STZ was obtained from Sigma-Aldrich Inc. (Sigma, S0130, USA). Analytical grade catalpol (product >99 % purity) was purchased from Liubobainiao Biotechnology Co., Ltd (Shijiazhuang, China). Blood glucose Span Diagnostic kit and Jinque test strips was obtained from Shanghai MicroSence Inc. (Shanghai, China). Blood lipid commercially available kits, CAT, GSH-PX, SOD and MDA commercially available kits were from Nanjing Jiancheng Bioengineering Institute (China).
Experimental animals and feeds
One hundred twenty male Wistar rats (200–250 g) were procured from the Experimental Animal Center, Chongqing Medical University, China. The rats were arbitrarily divided into four groups: normal group (n = 8), high-sugar and high-fat feed (high-fat, n = 8) group, STZ-diabetic animals with high-sugar and high-fat feed (STZ-fat, n = 12), and STZ-diabetic animals with high-sugar and high-fat feed treated with catalpol (STZ-fat-cat, n = 12).
Rats were kept in an air-conditioned room maintained at a constant temperature (24–26 °C) and humidity (50–60 %) under a 12-h light/dark cycle (07:00 on and 19:00 off). Standard rat feed and water were provided ad libitum. The rats were allowed to acclimatize to the laboratory environment for 7 days before the start of the experiment. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals in China (Permit: SCXK 2002A040), and the international guidelines on the ethical use of animals (NIH publications No. 80–23, revised 1996).
The high-sugar, high-fat diet was comprised of normal diet 54 kg, sucrose 16.5 kg, lard 8.31 kg, egg yolk 4.14 kg, and salt 83.4 g, with a total combined weight of 83.784 kg [14, 15]. The normal diet was purchased from Beijing China Fukang Biotechnology Co., Ltd (Beijing, China). The main components of the normal diet were crude protein ≥18 %, crude fat ≥4 %, crude fiber ≥5 %, crude ash ≤8 %, moisture ≤10 %, lysine ≥0.82 %, calcium 1–1.8 %, phosphorus 0.6–1.2 %, and salt 0.2–0.8 %.
Induction of diabetes in rats
Diabetes was induced by an initial high-sugar and high-fat feed for 3 weeks, followed by an intraperitoneal injection of STZ (30 mg/kg) for 3 days. STZ was dissolved in a freshly prepared 0.01 M citrate buffer (pH 4.5). The normal control group was injected with buffer alone. Rats with blood glucose ≥16.7 mmol/L for 3 weeks were considered as diabetic  and the death rates for modeling of STZ-fat were 22–28 %.
Experimental design and catalpol treatment of rats
At the first stage, catalpol was injected into the rat tail vein at a dose of 0, 5, 10, 20, or 50 mg/kg. Blood was collected after catalpol treatment for 14 days to assess the blood glucose-lowering effect of catalpol.
At the second stage, the most effective dose of catalpol was selected for further study. The plasma lipid level was detected in the normal, high-sugar and high fat feed, STZ-fat, and STZ-fat-cat groups at 14 days after treatment with catalpol. The plasma in each group was collected and then antioxidative enzymes such as GSH-PX, SOD, CAT, and the MDA oxidative products were detected with corresponding kits. Oral glucose challenge tests (OGCT) were then performed on experimental rats using a Roche blood glucose monitor (Roche Diagnostics, Rotkreuz, Switzerland.) with Jinque test strips. During the experiment, body weight, food intake, and water intake were recorded every 2 days. Finally, a splenic portion of pancreatic tissue was removed immediately after sacrifice to observe morphological changes of the pancreas.
Determination of plasma glucose
Blood samples were collected from the inner canthus using a capillary tube under chloral hydrate anesthesia at each time point. Concentration of blood glucose was determined by a glucose meter (Roche Diagnostics, Rotkreuz, Switzerland) with Jinque test strips (Span Biotech Ltd, India).
Plasma collection and biochemical determination
Blood samples were collected from the inner canthus using a capillary tube under chloral hydrate anesthesia after catalpol treatment for 14 days. The samples were centrifuged at 2810×g for 10 min at 4 °C within 1 h after collection, and then supernatants were collected. The concentration of plasma total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C); the activities of SOD, GSH-PX, CAT and the concentration of MDA were determined by a series of commercially available kits including SOD, GSH-PX, CAT, and MDA kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturers’ instructions.
Oral glucose challenge test
An OGCT was performed according to a method previously described with minor modifications [17–19]. Briefly, catalpol at the most effective dose, or the same volume of vehicle, was injected intravenously into rats for 14 days. Then a glucose dose of 2.5 g/kg was intragastrically administrated to the rats. Blood samples were collected sequentially from the tail vein before (0 h) and 0.5, 1, 2, 3, 5, 7, and 9 h post challenge. Plasma glucose changes at the indicated time were determined with a glucose meter (Accu-Chek, Switzerland) and Jinque test strips.
Histological evaluation of pancreas with hematoxylin and eosin (H&E) staining
A splenic portion of pancreatic tissue was removed immediately after sacrifice and rinsed with ice-cold saline. The tissue samples were fixed in 4 % buffered neutral paraformaldehyde solution overnight, embedded in paraffin, and deparaffinized using standard procedures [20, 21]. Thin Sections (5 µm) were dewaxed, dehydrated in a graded series of ethanol, and rehydrated, then stained with H&E for light microscopic examination (Leica, Germany). All histological analyses were performed in arbitrarily selected fields in sections by two investigators blinded to the identity of the treatment groups.
All data were analyzed by the SPSS statistical software (version 13.0, SPSS, Chicago, IL, USA), with P < 0.05 values were considered as statistically significant. Results were expressed as mean ± standard deviation (SD). Comparisons between the groups were assessed by paired-samples t test, and P value correction for multiple group comparison by LSD-t test. Dose-dependent manner of catalpol’s hyperglycemic action was visually determined from a dose–response curve.