Study design, and groupingAnimals

Twenty-four (24) male Wistar rats, aged 4–5 weeks and weighing between 100 and 115 g were housed in separate cages and kept at constant environmental conditions of temperature (22–26 °C), relative humidity (50–60%) and 12-h dark/light cycle. The rats were fed on a constant ration, and fresh, clean drinking water was supplied. All the rats were also acclimatized for a period of one week prior to the commencement of the study. The study was carried out in accordance with the world accepted best practices as written in the European Commission guidelines (Directives of 2010/63/EU as amended by Regulation (EU) 2019/1010) as well as the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines and was approved by the Ethical Review Committee, University of Ilorin, Ilorin, Nigeria (UERC/ASN/2018/357).

Chemicals

All the chemicals were of analytical grade. The chemicals used in the study include L-arginine, which was purchased from Bridge Biotech Laboratory, Ilorin, Kwara State. The L-arginine was dissolved in distilled water. It was prepared freshly and administered orally at a dose of 150 mg/kg body weight [17] for a period of 6 weeks (42 days).

High fat diet

A special high fat feed was formulated using the composition provided by Abdul Kadir et al. [18]. The high fat diet contains 414.0 kcal/100 g with 43% as carbohydrate, 17% as protein, and 40% as fat. The diet consists of 17,000 g of powdered rat feed, 1500 g of maize oil, 1500 g of ghee, and 5000 g of milk powder, giving a total of 25,000 g (25 kg) of high fat feed. The composition table is shown in (Table 1).

Table 1 Composition of high fat diet and normal rat chow dietAnimal grouping

After acclimatization to the laboratory conditions, the animals were randomly divided into four groups of six rats, placed in individual cages, and classified as follows; Control (CTR), rats received normal rat chow and 0.5 ml of distilled water daily, high fat diet-fed (HFD), rats received high fat feed only, high-fat diet and arginine (HFD + ARG), rats received a high fat diet and 150 mg/kg arginine orally, and arginine only (ARG), rats received a high fat diet and 150 mg/kg arginine orally, all for 6 weeks [18].

Collection of blood sample

After 6 weeks of treatment, animals were anesthetized with 0.8 mg Pentobarbital sodium intraperitoneally (50 mg/kg ip) and blood was collected by cardiac puncture. Blood was collected by cardiac puncture into plain tube, left for 30 min to clot in the tube and thereafter was centrifuged at 3000 rpm for 5 min at room temperature. Serum was stored frozen until needed for biochemical assay.

Fasting blood sugar test and oral glucose tolerance test (OGTT)

After 8 h of an overnight fast, the tails of the rats were pricked with a lancet blade for the FBS. Thereafter, 200 g/l of glucose in water, after proper dissolution, was given by oral gavage at a dose of 2 g/kg of body weight, and blood glucose was obtained at the tail tip at 30, 60, 90 and 120 min for glucose concentration using a handheld On-Call glucometer. This test was carried out on all rats, and the results were recorded.

Biochemical assaySerum insulin assay

Serum insulin concentrations were measured with an enzyme-linked immunosorbent assay kit (cat number:IS130D) procured from Ray Biotechnology, Inc. (Georgia, USA). The sensitivity of the assay was 0.5 mU/L, and the inter-assay coefficients of variation were < 7.5 and 9.3%, respectively.

Homeostasis model assessment of insulin resistance (HOMA-IR)

This parameter was derived by using the formula.

$$\left[ }\left( }/}} \right) \, \times }\left( }/}} \right)} \right] \, /.$$

Homeostasis model assessment of beta cell (homa-beta)

This parameter was derived by using the formula.

$$0}\left( }/}} \right) \, / \, \left( }\left( }/}} \right) \, - } \right)$$

Triglyceride-glucose (TyG) index

This parameter was derived by using the formula.

$$}\, = \,}\left[ } \left( }/}} \right) \, \times }\left( }/}} \right)} \right]/$$

Cardiac risk ratio

$$}\;}\;}\;\,}\;}\;\left( }} \right)\left( } - }} \right)$$

Atherogenic coefficient

$$}\;} = \, \left( }} - }} \right) \, /} - }$$

Atherogenic index of plasma

$$} = }\left( }/} - }} \right)$$

LDL-c

$$} = }}/\left( }/}} \right) \, \left[ } \right].$$

Serum leptin assay

Serum leptin concentrations were measured by the sandwich enzyme-linked immunosorbent assay (ELISA) method on a DSX ELISA automated analyzer. Protocols were run according to the manufacturer’s instructions. The analytical sensitivity for the leptin kit was 0.7 ng/ml, specificity for human leptin was 100% and the assay dynamic range was 0.7–100 ng/ml.

Phosphotylinosital 3 kinase assay

Phosphotylinosital 3 Kinase (also known as PI3K), assay was done using the Sandwich-ELISA kit purchased from Elabscience® with Cat.NoE0438Ra.

Statistical analysis

The results were expressed as mean ± standard deviation using graph pad prism 8. The data were analyzed using one-way ANOVA. Post-hoc Tukey test was used to determine the significance of mean values among the groups. The P-value (P < 0.05) was used to determine significant differences.