Dried A. mongholicus, P. montana and M. alba L. were purchased from Guangzhou University of Chinese Medicine Pharmacy Co., Ltd. A. mongholicus, P. montana, and Morus alba L. were powdered and mixed in a ratio of 1:2:1. The mixture was extracted with 60% ethanol (1:8, wt/wt) for 2 h. Extraction of filtered residue was repeated for three times. The extracting solution was mixed, filtered, and concentrated to 0.12 g/ml. The concentrated solution was purified through PIPO-OO, HPD-500 macroporous resin and 732 ion exchange resin. The APF powder was obtained after freeze-drying. The ingredients of APF were determined by high-performance liquid chromatography (HPLC) and HPLC evaporation light scattering detection (HPLC-ELSD). Three representative compounds including Astragaloside IV, pueparin and calycosin-o-β-D-glucopyranoside were identified and determined as quality control. The specific content and chromatographic condition were provided in previous Supplementary Data and Table 1.

Normal chow, high-fat-diet (60% fat) and 8-week-old male C57BL/6J mice were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China), and 8-week-old male B6.V-LepOb/LepOb (ob/ob) mice were obtained from Nanjing Biomedical Research Institution of Nanjing University (Nanjing, China). The mice were housed under pathogen-free conditions in a temperature-controlled room illuminated for 12 h every day and received humane care in accordance with the study guidelines established by the Guangzhou University of Chinese Medicine Laboratory Animal Holding Care. Following acclimation for 1 week, all C57BL/6J mice [except for 10 C57BL/6J mice as normal control (n = 10)] intraperitoneally received 120 mg/kg STZ once. After 3 weeks, the hyperglycemic mice were classified into four groups, namely, type 2 diabetic (n = 10), metformin (n = 10), 0.5 g/kg APF (n = 10), and 1.0 g/kg APF groups. The normal control group (NC) mice were fed with normal chow and treated with 5% acacia gum solution (p.o.). The T2DM, metformin, 0.5 g/kg APF, and 1.0 g/kg APF groups were fed with 60% HFD and treated with 5% acacia gum solution, 250 mg/kg metformin, 0.5 g/kg APF, and 1.0 g/kg APF (p.o.), respectively. After 12 weeks, all mice were sacrificed through cervical dislocation after anesthesia. Tissues were snap-frozen or fixed in formalin.

Type 1 diabetes (T1DM) was induced in mice by STZ. Except for 10 C57BL/6J mice as normal control (n = 10), all C57BL/6J mice intraperitoneally received five consecutive doses of 45 mg/kg STZ. After 2 weeks, the hyperglycemic mice were classified into T1DM (n = 6) and APF groups (n = 6) both fed with normal chow. Normal control (NC) and T1DM mice were treated with 5% acacia gum solution (p.o.). The APF group was treated with 1.0 g/kg APF (p.o.). After 6 weeks, all mice were sacrificed through cervical dislocation after anesthesia. Tissues were snap-frozen or fixed in formalin.

Ob/ob mice were designed into ob/ob (n = 8) and APF groups (n = 16) both fed with normal chow and individually treated with 5% acacia gum solution and 1.0 g/kg APF (p.o.), respectively. After 13 weeks, the APF mice were allocated to APF (n = 8) and APF + leptin groups (n = 8) that intraperitoneally received saline and 0.8 mg/kg recombinant rodent leptin, respectively, every 6:00 p.m. After 3 weeks, all mice were sacrificed through cervical dislocation after anesthesia. Tissues were snap-frozen or fixed in formalin.

All animal experimental protocols were approved by the Institutional Animal Ethics Committee of Guangdong Pharmaceutical University (GDPULACSPF No. 2012062) in compliance with the revised Animals (Scientific Procedures) Act 1986 in the UK.

Blood sample was collected from retinal vein plexus after the mice were fasted overnight or with feeding at 9:00 a.m. Mice were anesthetized by ether. Plasma was harvested after centrifugation. Plasma glucose (Glu), triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were determined using commercial kits from Rsbio (Shanghai, China). Non-esterified fatty acid (NEFA) was analyzed using NEFA assay kit from Wako (Osaka, Japan). Plasma insulin and leptin were examined using ELISA commercial kits from Cusabio (Wuhan, China). Hepatic TG and TC were extracted by isopropanol (1 mg tissue/20 μl isopropanol) allowed to stand at 4°C overnight after homogenization. Supernatants were harvested after centrifugation and determined using commercial kits.

