA and protein expression of NET in the brain and kidney of T2DM rats.Conclusion: Our findings confirm that JTW can play a hypoglycemic role by regulating the disorder level of neurotransmitter distribution in T2DM rats, which may have potential therapeutic implications for the treatment of T2DM.


Introduction
Type 2 diabetes mellitus (T2DM) is a serious and chronic disease resulting from genetic, environmental and metabolic risk factors, etc [1]. T2DM and its complications have become one of the most serious threats to human health [2]. Actually, the treatment of T2DM has made great progress, such as insulin secretagogues, insulin sensitizers and biguanides [3]. However, the long-term usage of these hypoglycemic drugs may induce nausea, vomiting, and gastrointestinal discomfort, etc [4]. Therefore, it is urgent to nd new drugs to treat T2DM and its complications.
Recent studies have reported that neurotransmitters in the body play an important role in the occurrence and development of diabetes [5,6]. For instance, the level of serotonin (5-HT) in peripheral organs increased signi cantly, which is correlated with the glycated hemoglobin (HbA1c) in diabetic patients [7]. The increased intestinal and plasma 5-HT levels were observed in diet-induced obese mice [8]. Moreover, the absence of 5-HT protects the mice under a high fat diet against the development of their metabolic syndrome [9]. Therefore, the regulation of neurotransmitter levels may play a crucial in effect the treatment of T2DM and its complications.
At present, the evidence on the e cacy of using Traditional Chinese Medicine (TCM) for T2DM prevention and treatment has been found [10,11]. Among these TCM, Jiao-Tai-Wan (JTW) is gaining a growing interest in the scienti c community. JTW, which consists of Rhizome Coptidis (Coptis chinensis Franch, Ranunculaceae) and Cortex Cinnamomi (Cinnamomum cassia Presl, Lauraceae) at a ratio of 10:1 (g/g), is a classical traditional Chinese prescription for treating insomnia. Its mechanism might be related with the modulation of the circadian clock and in ammation genes expressions in peripheral blood monocyte cells [12]. In recent years, numerous reports have demonstrated that JTW not only has the effect on insomnia alleviation, but also has the anti-diabetic effect [13]. JTW can signi cantly reduce fasting blood glucose in rodents, improve glucose tolerance, and enhance insulin sensitivity. Mechanistically, it may be related to the regulation of AMPK signaling pathway [11,14]. Nevertheless, the mechanism of JTW in the treatment of T2DM has not been clari ed. Based on the above problems, our study aims to explore the anti-diabetic effect of JTW in T2DM rats, and illustrate that adjusting the content of neurotransmitters in brain and peripheral organs may be its major mechanism.

