Xiao Ke Qing improves glycometabolism and ameliorates insulin resistance by regulating the PI3K/Akt pathway in KKAy mice

Abstract

Xiao Ke Qing (XKQ) granule has been clinically used to treat type 2 diabetes mellitus (T2DM) for 10 years in Chinese traditional medication. However, its mechanisms against hyperglycemia remain poorly understood. This study aims to investigate XKQ mechanisms on diabetes and diabetic liver disease by using the KKAy mice model. Our results indicate that XKQ can significantly reduce food and water intake. XKQ treatment also remarkably decreases both the fasting blood glucose and blood glucose in the oral glucose tolerance test. Additionally, XKQ can significantly decrease the serum alanine aminotransferase level and liver index and can alleviate the fat degeneration in liver tissues. Moreover, XKQ can ameliorate insulin resistance and upregulate the expression of IRS-1, PI3K (p85), p-Akt, and GLUT4 in the skeletal muscle of KKAy mice. XKQ is an effective drug for T2DM by ameliorating insulin resistance and regulating the PI3K/Akt signaling pathway in the skeletal muscle.

Keywords

XKQ / type 2 diabetes mellitus / KKAy mice / PI3K/Akt pathway / diabetic liver disease

Introduction

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is pathophysiologically characterized by a relative lack of insulin secretion and resistance (IR) [¹,²]. IR involves a series of clinical manifestations caused by decreasing the sensitivity to insulin in the liver, muscles, and adipose tissues, which diminishes the effectiveness of insulin in lowering blood sugar [³,⁴]. The skeletal muscle mainly accounts for insulin-stimulated glucose disposal. In healthy people subjected to a euglycemic and hyperinsulinemic clamp, approximately 80% of the glucose is taken up by the skeletal muscle for glycogen synthesis and glycolysis [⁵,⁶]. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway plays a vital role in insulin signal transduction and insulin-mediated glucose metabolic pathways [⁷–⁹].

Diabetic liver injury is one of the most common complications in diabetes development, which is fatal for diabetic patients with early signs of hepatic fat degeneration, deposition, or even hepatic dysfunction. The development of fatty liver can cause metabolic disorders of glucose and fats, or a more serious form of IR, which worsens diabetes and contributes to the formation of a vicious cycle [¹⁰,¹¹].

Xiao Ke Qing (XKQ) granule has been approved by the China Food and Drug Administration to clinically treat T2DM for 10 years. XKQ can reduce fasting blood glucose (FBG), postprandial blood glucose, hemoglobin A1c, and plasma triglyceride levels in T2DM [¹²,¹³]. XKQ can reduce the blood glucose in streptozotocin-induced diabetic rats or in alloxan and epinephrine-induced diabetic mice. However, the hypoglycemic mechanisms of XKQ remain poorly understood [¹⁴,¹⁵].

Mangiferin, timosaponins, and berberine are the main components of XKQ [¹⁶]. Mangiferin regulates glucose in diabetic rats [¹⁷], eliminates liver steatosis and prevents adiposity in mice [¹⁸], and improves serum lipid profiles by reducing serum triglycerides and free fatty acids in overweight patients with hyperlipidemia [¹⁹]. Timosaponins can significantly improve the glucose tolerance and reduce the FBG in diabetic mice [²⁰]. Berberine is efficient in the treatment of T2DM. Studies on insulin resistance of human patients [²¹] and in animal or cell models [²²–²⁶] have established the hypoglycemic effect of berberine. Kong et al. [²⁷] investigated the molecular mechanism of berberine against insulin resistance, which could reduce the level of FBG and fasting serum insulin by upregulating the expression of insulin receptor (InsR) both in vitro and in vivo. Berberine decreases insulin resistance through the PI3K signaling pathway in diabetic rats [²⁸].

Based on previous findings, the hypoglycemic effect and mechanisms of XKQ on T2DM KKAy mice were investigated. The effect of XKQ on diabetic liver disease was also observed.

Materials and methods

Animals

A total of 60 8-week-old KKAy and 10 age-matched C57BL/6J female mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All mice were housed in a temperature-controlled (24±1 ℃) and constant-humidity (55%±5%) facility with dark/light circulation for 12 h and provided with food and water ad libitum.

