Oleanolic acid and moderate drinking increase the pancreatic GLP-1R expression of the β-cell mass deficiency induced hyperglycemia

Animals and experimental procedure

Male Sprague Dawley (SD) rats (7–8 weeks of age) were obtained from the Experimental Animal Center of Xi’an Jiaotong University (SCXK 2012-003). The animals were maintained in a 12 h/12 h light and dark cycle at the temperature of (23 ± 2) °C and free access to food and water. All animal experiments were carried out following the National Institutes of Health guide for the care and use of Laboratory animals and the guidelines of the Experimental Animal Ethics Committee of Shaanxi University of Chinese Medicine (SUCMDL20160113001).

Fifty rats were injected with 55mg/kg STZ to generate hyperglycemia according to our previous method (Xu, Jois & Cui, 2022). Rats with blood glucose levels ≥16.7 mmol/L were selected for further treatment. Hyperglycemia rats were randomly divided into the hyperglycemia group (10 rats), OA group (10 rats), EtOH group (8 rats), and OA+EtOH group (10 rats). 10 rats were injected with an equal dose of 0.1 M sodium citrate buffer (pH = 4.4) as the control group. Control and hyperglycemia groups were treated with normal saline (10ml/kg/d), the OA group was administered with 100 mg/kg/d of OA suspension, the EtOH group was administered with 5% V/V of ethanol (10 ml/kg/d) which was based on previous research (Adaramoye & Oloyede, 2012; Hu et al., 2014), and the OA+EtOH group was treated with 100 mg/kg OA mixed in 5% V/V of ethanol. The body weight and FBG concentrations were measured every week, food intake was recorded regularly. Rats were anesthetized with 2% isoflurane after 6 weeks of treatment, then blood, livers, and pancreas were collected. The pancreas tissue used for qPCR, was immediately snap-frozen in liquid nitrogen and kept in RNase free tubes at −80 °C before RNA extraction. The liver and pancreas used for the sections were firstly rinsed with ice-cold PBS, then fixed with 4% paraformaldehyde for 12 h. After sample collection, all animals were euthanized with 5% of isoflurane.

HE staining

Paraffin embedded pancreatic tissue was sectioned into 3 µm and placed onto positively charged glass slides. After de-waxed and rehydrated sections were stained in hematoxylin solution for 5 min. Followed by eosin red staining for 15 s, samples were dehydrated and sealed with neutral balsam.

Immunohistochemical staining

Pancreatic sections were de-waxed and rehydrated, then incubated with 3% hydrogen peroxide for 20 min. After antigen retrieval, sections were blocked with a 5% BSA for 30 min. Rabbit anti-insulin (1:50; BM4310; Boster, Wuhan, China) or rabbit anti-GLP-1R (1:150; bs-1559R; Bioss, Beijing, China) was applied as the primary antibody to detect insulin and GLP-1R expression. The labeled avidin-biotinylated rabbit IgG kit (SA1022, Boster, Wuhan) was selected to amplify the antibody signal. The antibody-antigen complex was visualized by incubating the samples in DAB chromogen (AR1022, Boster, Wuhan) and counterstained with Harris hematoxylin.

Immunofluorescent staining

Sections were firstly incubated with primary antibodies rabbit anti-insulin (1:50; BM4310; Boster, Wuhan, China) and mouse anti-glucagon (1:200; BM1621; Boster, Wuhan). After thoroughly washing with TBST, samples were then incubated with FITC conjugated goat anti-rabbit IgG (1:50; BA1105; Boster, Wuhan, China) and Cy3 conjugated goat anti-mouse IgG (1:50; BA1031; Boster, Wuhan, China). Finally, sections were cover-slipped with Boster AR1109 (Boster, Wuhan, China) mounting medium.

PAS staining

Liver glycogen was measured using a Glycogen Periodic Acid Schiff(PAS/Hematoxylin) Stain Kit (G1281; Solarbio, Beijing, China) and the assay was performed according to the manufacturer’s instructions.