Preparation and analysis of JTW extracts
Rhizoma Coptidis and Cortex Cinnamon were purchased from Beijing Heyanling Pharmaceutical Development Co. LTD (Beijing China), certi ed by Professor Liu Chunsheng of School of Chinese Pharmacy, Beijing University of Chinese Medicine. First, the ratio of 10:1 Rhizoma Coptidis (800 g) and Cortex Cinnamon (80 g) was soaked in 10 times deionized water for 30 min. Secondly, the mixture of Rhizoma Coptidis and Cortex Cinnamon was extracted twice for 2 hours each time. Next, the JTW extract is ltered and concentrated to 6.83 g·mL − 1 . Then, a certain volume of JTW extract was added with 10 times deionized water, which was equivalent to 3.3 g·mL − 1 of raw material per mL.
JTW dry powder was obtained by freeze-drying part of the extracts. The JTW dry powder was accurately weighed and ultrasonically dissolved in methanol, and then ltered to be tested. The chromatographic separation was performed on an Agilent HPLC 1290 series system (Agilent, Waldbronn, Germany). The detection conditions of HPLC were as follows: Agilent Extend-C18 column (4.6 mm×250 mm, 5 µm); The mobile phase was composed of acetonitrile (A) and 0.05 mol·L − 1 potassium dihydrogen phosphate solution (B) (A: B = 45: 55); The ow rate was 1.0 mL·min − 1 ; The column temperature was 30℃ and the injection volume was 5 µL. The main components and contents of JTW were shown in Fig. 1a  The rats were randomly divided into two groups fed with normal control diet (n = 8) or a high-fat diet (including basic feed 72.7% (w/w), lard 20% (w/w), egg yolk powder 5% (w/w), cholesterol 2% (w/w), bile salt 0.3% (w/w)) for 4 weeks. After 4 weeks of dietary intervention, rats on high-fat diet were injected with streptomycin (STZ, sigma, USA) dissolved in citric acid buffer (0.1 mmol·L − 1 , pH = 4.5, 4℃), with a dose of 35 mg·kg − 1 . Three days after injection, fasting blood glucose (FBG) of those rats was measured. The rats with fasting blood glucose level ≥ 11.1 mmol·L − 1 were used in the further experiments, which were fed with high-fat diet throughout the whole study.
Thirty-two rats were randomly allocated into four equal groups as follows: normal control group, TZDM group (Model), JTW extract (0.683 g·mL − 1 ) treatment group (JTW) and metformin (183 mg·kg − 1 ) treatment group. Rats in normal control group and model group were given the equal amount of deionized water by gavage. All rats were administered once a day continuously for 4 weeks. The body weight, food intake, water intake and blood glucose of all rats were monitored weekly. In addition, urine was collected before the end of the experiment.

Oral glucose tolerance test (OGTT)
After 3 weeks of feeding, the rats of all groups were fasted overnight and orally loaded with glucose (2.0 g/kg). Blood glucose were measured in blood collected by venous bleeding from the tail vein immediately before and, 30, 60, 120 min after glucose administration.

Biochemical analysis
The serum insulin level was measured by rat insulin ELISA Kit (Jiangsu Kete, China) according to the manufacturer's instructions. The homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated as [FBG (mmol/L) × FIN (mU/L)] / 22.5 to assess insulin resistance.

Biosample preparation
The tissue samples (heart, liver, brain, kidney, adrenal gland, intestinal) were homogenized in cold watermethanol (8:2) solution at a ratio of 1:2 (m: v). The protein concentration of tissue samples was determined by BCA protein detection kit (Beyotime, China). The serum was mixed with twice the amount of water-methanol (8:2), and urine with 9 times water-methanol (8:2). Above all biological samples were performed as follows. Biological samples were centrifuged at 12000 rpm for 15 min at 4 ℃, and then the supernatant was taken for 400 µL. Both 100 µL internal standard (4 µg·mL − 1 ) and 1.5mL 0.1% formic acid-acetonitrile (4:6) were added to the supernatant, which centrifuged at 12000 rpm for 15 min at 4 ℃.
The supernatant was further used for UPLC-MS/MS analysis. The optimal conditions of the APESI source of positive ion mode were employed as follows: capillary voltage of 4 kV, ion source temperature of 400℃, degassing ow rate of 800 L·hr − 1 , cone ow rate of 150 L·hr − 1 , spray pressure of 7.0 bar. The parameters and ion pairs of each neurotransmitter mass spectrum were shown in Table 1.

Histopathological analysis
Liver and kidney tissues xed with 10% formalin were sectioned after being embedded in para n. Liver and kidney sections were prepared and stained with hematoxylin and eosin (H&E). Images were acquired using an inverted microscope (Olympus, Japan).

Quantitative real-time PCR analysis
The total RNA from brain and kidney tissues was extracted with Trizol reagent (Tiangen, China). One microgram of total RNA was converted to cDNA by using the Thermo sher RT-PCR kit (Thermo, USA).
Real-time quantitative PCR (qPCR) was performed with Power SYBR® Green PCR Master Mix (Thermo, USA). The PCR reaction was run in triplicate for each sample using the Step One Real-Time PCR System (Applied Biosystems, USA). The details of primer sequences were shown in Table 2.