Glucose (2 g/kg) was intragastrically administered to mice that fasted overnight. Insulin (1 U/kg) was intraperitoneal injected to mice that fasted for 6 h. Insulin sensitivity was evaluated using the homeostatic model assessment of insulin resistance [HOMA-IR, fasting blood glucose (mmol/L) × fasting serum insulin [(mIU/L)/22.5].

Livers, pancreas, and adipose tissues were fixed in formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. For Oil Red O staining, hepatic tissues were embedded in optimal cutting temperature compound, sectioned, and stained with Oil Red O.

Total RNA was isolated by homogenizing tissues in Tiangen TRIzol reagent (Beijing, China), and single standard cDNA was synthesized by using Tiangen cDNA kit (Beijing, China). Quantitative real-time PCR was performed with Thermo Scientific PikoReal 96 Real-Time polymerase chain reaction (PCR) System (Waltham, MA, United States). Primer sequences are listed in Table 2.

Total protein extracts were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 for 2 h at room temperature and incubated with anti-PTP1B (Abcam, United States), anti-TCPTP, and anti-GAPDH (Cell Signaling Technology, United States) at 4°C overnight, rinsed three times with TBST, and incubated with respective secondary antibodies for 2 h at room temperature. Protein bands were visualized with Thermo Fisher Scientific SuperSignal West Femto Maximum Sensitivity Substrate (Rockford, United States) and captured using an Image Quant LAS4000 imaging system (Shanghai, China).

All results were expressed as means ± standard error of the mean. Data were analyzed by Kolmogorov-Smirnov and Mann-Whitney U test for normal distribution analysis. Data from more than two groups were analyzed by one-way ANOVA. Student’s t test was performed to identify differences between two groups. p < 0.05 was considered significant.

After feeding with HFD 10 weeks, the T2DM group exhibited hyperglycemia, hyperlipidemia, hyperinsulinemia, impaired glucose tolerance, and elevated HOMA-IR index (Figures 1A–E; Supplementary Figures S1A,B), indicating the strong development of T2DM. However, the levels of fasting glucose, insulin, and HOMA-IR were normalized by treating with 0.5 g/kg APF, 1 g/kg APF, and metformin, respectively (Figures 1A–C). Meanwhile, STZ administration elicited the injury of morphology of pancreas in T2DM group (Supplementary Figure S1C), which was attenuated by 0.5 and 1 g/kg APF treatment (Supplemental Figure S1C). These data suggested that APF reshapes the STZ-injured pancreas and reduces the compensatory secretion of insulin. Therefore, the HFD-induced insulin resistance and hyperinsulinemia are alleviated by APF treatment.

STZ administration significantly reduced the body weight of mice (Figure 2A). After feeding with HFD, the T2DM group had higher body weight gain than the NC group (Figure 2B). Fat mass weight was also monitored, and the results showed that epididymal fat, subcutaneous fat, and total lipid mass were significantly enhanced after HFD feeding (Figures 2C–E). Plasma leptin levels proportional to the fat mass also increased with HFD feeding (Figure 3A). In addition to body mass, liver weight, and hepatic TC, the TG content was significantly higher than that in the NC group (Figures 3B–D). Histological staining also displayed substantial amounts of hepatic macrovesicular steatosis in the T2DM group (Supplementary Figure S2). These results showed that the T2DM group turned obese and developed NAFLD accompanied with insulin resistance. With regard to the biological function of leptin in anti-ectopic lipid accumulation, the T2DM group showed insulin and leptin resistance.

APF treatment significantly inhibited the increase in body weight gain (Figure 2B). Epididymal fat mass and total lipid mass also deceased with APF intervention (Figures 2C,E). Moreover, 1 g/kg APF treatment diminished the diameter of the adipocyte and might have contributed to improving the endocrine function of adipose tissues (Supplementary Figure S3). Thus, the hyperleptinemia of T2DM mice was normalized by 1 g/kg APF treatment (Figure 3A). Finally, APF administration reduced the hepatic TC and TG contents and remarkably diminished the hepatic macrovesicular steatosis without effects in daily diet consumption (Figures 3C–E; Supplementary Figure S2). The results indicated that APF attenuates obesity, hyperleptinemia and NAFLD. Based on the effects of APF in insulin sensitivity. We considered APF reversed the insulin-leptin resistance though regulating the adipoinsular axis.

