Chemicals and materials

Tetrachloroauric acid and streptozotocin (Sigma-Aldrich, USA), glucose GOD-PAP (Fortress diagnostics, UK), total RNA purification kit (Jena Bioscience, Germany), PrimeScript™ RT master mix (Takara, Japan), BlasTaq™ 2X qPCR MasterMix (Applied Biological Materials, Canada), protein quantification kit BCA assay, (Abbkine, China), phosphoenolpyruvate carboxykinase activity assay kit (MyBioSource, USA).

Synthesis and characterization of the gold nanoparticles

Biosynthesis of AuNPs was conducted using dried leaf extract of D. viscosa, collected in October 2020 from North of Jordan. Leaves of D. viscosa were dried in an oven at 37 °C overnight and were stored at room temperature away from light.

Leaf extract was prepared by adding 1 g of the dried plant to 100 ml of boiling water with continuous stirring for 15 min and then it was kept cooling at room temperature and filtered using Whatman No. 1 filter paper. The extract was then added to 1 mM aqueous HAuCl4 solution at a volume ratio of 1:5 followed by measurement of absorbance at a resolution of 2 nm and wavelength range between 240 and 800 nm at 4-h intervals in a quartz cuvette with a path length of 1 cm. After the reaction had completed, the solution was centrifuged at 10,000 rpm for 20 min and the pellet was weighed and resuspended in distilled water.

Surface charge measurement and the formed NPs hydrodynamic diameter were determined using Zetasizer Nano ZS90 (Malvern Panalytical, UK), where 100 µl of the solution was suspended in 900 µl of distilled water. Measurements were taken under the following conditions: 25 °C, 1.33 dispersant refractive index, and 0.8872 cP viscosity. The shape of the formed nanoparticles was determined by TEM using a Titan FEI microscope, where the sample was first dispersed in water, deposited on a carbon grid, and allowed to dry prior to imaging.

Animal studies

All experimental animal procedures were carried out according to the National Institutes of Health (NIH) guide for the care and use of laboratory animals and approved by the animal ethics committee at Yarmouk University (ACUC/2021/10).

Induction of type 2 diabetes in rats and treatment

Adult male Sprague–Dawley rats, 3 months of age and weighing between 150 and 200 g, were obtained and kept at the animal in Yarmouk University. Rats were divided into three groups (n = 6–8/group): control (non-diabetic), diabetic without any treatment, and diabetic treated with a daily intraperitoneal injection of AuNPs at a dose of 2.5 mg/kg for 21 days [9]. Type 2 diabetes was induced by maintaining the rats on HFD for 2 weeks; the diet consisted of egg yolk and butter mixed with feed. This was followed by a single intraperitoneal injection of freshly prepared STZ (45 mg/kg) dissolved in 50 mM sodium citrate buffer (pH 4.5). Three days following the injection, blood glucose levels were measured in fasting rats using a glucometer (GlucoLab, Infopia, Korea); rats with blood glucose levels higher than 150 mg/dl were considered diabetic.

Blood glucose measurement

Blood samples were collected in the fed state by heart puncture and were placed in plain tubes for serum collection and kept undisturbed for 15 min, followed by centrifugation at 10,000 rpm for 10 min. According to the manufacturer’s protocol, glucose levels of serum samples were determined using the GOD-PAP colorimetric method (Fortress diagnostics, UK).

Measurement of PEPCK enzyme activity

Liver tissue homogenate was prepared by homogenizing the liver sample with ice-cold phosphate buffer saline (PBS) at a ratio of 1 g tissue:9 ml PBS on ice. The homogenates were then centrifuged at 5000 g and 4 °C for 5 min to get the supernatant. The protein concentration was determined for all samples with a protein quantification kit using the bicinchoninic acid (BCA) assay method according to the manufacturer’s protocol (Abbkine, Inc. China).

The homogenates were diluted with PEPCK assay buffer (1:2) for the measurement of the enzyme’s activity. According to the supplier’s protocol, a colorimetric detection method was used (MyBioSource, Inc., USA). Final absorbance was measured in kinetic mode at 570 nm and 37 °C, for 55 min at 5-min intervals. Two-time points were selected for all the samples to calculate PEPCK activity.

Real-time PCR

The quantitative gene expression of hepatic PEPCK was determined using RT-PCR. First, total RNA was extracted from liver samples using a spin column-based method based on the instructions of the supplier’s protocol (Jena Bioscience). RNA concentration was quantified by measuring absorbance at 260 nm using µDrop plate, Multiskan GO, and SkanIt software (Thermo Scientific, USA). The RNA was then reverse transcribed into cDNA using a commercial kit (Takara, Japan). The reaction mixture was placed in a thermocycler at 37 °C for 15 min, followed by inactivation of reverse transcriptase at 85 °C for 5 s.

BlasTaq™ 2X qPCR MasterMix was used for the RT-PCR step. The reaction components included the cDNA sample, forward and reverse primers, BlasTaq™ 2X qPCR MasterMix, and nuclease-free water with a final volume of 20 µl. The samples were placed in the RT-PCR thermocycler LineGene 9600 (Bioer Technology Co., China), and the reaction conditions were as shown in Table 1. The cycling parameters were as follows: 95 °C for 3 min and 45 cycles of 95 °C for 3 s and 60 °C for 30 s. Relative mRNA concentrations were normalized to the housekeeping gene GAPDH. The fold changes in mRNA expression were determined using the 2−ΔΔCT method [19]. The sequence of the primers used is shown in Table 1.

Table 1 Sequences of primers used for quantitative real-time RT-PCRStatistical analysis

One-way analysis of variance (ANOVA) test was performed for data analysis using SPSS software version 23 (SPSS Inc., Chicago, IL), and graphs were created using GraphPad Prism version 9.0.0. Statistical significance was considered when the P-value was less than 0.05.