Statistical analysis
Statistical analysis was performed by SAS 8.2. All values were expressed as means ± standard deviation.
Comparisons among the three groups were achieved via one-way analysis of variance (ANOVA) followed by Dunnett's test., and the independent sample t-test was used for the comparison between the two groups. P < 0.05 was considered as a signi cant difference.

Results
3.1 JTW improves the weight body, food and water intake, blood glucose and insulin resistance of T2DM rats The metabolic effects of JTW were evaluated by administering the compound to T2DM rats. As shown in Fig. 2a, after 4 weeks of JTW and metformin treatment, the rats were obviously protected from weight reduction. Further studies showed that compared with the normal group, the food and water intake of the model group rats were signi cantly increased, while JTW and metformin inhibited the food and water intake of T2DM rats, compared with the model group ( Fig. 2b and Fig. 2c).
We next measured the blood glucose of all rats (Fig. 2d). As a result, the blood glucose of rats in the model group was signi cantly higher than that in the normal group. However, JTW and metformin treatment signi cantly reduced the blood glucose of rats compared with model group rats. To examine whether glucose metabolism of T2DM rats by JTW treatment, we investigated glucose homeostasis by oral glucose tolerance test (OGTT). JTW treatment greatly improved glucose homeostasis in T2DM rats compared with model group rats ( Fig. 2e and Fig. 2f). Based on the above ndings, we speculated that JTW improves insulin resistance. As expected, JTW signi cantly reduced the serum insulin level and HOMA-IR of T2DM rats compared with the model group ( Fig. 2g and Fig. 2h). These results indicate that JTW ameliorates glucose homeostasis and insulin resistance in T2DM rats.