RNA extraction and qRT-PCR

Total RNA was extracted from pancreas tissue using the Takara RNAiso Plus reagent (D9109, Takara, Japan). The concentration of total RNA was determined by spectrophotometry (Nanodrop 2000; Thermo Scientific, Waltham, MA, USA). Reverse transcription for cDNA was synthesized from total RNA using a PrimeScript RT Master Mix Kit (RR037A; Takara, Shiga, Japan). The real-time PCR was carried out on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with an SYBR Green premix EX Taq II kit (RR820A; Takara). The results for each specific gene were normalized to the ribosome 18s RNA gene. The expression level of each gene was calculated by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). The primer sequences used in this study were:18sRNA (F) GAC TCA ACA CGG GAA ACC TCA CC and (R) ACC AGA CAA ATC GCT CCA CCA AC; Ins1 (F) GGA CCC GCA AGT GCC ACA AC and (R) TGA TCC ACA ATG CCA CGC TTC TG; Gcg(F) ACC GTT TAC ATC GTG GCT GGA TTG and (R) TCT GGC GTT CTC CTC CGT GTC.

Image acquisition and statistical analysis

An Olympus BX41 microscope (Olympus, Tokyo, Japan) was used to image HE staining, PAS staining, and Immunohistochemical staining (IHC). Immunofluorescent staining (IFC) pictures were acquired by a Zeiss LSM 5 Pascal confocal microscope (Jena, Germany).

Cell counting and positive staining area selection method was used as our previous work (Xu, Jois & Cui, 2022). Randomly selected images from 3–5 animals of each group were used for analysis with image J software. For PAS staining the glycogen expression level was demonstrated semi-quantitatively as the percentage of the PAS positive staining area/liver area. For IHC and IFC staining, insulin, glucagon, and GLP-1R positive cells were counted from the captured islets. The liver tissue pictures or islet number used for analysis was marked as per figure notes.

All the data were reported as the mean ± SEM. The statistics were processed using the GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). If the data passed the normality test or data set ≥30, two-tailed t-test was used for comparison between the two groups, and the multiple groups’ variance analysis was used one-way ANOVA with post hoc Tukey–Kramer test. Nonparametric Mann–Whitney test was performed for data which not passed the normality test. A p value less than 0.05 was considered significant difference.

OA and moderate drinking alone or in combination suppressed STZ induced hyperglycemia

STZ injection significantly increased the FBG level as compared to the control group (p < 0.001); the average FBG level in OA, EtOH, and OA+EtOH groups fluctuated from 11.3–28.8 mmol/L throughout the treatment but were lower than in the hyperglycemia (25–32.1 mmol/L) group (Fig. 1A). When counted the 6 weeks together, the average FBG level of OA, EtOH and OA +EtOH groups were significantly decreased compared to the hyperglycemia group (p < 0.05, Fig. 1B). The body weight in the hyperglycemia group was dramatically decreased compared to the control group, but it did not differ from all the other treatment groups (Fig. 1C). Diabetic rats consumed more food than control, whereas the food intake of OA, EtOH and OA+EtOH were not different from the hyperglycemia group (Fig. 1D).

HE staining showed that the islets in the control group were intact and in oval shape, but the islets in the hyperglycemia group were destroyed and irregular in shape. All the 3 treatment groups affected the islet size. The islets in the OA group were small and deformed; the islets size of EtOH group was augmented and the islets in OA+EtOH group were irregular in size and tend to be bigger than that in hyperglycemia group (Fig. 1E, dotted circle).

OA enhanced the moderate drinking induced liver glycogenolysis of STZ induced hyperglycemia

During the fasting state, hepatic glycogenolysis is crucial to maintain blood glucose level, so glycogen level can represent the status of liver glycogenolysis. To determine the live glycogen changes, PAS staining was performed. The PAS positive staining in the control group was located in the hepatocellular soma and sparsely distributed, after STZ treatment the PAS positive signal was intensively expressed in the liver (Fig. 2A, star). The PAS staining pattern in the OA group was similar to the hyperglycemia group; PAS positive signal was mainly found around the portal area and central vein of the EtOH group, while very less PAS staining could be observed in the OA+EtOH group (Fig. 2A). The concentration of liver glycogen level was calculated as the percentage of PAS positive area. Rats in the hyperglycemia group had notably elevated glycogen level compared to the control group; OA treatment significantly increased the glycogen level; EtOH treatment down-regulated the heightened glycogen level and OA+EtOH neutralized the hyperglycemia boosted glycogen expression (Fig. 2B).