Drugs and reagents

XKQ extractum, with a batch number of 20140403, was offered by Tianjin Tasly Pharmaceutical. Pioglitazone hydrochloride at 15 mg/piece, with a product batch number of 169A and a sub-package number of 10991315, was produced by Japan’s Takeda Pharmaceutical Company Limited and sub-packed by Tianjin Takeda Pharmaceutical Company Limited. The AST test kit, with R1 and R2 batch number of EL157 and EL158, respectively, and the ALT test kit, with R1 and R2 batch number of EK437 and EK438, respectively, were provided by Japan’s Wako Pure Chemical Industries Limited. The IRS-1 and GLUT4 monoclonal antibodies were purchased from the Santa Cruz Company in the US. The GAPDH poly clonal antibody was bought from America’s Bioworld Company. The PI3K (p85) and p-Akt (ser473) monoclonal antibodies were purchased from the Abcam Company in the US. The anti-mouse and anti-rabbit IgG (H&L) of goat were purchased from China’s PTG Company.

Animal grouping and drug administration

C57BL/6J mice were fed with standard chow diet, whereas KKAy mice were fed with high-fat diet during the experimental period. T2DM KKAy mice (fed with high-fat diet for 4 weeks) with FBG level higher than 12 mmol/L were selected. Fifty T2DM KKAy mice were then randomly grouped into five: diabetic model, pioglitazone hydrochloride treated (Piog), the three XKQ treated (XKQ high, medium, and low), and control groups with age-matched C57BL/6J mice. All drugs were dissolved in 0.9% sodium chloride and given through intragastrical administration. Pioglitazone was given at a dose of 12.3 mg/kg/day. The XKQ extractum was given at a dose of 9.91, 4.96, or 2.47 g/kg/day for 8 consecutive weeks. The control and model groups were treated with same volume of saline.

Body weight (BW) and food and water intake tests

BW was measured using an electronic weighing scale weekly. Food and water intake were measured in weeks 1, 3, 5, and 7 by using an electronic weighing scale during the treatment period. The mice were given a certain amount of food and water at 16:00 the day before, and the remaining food and water were weighed on the following morning. Their difference was recorded as the amount of food and water intake.

FBG test

FBG was measured between 13:00 to 13:30 on the 2nd day of each week during the treatment period after fasting for 4 h. Blood samples were obtained from the tail vein and monitored using a standard glucometer (BIOSEN 5030, Germany).

Oral glucose tolerance test (OGTT)

OGTT was carried out in the 8th week of treatment. To obtain the glucose levels, the mice were orally administrated with 2 g/kg body weight of glucose after fasting for 4 h. Blood samples were drawn from the tail vein before the glucose loading (time 0) and after 30, 60, and 120 min of glucose administration. Plasma glucose was monitored using a standard glucometer (BIOSEN 5030, Germany). The glucose tolerance was evaluated using the area under the curve (AUC). OGTT was applied by calculating the AUC with blood glucose (BG) as follows: AUC=1/4 × (BG0) + 1/2 × (BG30) +3/4 × (BG60) + 1/2 × (BG120).

Serum fasting insulin (FIN) test and insulin resistance index (pre-experiment)

In the pre-experiment phase, after 4 weeks of drug administration with the same dose as in the present study, the mice were fasted for 12 hours, and blood samples were collected by eye removal. The serum was separated via centrifugation at 3000 rpm for 10 min. The serum FIN levels were determined through a mouse insulin enzyme-linked immunosorbent assay kit (R&D Systems, USA). FBG was monitored using a standard glucometer (BIOSEN 5030, Germany). IR was calculated using the homeostasis model assessment index for IR (HOMA–IR). HOMA–IR index= FBG (mmol/L) × FIN (µU/ mL)/22.5.

Western blot analysis

The thigh muscle tissues of the mice were used for the Western blot analysis. The skeletal muscles were lysed in the radio immunoprecipitation assay buffer and supplemented with a complete protease inhibitor cocktail. Protein content was determined through bicinchoninic acid assay. Equal amounts of protein (40 µg) were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred into a polyvinylidene fluoride membrane by wet transfer. The membranes were blocked using 5% skimmed milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature and then incubated with a primary antibody against PI3K, p-Akt, IRS-1, or GLUT-4 at 4 ℃ overnight. The next day, the membranes were rinsed with TBST and incubated with HRP-conjugated secondary antibodies for 60 min at room temperature. The signals were detected using the ECL kit according to the standard procedures and then quantified by densitometry by using ChemiDoc XRS (Bio-Rad, USA). GAPDH was used as the loading control.