JTW reduces lipid levels in serum and liver of T2DM rats
T2DM is usually closely associated with hepatic steatosis. To investigate the bene cial effects of JTW on hepatic steatosis, we rstly observed the liver morphology of T2DM rats treated with and without JTW by H&E staining. The H&E staining of liver revealed that compared with the normal group, the model rats had more hepatocellular edema and lipid dripping. After being treated with JTW, the edema and steatosis of rat hepatocytes were reduced compared with the model group (Fig. 3a). In addition, we also determined the content of TC and TG in liver tissue ( Fig. 3b and Fig. 3c). Consistently, compared with the model group, JTW signi cantly reduced the levels of TC and TG in the liver of T2DM rats, which suggests that JWT may exert some protective effects on the liver.
Moreover, the serum analysis shows that JTW and metformin treatment reduced TG, TG and LDL-C levels while increasing HDL-C levels of the serum in T2DM rats (Fig. 3d, 3e, 3f and Fig. 3g). Taken together, these results indicate that JTW can effectively ameliorate lipid metabolism in T2DM rats.
3.3 JTW improves neurotransmitter endocrine disorder in T2DM rats 5-hydroxytryptamine (5-HT), also known as serotonin, is a major excitatory neurotransmitter and a strong vasoconstrictor. It is abundant in brain tissue, intestine and platelets, and plays a key role in learning, memory and emotion regulation [15]. It has been demonstrated that intestinal microorganisms can affect the host's glucose homeostasis through 5-HT biosynthesis in intestinal chroma n cells; When the level of 5-HT in serum and colon decreased, the glucose tolerance of mice increased signi cantly [16]. However, it has been nding that low brain serotonin levels are associated with poor memory and depressed mood [17]. In this study, we measured the contents of 5-HT, 5-HIAA, DA and other neurotransmitters in the central and peripheral of T2DM rats (Fig. 4a-4h). Compared with the normal group, the level of 5-HT in the brain of T2DM rats decreased signi cantly, while JTW treatment can dramatically increase the level of 5-HT in the brain of T2DM rats compared with the model group. Interestingly, the 5-HT levels in the adrenal glands, intestines, and serum of T2DM rats were markedly higher than those of normal control rats. After JTW treatment, the levels of 5-HT in the adrenal gland, intestine and serum of T2DM rats decreased signi cantly, which suggested that JTW could increase glucose tolerance in T2DM rats by regulating the level of peripheral 5-HT. 5-HIAA, or 5-hydroxyindole acetic acid, is an inactive acidic metabolite produced by serotonin (5-HT). The change of 5-HIAA content can indirectly re ect the change of 5-HT content [18,19]. Consistent with the results of 5-HT content determination, after JTW treatment, 5-HIAA levels in the intestine and serum of T2DM rats dramatically decreased compared with the model group.
It is now recognized that norepinephrine (NE), which is a neurotransmitter in the sympathetic nervous system, is stored in the intracellular membrane-bound particles of adrenergic nerve endings [20].
Abnormal sympathetic activity is a predictor of insulin resistance, diabetes and cardiovascular events [21]. It has been found that the depletion of the norepinephrine stores in the heart in diabetic patients may in part be responsible for their reduced survival rate in acute myocardial infarction [22]. Moreover, Champaneri et al reported that men with diabetes had signi cantly lower urinary catecholamines included epinephrine, norepinephrine, and dopamine compared with those without diabetes [23]. In this study, compared with the normal group, the NE levels in the heart and urine of the model group rats were signi cantly lower. After the treatment with JTW, the NE levels in the heart and urine of the rats were signi cantly higher than that in the model group. However, the results of NE determination showed that the NE level in serum of T2DM rats was dramatically higher than that in the normal control group. While compared with the model group, the JTW-treated signi cantly reduced the NE level in the serum of T2DM rats, which is consistent with the results of previous studies [21].
Epinephrine is now increasingly recognized as an important metabolic hormone that helps mobilize energy storage in the form of glucose and free fatty acids [24]. It was found that the levels of neurotransmitters in diabetic animals changed in varying degrees; Importantly, adrenaline was found to increase signi cantly in adrenal tissue and decrease in serum [25]. Strikingly, in this study, we also found that the levels of epinephrine in brain, kidney, blood and urine of T2DM rats were notably decreased, while those in adrenal gland were signi cantly increased compared with normal rats. While compared toT2DM rats, supplementation of JTW showed the levels of epinephrine in brain, kidney, blood and urine of T2DM rats were notably increased, meanwhile those in adrenal gland were remarkably decreased.
Dopamine is the main catecholamine neurotransmitter in the mammalian brain. It controls a variety of functions, including motor activity, cognition, mood, active reinforcement, food intake, and hormonal regulation [26]. Several lines of research indicate that abnormal dopaminergic neurotransmission could be involved in pathophysiological processes leading to obesity and diabetes [27]. Studies demonstrated the intrarenal dopaminergic system was crucial for modulating the development and progression of diabetic kidney injury, and the decrease of renal dopamine production had negative effect on diabetic nephropathy [28]. Moreover, the increase of dopamine levels in the heart and brain could inhibit the occurrence and development of T2DM [29,30]. Compared with the normal group, the levels of DA in the heart, brain kidney and urine of T2DM rats decreased signi cantly, while JTW treatment can dramatically increase the levels of DA above those of T2DM rats compared with the model group.
GABA is the main neurotransmitter of the central nervous system, and it also plays an important role in the peripheral nervous system [31]. Previous studies also shown that GABA plays a role in endocrine pancreas and diabetes mellitus [32]. In addition, more recent studies have revealed that GABA promotes human β-cell proliferation and improves glucose homeostasis [33,34]. In this study, the levels of GABA in the liver, brain, adrenal gland, intestine and serum of the model group rats exhibited a remarkable reduction compared to those in the normal group. Importantly, intervention with JTW for 4 weeks obviously reversed the decrease of GABA levels in those of T2DM rats compared with the model group.
These results suggest that JTW may promote islet β-cell proliferation and ameliorate glucose homeostasis by increasing the levels of GABA of T2DM rats.