Ethanol neutralized OA raised islet β-cell ratio of STZ induced hyperglycemia

To detect the islet β-cell number, pancreatic tissues were immunohistochemically stained for insulin. As expected, the insulin positive staining was distributed all over the pancreatic islets of the control group, and there were few dark stained β-cells randomly located in the islets’ residue of hyperglycemia group. The insulin expression pattern in the islets of the OA group was similar to the hyperglycemia group, except few insulin positive cells in acinar cells outside of islets. In the EtOH and OA+EtOH group, the islets were larger and the insulin-positive cells were irregularly distributed among the islets (Fig. 3A, red dotted circle).

The islet β-cell number dramatically decreased after STZ injection and OA, EtOH or OA+EtOH treatment did not significantly affects the β-cell mass of hyperglycemia rats (Fig. 3B). The total islet cell number in hyperglycemia group was significantly decreased compared to the control group. EtOH and OA+EtOH treatments significantly increased total islet cell number compared to OA (Fig. 3C). In the control group, the proportion of β-cell to total islet cell was about 72%. The β-cell ratio for the hyperglycemia group was only 1/3 as in the control group. OA significantly upregulated the β-cell to total islet cell ratio compared to the hyperglycemia and EtOH group (Fig. 3D), while EtOH increased the islet area as compared to the OA and hyperglycemia group (Fig. 3E).

OA and moderate drinking had opposing effects on the α-cell ratio of STZ induced hyperglycemia

The two major islet cell types α-cell and β-cell secrete insulin or glucagon to maintain glucose homeostasis. Double staining of insulin and glucagon was performed to investigate the α-cell and β-cell expression patterns in the islet. In the control group, the β-cells were dominantly expressed in the center of the pancreatic islet which was surrounded by a ring of α-cells. There were only a few β-cells surrounded by the massively expressed α-cells, in the islet of the hyperglycemia group. The general expression pattern of α-cell and β-cell in OA, EtOH, or OA+EtOH group was similar to hyperglycemia group based on the observation of images (Fig. 4A).

STZ induced hyperglycemia decreased the number of β-cell in the islet and OA, EtOH, or OA+EtOH treatment did not significantly alter the β-cell cell expression (Fig. 4B). Interestingly, the combined cell number of α and β-cells in EtOH and OA+EtOH groups were raised when compared to the OA group (Fig. 4C), which could either be a result of the increase in α-cell number (Fig. 4E) or α-cell/ β-cell ratio (Fig. 4F). The β-cell to α-cell plus β-cell ratio was increased in the OA group when compared to hyperglycemia or EtOH group (Fig. 4D) and it was inversely related to α-cell to the β-cell ratio (Fig. 4F) or α-cell to α-cell plus β-cell ratio (Fig. 4G).

OA and moderate drinking increased the GLP-1R positive cell ratio of STZ induced hyperglycemia

GLP-1R in the islet contributes to glucose regulation by improving β-cell function and promoting islet α-cell to β-cell trans-differentiation (Lee et al., 2018). To test the effects of OA, EtOH, or OA+EtOH on pancreatic GLP-1R expression, pancreases were immuno-histochemically stained for GLP-1R. GLP-1R was expressed all over the islet of the control group. In the islet of hyperglycemia group, GLP-1R was stained with brown color and randomly distributed in some cell soma. The expression pattern of GLP-1R in the OA group was similar to hyperglycemia group but stained in a darker color. GLP-1R positive cells were disorderly located in the expanded islet of the EtOH and OA+EtOH group (Fig. 5A).

In comparison with hyperglycemia group, OA, EtOH or OA+EtOH treatment increased the number of positive GLP-1R cells (Fig. 5B) as well as GLP-1R positive cell to total islet cell ratio (Fig. 5D). Although the total islet cell number of EtOH or OA+EtOH was augmented when compared to the OA group (Fig. 5C), only EtOH had a larger islet size compared to OA and hyperglycemia group (Fig. 5E).

OA and 5% ethanol rescue hyperglycemia suppressed Ins1 mRNA expression

To verify the gene expression pattern of insulin and glucagon, Ins1 and Gcg mRNA expression levels were tested. STZ treatment significantly suppressed the Ins1 expression, and OA, EtOH, or OA +EtOH treatment upregulated the Ins1 expression, but only EtOH and OA +EtOH groups were significantly different from hyperglycemia group (Fig. 6A). Compared to the control, hyperglycemia dramatically increased Gcg expression; the OA group had a lower Gcg level, but EtOH and OA+EtOH groups had higher Gcg levels than hyperglycemia; these groups were not statistically different from each other (Fig. 6B).