Potential probiotic isolates Lact. fermentum MCC2759 and Lact. fermentum MCC2760 identified in our laboratory were used in this study. Viable count of overnight grown cultures (1.5 OD) was taken by serial dilution and plating on MRS agar. Lact. fermentum MCC2759 and Lact. fermentum MCC2760 had approximately 109 CFU/ml. The cultures were propagated in MRS medium overnight, and 109 CFU/ml of cells were suspended in phosphate-buffered saline (PBS) for oral administration in rats.
Animal Maintenance and Diet
Wistar rats (female) weighing 120–160 g were housed at the institute Animal House Facility, CSIR-Central Food Technological Research Institute, Mysuru. The animals were maintained under controlled temperature and humidity with a 12-h light and dark cycle in standard polypropylene cages. The rats were acclimatized for a week, fed with a normal diet (AIN 93G), and had access to food and water ad libitum. The experimental procedures were followed as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, and approved by the Institute Animal Ethical Committee (IAEC), CSIR-CFTRI, Mysuru, following IAEC regulations (Approval no. 335/14).
Study 1: High-Fat Diet Feeding and Grouping
All animals were fed a normal diet (AIN 93G) for a week before dietary manipulation. Thereafter, normal control rats were fed with a normal diet, while experimental groups received a high-fat diet (Supplementary data 1) for an initial period of 4 weeks. The animals had access to water ad libitum. Subsequently, the rats were divided into the following groups (n = 6 rats per group): (1) normal control (NC), (2) high-fat diet control (HFDC), and (3) and (4) high-fat diet-fed groups treated intragastrically with potential probiotic L. fermentum MCC2759 and MCC2760 (109 CFU/ml suspended in PBS) once daily for 4 weeks, respectively. The total duration of the study was 8 weeks.
Study 2: Induction of Type 2 Diabetes and Grouping
A total of 40 rats (120–160 g) were taken, control rats were fed a normal diet, while the remaining rats were fed an HFD for 4 weeks as described above. Overnight fasting HFD rats were injected with a single low dose of STZ (Sigma-Aldrich Pvt. Ltd., Bengaluru, India) at 45 mg/kg body weight dissolved in 0.1 M citrate buffer (pH 4.5) intraperitoneally. Normal or non-diabetic rats (n = 10) were administered with only citrate buffer as a vehicle. STZ-induced diabetic rats were given 5% glucose solution for the first 24 h to surmount drug-induced hypoglycaemia. Diabetic rats with fasting blood glucose of > 200 mg/dL were selected 7 days after streptozotocin injection. The rats (n = 10/group) were randomly divided into the following groups: (1) normal control (NC), (2) diabetic control (DC), and (3) and (4) diabetic rats treated with potential probiotic Lact. fermentum MCC2759 and Lact. fermentum MCC2760 (109 CFU/ml) intragastrically for 4 weeks, respectively.
Food Intake, Weight Gain, and Organ Weight
Food intake and gain in body weight were monitored weekly during the experimental period (4 weeks for type 2 diabetic study and 8 weeks for HFD fed study). The weight of different organs was taken post-necropsy.
Oral Glucose Tolerance Test
Oral glucose tolerance test (OGTT) was performed after 8 weeks and 4 weeks of study 1 and study 2, respectively. Rats were starved for 12–14 h before intragastric gavage of glucose (200 g/L in solution; 2 g/kg body weight). Blood was taken by pricking the tail vein and measured using a glucometer (Accu-Chek, Roche Diabetes Care India Pvt. Ltd., Mumbai) at 0, 30, 60, and 120 min post glucose administration. Area under curve was calculated from the blood glucose values (mg/dL) to determine the glucose excursion or tolerance.
Animals were euthanized after 4 weeks of experiment duration. Blood was collected by cardiac puncture and allowed to clot at 4 °C for 2 h followed by centrifugation at 757 g-force for 20 min at 4 °C, and serum was collected. The serum was stored at − 20 °C until further analysis. Tissues of the intestine, liver, MAT (mesenteric adipose tissue) and muscle were collected and stored in RNA later solution (Sigma-Aldrich Pvt. Ltd., Bengaluru, India) until further use.
Serum Biochemical Analysis
Levels of glucose, cholesterol, high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), triglycerides, cholesterol, total protein, uric acid, urea, creatinine, albumin, SGPT, and SGOT were analyzed in the serum using standard analytical kits (Agappe India Pvt. Ltd., Kerala, India). Plasma insulin was determined using ELISA as per the manufacturer’s instructions (Mercodia, Uppsala, Sweden).
Evaluation of Marker Genes by Real-Time qPCR
Intestine, liver, MAT, and muscle tissues (1 g) were homogenized in a tissue homogenizer. The homogenate was subjected to RNA isolation using 1-ml Trizol reagent (Sigma-Aldrich, Bengaluru, India) according to the manufacturer’s instructions. RNA was reversed transcribed into cDNA using Transcriptor High Fidelity cDNA Synthesis Kit (Bionova-Roche Chemicals, Bengaluru, India) as per manufacturer’s instructions. Real-time quantitative polymerase chain reaction (qPCR) (Biorad CFX-96, Bengaluru, India) was carried out using diluted cDNA (1:25) as a template, SYBR Green Jumpstart Taq ReadyMix (Sigma-Aldrich, Bengaluru, India), and gene-specific primers. The PCR program consisted of an initial denaturation at 94 °C for 3 min followed by 40 cycles of denaturation (94 °C for 30 s), annealing (60 °C for 30 s), and elongation (72 °C for 30 s). Primer specificity and efficiency were evaluated from the melt curves. Data were obtained from triplicate samples run along with no-template control. Oligonucleotide primers were designed using the Primer3 software (http://primer3.ut.ee/) by obtaining consensus sequences belonging to rat (Rattus norvegicus) genes from NCBI and later synthesized from Eurofins Genomics India Pvt Ltd (Bengaluru, India). The primers related to inflammatory cytokine genes and other markers are listed in Supplementary data 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control for normalization of gene expression. Gene expression results were expressed as relative normalized expression (fold change) calculated using the 2−ΔΔCt method.
Tissues of liver, kidney, muscle, pancreas, and intestine were sectioned and stained with hematoxylin and eosin (H&E) stain for histopathology evaluation. The stained tissue slides were observed under a bright-field microscope (Labomed, Burlington, NC, USA).
Data were statistically analyzed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com). All data were expressed as mean ± SD/SEM (n = 6). One-way ANOVA was used for the analysis of a single parameter, and two-way ANOVA analysis was used for comparison between groups. p < 0.05 and p < 0.001 were considered significant for one-way and two-way ANOVA, respectively.