PCA analysis of neurotransmitter distribution levels in Tissues, blood and urine
In view of the above result, the overall structural changes of neurotransmitters in tissues (heart, liver, brain, kidney, adrenal gland, intestinal), blood, and urine among the groups were analyzed by unsupervised principal component analysis (PCA). The results of the PCA were shown in Fig. 5a-5h. JTW treatment revised the variations of the levels of 6 neurotransmitters in the brain, kidney, adrenal gland, intestine, blood and urine of T2DM rats along [t1] to some extent, while moving the structure along [t2] markedly. Taken together, JTW treatment signi cantly restored the relative abundance of several neurotransmitters disturbed by T2DM, which was closely related to metabolic disorders.

JTW regulates the expression of neurotransmitter related transporter in brain tissue of T2DM rats
In accordance with the above ndings, to explore whether JTW can regulate neurotransmitters to play a hypoglycemic effect, we also detected the expression of neurotransmitter related genes and proteins in brain tissue. The results of PCR and WB showed that compared with the normal group, the mRNA and protein levels of SERT, DAT and GAT-1 in the model group rats were signi cantly increased, while the mRNA and protein levels of NET were signi cantly decreased; After JTW treatment, the mRNA and protein levels of SERT, DAT and GAT-1 were signi cantly decreased, while the mRNA and protein levels of NET were signi cantly increased (Fig. 6a-6i).
3.6 JTW regulates the expression of neurotransmitter related transporter in kidney tissue of T2DM rats According to the distribution level of neurotransmitters in different tissues, we found that neurotransmitters changed most signi cantly in brain and kidney tissues of T2DM rats. Therefore, we also detected the expression of neurotransmitter related gene and protein in renal tissue. Furthermore, we also observed the pathological morphology of rat kidney. Compared with the normal group, the basement membrane of the kidney tissue of type 2 diabetic rats was diffusely thickened, and part of the glomerular structure was destroyed. After treatment with JTW, the pathological damage of the kidney tissue was improved (Fig. 7a). Consistent with the expression of neurotransmitter related genes and proteins in brain tissue, the results of PCR and WB in kidney tissue showed that compared with the normal group, the mRNA and protein levels of SERT, DAT, and GAT-1 in the model group were markedly increased, and the mRNA and protein levels of NET were notably reduced; after JTW treatment, SERT, DAT, and GAT-1 were markedly increased, and the mRNA and protein levels of NET are notably increased (Fig. 7b-7j).