Measurement of serum alanine (ALT) and aspartate aminotransferase (AST) and liver index

At the end of the experiment, the mice were sacrificed, and blood samples were collected from the retro-orbital sinus to obtain the serum. The liver function-related ALT and AST were measured using an automatic biochemical analyzer (Hitachi, Japan). Liver samples were harvested and rinsed with saline. After being drained by using filter paper, the samples were weighed. Liver index=liver weight/body weight×100%.

Liver histopathological analysis

At the end of the experiment, the mice were sacrificed, while their liver was removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections (4-µm thick) were obtained by cutting and staining with hematoxylin and eosin (H&E). The stained sections were visualized under a light microscope for histological evaluation.

Statistical analysis

The data for each experimental group were presented as mean±standard deviation (SD). The differences between the two sets of data were evaluated using one-way analysis of variance by using the SPSS software; P<0.05 was considered significant.

Results

Effects on BW, food and water intake

The BWs of the mice gradually increased for all experimental groups (Fig. 1A). The BW of the model group was higher than that of the control group throughout the experimental period. The BWs of the treatment groups had no significant changes as compared with the model group.

The daily food and water intake levels of the model group were significantly higher than those of the control group (Fig. 1B–1C, P<0.01). The daily food intake level of the Piog group and XKQ high/medium/low-dose groups increased by about 52.6%, 41.3%, 47.9%, and 36.9% and daily water intake rate increased by 85.9%, 63.5%, 49.5%, and 42.3%, respectively, compared with that of the model group.

Effect on the FBG of the diabetic model mice

Compared with the model group, the Piog group showed a significant reduction in FBG. XKQ administration reduced the FBG to some extent. The FBG levels at high and medium dose of XKQ were lower than that in the model group (P<0.05, Fig. 2). The FBG at low dose of XKQ also significantly decreased compared with the model group (P<0.05) starting from the 4th week of administration.

Effect on the OGTT of the diabetic model mice

OGTT was conducted in the eighth week, and the results are shown in Fig. 3. In contrast with the control group, the AUC in blood glucose of the model group was higher (P<0.01). The AUC of the XKQ treated and Piog groups were significantly lower (P<0.05) than that of the model group, which indicates that the glucose tolerance of the T2MD KKAy mice had been greatly improved in all treatment groups.

Effect on the HOME–IR of the diabetic model mice

The pre-experimental results are shown in Fig. 4. Compared with the control group, the FIN, FBG, and HOMA–IR index for the model group were significantly higher (P<0.01, Fig. 4). No significant difference was observed in the FIN level between the treatment and model groups. Compared with the model group, the FBG and HOMA–IR index of the treatment groups were significantly decreased (P<0.05, P<0.01).

Effect on the serum ALT and AST and liver index of the diabetic model mice

As shown in Fig. 5A, the levels of ALT and AST were significantly increased in the model group compared with the control group (P<0.01). All XKQ treated groups showed a significant decrease in the ALT level compared with the model group (P<0.05). Similarly, the XKQ treatment groups showed a tendency toward reduced AST levels compared with the model group. However, no significant difference was found between the Piog and model groups.

For the liver index, it was significantly higher in the model group than in the control group (P<0.01, Fig. 5B), whereas all XKQ treated groups showed a decreasing trend compared with the model group. Moreover, the liver indices for the high and middle XKQ dose groups were significantly lower (P<0.05).

Pathological examination

The pathological examination (Fig. 6) shows that the liver for the control group was characterized by orderly composed cells, the hepatic sinusoid was free from blood stasis, and the portal area was free from inflammatory cell infiltration and fibroblastic proliferation. In contrast, serious fatty degeneration was observed in liver cells with a massive accumulation of fat vesicles in the cytoplasm of the model group. The XKQ and Piog treatment reduced the hepatic lipid accumulation in the KKAy mice, while the high-XKQ dose group displayed the best effect.

Effect on the protein expression of IRS-1, PI3K, p-Akt, and GLUT4 in the muscle tissue of diabetic model mice

The PI3K/Akt signaling pathway is closely related to the development of diabetes and the mechanism for decreased insulin sensitivity. As shown in Fig. 7, the protein expression levels of IRS-1, PI3K, p-Akt, and GLUT4 in the diabetic model group were much lower (P<0.01) than that of the control group. After 8 weeks of XKQ treatment, all protein expression levels improved (P<0.05 or P<0.01) compared with those of the model group. The results show that XKQ treatment can increase the protein expression levels of IRS-1, PI3K, p-Akt, and GLUT4 in diabetic mice. Therefore, XKQ had a positive effect on the PI3K/Akt signaling pathway.