Leptin-deficient ob/ob mice were selected to validate whether leptin is essential for APF improve insulin sensitivity by regulating adipoinsular axis (Supplementary Figure S4C). In the status of absent of leptin, APF could not normalize the hyperglycemia and hyperinsulinemia of ob/ob mice (Figures 4A,B). APF even increased the levels of plasma insulin and HOMA-IR index (Figures 4B,C). We considered that APF may still influence the endocrine function of pancreas, but the effects are unpredictable without leptin. Monitoring revealed that insulin resistance was robust in ob/ob mice with APF treatment (Figures 4D,E).

Based on the physiological effects of adipoinsular axis, the pathological features of obesity and NAFLD in ob/ob mice with APF administration were examined. Different from that in STZ + HFD induced T2DM mice, the weight-loss effects of APF were not observed in ob/ob mice (Figures 5B,D). Meanwhile, the hepatic macrovesicular steatosis of ob/ob mice was not reversible by APF intervention (Supplementary Figure S5). Although APF reduced the hepatic weight, APF failed to decrease the levels of hepatic TG and TC (Figures 6A–C). On the basis of body weight, the fat mass of ob/ob mice was examined. APF could not reduce the subcutaneous fat mass and even enhanced the epididymal fat mass in ob/ob mice (Figures 6D,E). As implied by these data, the destruction of adipoinsular axis in the leptin side would abolish the effects of APF. APF lost its effects of anti-insulin resistance, obesity, and NAFLD without leptin regulation but still had minimal influence on pancreas and adipose tissues.

Ob/ob mice were administered with recombinant rodent leptin (i.p.) after APF treatment for 13 weeks (Supplementary Figure S4C). Hyperglycemia, hyperinsulinemia, HOMA-IR, and insulin tolerance were normalized after leptin treatment (Figures 4A–E). Leptin supplementary treatment significantly inhibited their daily diet consumption and body weight (Figures 5A–D). For NAFLD and fat mass, leptin administration attenuated hepatic steatosis and reduced hepatic TC and TG levels (Supplementary Figure S5; Figures 6A–C). Therefore, fat mass, including epididymal and subcutaneous fat, was decreased by leptin supplement (Figures 6D,E). Leptin also diminished the diameter of ob/ob mouse adipocytes, which was the same effect as APF in STZ + HFD-induced T2DM mice (Supplementary Figures S3, S6). All these data were confirmed by previous studies of leptin and validated the effects of adipoinsular axis from another point of view.

We hypothesized that APF ameliorate diabetes by regulating adipoinsular axis. Except for the leptin, insulin also must be essential for the hypoglycemic effects of APF. As expected, APF could not ameliorate diabetes and metabolic syndromes without leptin presence in ob/ob mice. Then, a classic T1DM mice model was established to investigate the anti-diabetic effects of APF in absence of inuslin. After STZ administration, the mice showed body loss, hyperglycemia, and insulin deficiency (Figures 7A–C). However, the hypoglycemic effects of APF in T2DM mice was not observed in T1DM mice (Figure 7B). In addition, the levels of plasma insulin was hardly detected with APF treatment (Figure 7C). Meanwhile, APF also had no effects in TC and TG in T1DM mice (Figure 7D, E). Therefore, both hormones (leptin and insulin) are indispensable in regulating the adipoinsular axis by APF. Abolishment of each hormone would disrupt the effects of APF for anti-diabetes, obesity, and NAFLD.

APF ameliorated insulin resistance and NAFLD by improving the adipoinsular axis. The underlying mechanism of these effects was then investigated. Hepatic lipid metabolic gene expression was determined based on the decreased hepatic lipid accumulation by APF. APF treatment significantly suppressed the expression of lipogenic gene, fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD1) (Figure 8A). Both genes contribute to hepatic de novo lipogenesis (Wang et al., 2015). On the contrary, APF alleviated the hepatic expression of gluconeogenesis gene phosphoenolpyruvate carboxykinase (PEPCK), a target gene for leptin to improve insulin sensitivity (Burcelin et al., 1999). These data suggested that APF improves the hepatic biological effects of insulin–leptin and suppresses the expression of hepatic lipogenic and gluconeogenic genes. Protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) are negative regulators of insulin and leptin reaction (Thareja et al., 2012; Zhang et al., 2015). Thus, their expression levels were determined by real-time PCR and Western blot. The genetic and protein expression levels of PTP1B in STZ + HFD-induced T2DM mice were significantly enhanced (Figures 8B,C), and this finding was consistent with the phenotype of dysfunction of adipoinsular axis in mice. However, APF treatment significantly reversed the overexpression of PTP1B but not TCPTP (Figures 8B–D), implying that APF improves the adipoinsular axis balance by suppressing PTP1B and consequently contributes to the attenuation of obesity, NAFLD, and T2DM.