Discussion
T2DM is an increasingly serious global health problem, which is closely related to environmental factors and genetic factors, etc [35]. T2DM can cause serious complications, such as depression, anxiety [36], non-alcoholic fatty liver [37] and diabetic nephropathy [38], etc. Interestingly, neurotransmitter levels play a vital role in the pathogenesis of T2DM and its complications [39]. In diabetes, hyperglycemia and insulin resistance can lead to disorders of neurotransmitter levels [40]. Therefore, we speculate that regulating the level of neurotransmitters can be used to prevent and treat T2DM and its complications.
In this study, we investigated the effects of JTW on glycolipid metabolism and neurotransmitter levels in T2DM rats. It is demonstrated that JTW reduced the fasting blood glucose level, insulin resistance, food intake, water intake, serum lipids and hepatic lipids, and improved insulin sensitivity in T2DM rats. The above results are consistent with previous other studies [14,41]. Hence, these results clearly showed that JTW has a potent anti-diabetic effect.
Our study showed that neurotransmitter levels were disordered owing to the high fat diet feeding in T2DM rats, which was dramatically improved by JTW treatment. In particular, the level of 5-HT in the brain of T2DM rats decreased signi cantly compared with the normal group, After the JTW treatment, the levels of 5-HT in the brain T2DM rats increased signi cantly. In contrast, the 5-HT levels in the adrenal glands, intestines, and serum of T2DM rats were markedly higher than those of normal control rats, While JTW dramatically decreased the level of 5-HT in the adrenal gland, intestine and serum of T2DM rats decreased signi cantly, which suggested that JTW could increase glucose tolerance in T2DM rats by regulating the level of peripheral 5-HT. These neurotransmitter disorders caused by HFD and T2DM have been reported in other studies [42,43]. In addition, high plasma levels of 5-HT have been reported in T2DM patients, although the potential effect on insulin secretion is unclear [44]. It has been demonstrated that the nervous system can regulate insulin secretion from pancreatic β-cells through neurotransmitters and their respective receptors expressed in β-cells [45]. Importantly, the increased expression of 5-HT receptor 2C in pancreatic β-cells might inhibit insulin secretion of db/db mice [43].
Norepinephrine (NE), a neurotransmitter, plays an extensive role in the regulation of stress physiology, blood glucose levels and blood pressure [46]. However, the mechanism by which noradrenergic system is involved in the pathophysiology of diabetes has not yet been elucidated. Importantly, all diabetic rats, plasma norepinephrine was elevated and norepinephrine reuptake transporter expression reduced compared to non-diabetics [21]. Consistent with the above results, our results showed that the NE level in serum of T2DM rats was dramatically higher than that in the normal control group. Compared with the model group, JTW signi cantly reduced the NE level in the serum of T2DM rats. Moreover, we also measured NE levels in the heart and urine. Interestingly, the NE levels in the heart and urine of the model group rats were signi cantly lower compared with the normal group. After the treatment with JTW, the NE levels in the heart and urine of the rats were signi cantly higher than that in the model group. Meanwhile, we also determined the content of 5-HIAA, epinephrine, dopamine and γ-aminobutyric acid in brain and peripheral tissues of T2DM rats. However, the levels of these neurotransmitters change differently in different tissues. Therefore, we urgently need the further study to clarify its mechanism.
The high-a nity neurotransmitter transporter (5-HT [47], DAT [48], GAT-1 [49] and NET [50]) is the primary mechanism by which neurotransmitter (5-HT, DA, GABA and NE) synaptic transmission is terminated, which can control the duration and intensity of neurotransmitter signal through synaptic reuptake. Based on the result that changes in neurotransmitter levels are the most obvious in T2DM rats, we determined the genes and proteins expression of their related neurotransmitter transporter in the brain and kidney tissues. The results showed that compared with the normal group, the mRNA and protein levels of SERT, DAT, and GAT-1 in the model group were markedly increased, and the mRNA and protein levels of NET were notably reduced. After JTW treatment, SERT, DAT, and GAT-1 were markedly increased, and the mRNA and protein levels of NET are notably increased. These results are consistent with previous studies [47][48][49][50]. In addition, the levels of neurotransmitters in the central and peripheral are also affected by other factors such as synthetic enzymes and receptors, etc [51]. Hence, we need further research to reveal the mechanism of JTW on diabetes.

Conclusion
In summary, our study demonstrated that JTW ameliorated glucose homeostasis, insulin resistance, and lipid metabolism in T2DM rats by regulating the disorder level of neurotransmitter distribution in the brain, kidney, intestine, adrenal gland, blood, and urine of T2DM rats.

Funding
This work was funded by the National Natural Science Foundation of China (81673680).

Availability of data and materials
The datasets generated and/or analysed during the current study are not publicly available due to limitations of ethical approval involving the patient data and anonymity but are available from the corresponding author on reasonable request.   Effects of JTW on body weight, food intake, water intake, blood glucose and serum insulin in T2DM rats.   Norepinephrine (NE); Epinephrine (E); Dopamine (DA); γ-aminobutyric acid (GABA). Data were shown as mean ± SD (n = 8). *P < 0.05, **P < 0.01 vs. Control group. #P < 0.05, ##P < 0.01 vs. T2DM group.