Discussion

KKAy mice are a popular T2DM animal model [²⁹], with syndromes including polyphagia, polydipsia, polyuria, hyperglycemia, IR, and obesity. Our results show that XKQ treatment significantly decreases FBG and the food and water intake in T2DM KKAy mice. These findings are consistent with clinical studies [¹³].

T2DM disorders are typically associated with impaired glucose tolerance and IR [⁵]. Reduced sensitivity to insulin in peripheral target tissues such as the liver, muscles, and adipose tissues leads to abnormal insulin secretion, ultimately resulting in hyperglycemia. The IR in peripheral target tissues, particularly in the skeletal muscles, is a major cause and the main therapeutic target of IR in T2DM [⁶]. In the present study, XKQ improved the glucose tolerance in diabetic KKAy mice. In addition, XKQ can significantly decrease the HOMA–IR index in KKAy mice (Fig. 4).

To further investigate the hypoglycemic mechanisms of XKQ in diabetic KKAy mice, the expression of some key proteins in the insulin signaling pathway were analyzed. The PI3K/Akt pathway is the main signal transduction pathway downstream of InsR, which is closely related to the development of diabetes and decrease in insulin sensitivity [³⁰]. The skeletal muscle InsR substrate (IRS)-1 serves as the major docking protein and undergoes tyrosine phosphorylation through the activated InsR in regions containing specific amino acid sequence motifs. The phosphorylated tyrosine residues of IRS-1 mediate an association with the 85-kDa regulatory subunit of PI3K, thereby activating other downstream enzymes. The activated PI3K and Akt facilitate the translocation and fusion of GLUT4-containing vesicles to the membrane, increasing the GLUT4 transporters and glucose intake on the membrane [³¹,³²]. When IR occurs, the activation of the PI3K/Akt pathway may be blunted [³³,³⁴]. Our preliminary results show that XKQ can significantly reduce the IR in KKay mice. In the present study, XKQ can upregulate IRS-1, PI3K (p85), p-Akt, and GLUT4 protein levels in the muscle tissues of KKAy mice. Thus, XKQ might reduce IR by regulating the PI3K/Akt pathway in KKAy mice.

Diabetic liver injury is a common complication of diabetes, as about 50%–75% of T2MD patients suffer from non-alcoholic fatty liver disease (NAFLD) [³⁵]. The coexistence of NAFLD and diabetes mellitus increases the risk of developing severe forms of NAFLD and chronic vascular complications of diabetes mellitus [³⁶]. The fat levels in the liver are closely associated with IR [¹⁰]. More evidence indicates that berberine [³⁷], a main component of XKQ, has beneficial effects on NAFLD. Increased insulin sensitivity, regulation of the adenosine monophosphate-activated protein kinase pathway, improvement of mitochondrial function, and the alleviation of oxidative stress are the major targets of berberine in the treatment of NAFLD. In addition, mangiferin, which is another main component of XKQ, eliminates liver steatosis and prevents adiposity in the mice. The same beneficial effects on the liver were found in the present study, where KKAy mice developed hepatic steatosis and liver function abnormality. The results also indicate that the levels of ALT and AST were significantly increased in the model group. Moreover, treatment with XKQ not only decreases the serum ALT and liver index but also ameliorates hepatic steatosis and liver lesions in obese diabetic KKAy mice. As a new concept, caloric restriction rapidly lowers hepatic fat content and improves insulin sensitivity in T2DM patients [³⁸]. The combined implications of these studies are that excess fat in the liver, regardless of whether saturated or unsaturated, causes IR. Therefore, future therapeutic strategies targeting hepatic IR should be aimed at lowering hepatic fat. Hence, XKQ may reduce hepatic IR by reducing fat levels in the liver.

In summary, XKQ treatment can reduce FBG and alleviate hepatic fatty degeneration in diabetic KKAy mice. The hypoglycemic effect of XKQ may be caused by the improvement of the PI3K/Akt signaling pathway to reduce IR. These findings can help us to understand the mechanism of XKQ and support that the XKQ is an applicable therapeutic treatment for T2DM.

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