For analyzing plasma glucose and insulin, commercially available kits were procured from Stanbio Laboratory, USA and Bio-Merieux, RCS, Lyon, France, respectively. Plasma was used for analyzing total cholesterol, triglycerides, phospholipids, free fatty acids, VLDL and HDL by colorimetric methods using kits (Nicholas Piramal India Ltd., Mumbai). Orlistat (Cat No 04139) was obtained from Sigma-Aldrich. All other reagents used in the experiments were of analytical grade and of high purity. Trireagent and cDNA preparation kits were obtained from Qiagen.
Animals and treatments
Male SD rats weighing 180–200 g were randomly divided into six groups of six each. Normal control rats were fed with normal pellet diet as per AIN-93 guidelines, while the other groups of animals were fed with HFD for 22 weeks. The composition of HFD is as previously described by us . Piperonal was supplemented to selected HFD groups for 6 weeks from 16th week, as mentioned in the experimental design below. Experiments were performed at the National Center for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad, India (Regd. No. 154/RO/C/1999a/CPCSEA). Animals were placed in cages at 22 ± 2 °C, with 14–16 air changes per hour with a relative humidity of 50–60% and 12 h light/dark cycle. All procedures involving laboratory animals were in accordance with the Institute Animal Ethical Committee (IAEC No: 36 a/2012(i)/a/−CPCSEA/−IAEC/SVU/MB; Dt.01.07.2012).
Isolation and characterization of piperonal
Piperonal was isolated at the Natural Products Division, Indian Institute of Chemical Technology, Hyderabad, India, from ethylacetate extract of black pepper seeds and purity was checked by HPLC equipped with Phenomenex Luna C18 column (150–4.6 mm, 5 m i.d.). Acetonitrile-water system was selected as mobile phase, PDA detector was used to measure the wavelength and peak position and structure was confirmed by 1H NMR and 13C NMR. The procedures used are in line with Rao et al. .
Piperonal weighing 1.6, 1.8 and 2.4 g, each mixed with 4 kg of HFD, was prepared to get a dose of 20, 30 and 40 mg/kg b.wt. Twenty grams of diet/day/rat were given to the groups as shown in the experimental design. The exact dose of piperonal consumed was calculated from daily food intake.
Normal diet control
High-fat diet control (HFD control)
: HFD + Orlistat (5 mg/kg b.wt)
HFD + piperonal (20 mg/kg b.wt)
HFD + piperonal (30 mg/kg b.wt)
HFD + piperonal (40 mg/kg b.wt)
Body weight and feed efficiency ratio
The body weight of the experimental rats was assessed once a week, while the food consumed and leftover was measured daily. For the measurement of nutrient metabolites like food intake, water intake, urine volume and fecal weight, both control and experimental rats were placed in the metabolic cages for 72 h (Techniplast, Italy).
Body composition by Total body electrical conductivity (TOBEC)
At the end of the experiment, body composition which includes lean mass, fat-free mass, fat%, total fat (g), total body Na, K levels and water content was measured in all experimental groups by Total Body Electrical Conductivity (TOBEC) using small-animal body composition analysis system (EM-SCAN, Model SA-3000 Multi detector, Springfield, USA) as described by us previously . The following body composition parameters were obtained mathematically, where E stands for total electrical conductivity. i) Total body fat: total body weight – lean body mass; ii) Fat percentage: (total body fat × 100)/total body weight; iii) Lean mass: 0.5 × E + 0.3 × total body weight; iv) Fat-free mass: 16.28 + 0.4 x E; v) Total body sodium (TB Na) (mg): (2.5 × E) + 49.1; vi) Total body potassium (TB K) (mg): (4.95 × E) + 164.4; vii) Total body water (TBW): 31.5 + 1.8 × E.
The above prediction equations have been reported to be very accurate for Wistar, Sprague Dawley, F-344 N, CFY, WKY and Holtzman rats .
Estimation of BMC and BMD by DXA
The body composition of the experimental animals was assessed at the end of the experiment by Dual-X ray absorptiometry (DXA) using body composition analysis system (Halogen 1000 series). DXA data were used to compare the levels of body adiposity, bone mineral concentration (BMC) and bone mineral density (BMD) between the control and experimental groups, and calculations were made according to manufacturer’s protocols .
Plasma lipid profile
At the end of the experiment, blood was collected from overnight-fasted rats by retro-orbital puncture method. Plasma was separated by centrifugation at 2500 rpm for 15 min and stored at 80 °C for further biochemical analysis. Total cholesterol (TC), HDL, VLDL, LDL and triglyceride levels (TGs) were estimated by CHOD-PAP method, GPO-PAP method; phospholipids (PLs) and free fatty acids (FFAs) were assessed as described by us previously .
Blood glucose, insulin and insulin resistance
Plasma glucose was estimated using kits (Cat No 1060–500, Stanbio Laboratory, USA). Plasma level of insulin was determined using kits from Bio-Merieux, RCS, Lyon, France. Insulin resistance was calculated using the homeostasis model assessment.
Oral glucose tolerance test (OGTT)
Oral glucose tolerance test was performed at the end of the experiment; after overnight fasting, glucose was administered orogastrically at a dose of 2 g/kg body weight and blood samples were collected from supraorbital sinus at intervals of 0, 30, 60, 90 and 120 min and glucose level was estimated .
Estimation of leptin, adiponectin, lipase and α-amylase
Plasma leptin and adiponectin levels were measured by using enzyme-linked immunosorbent assay kits (Crystal Chem, Downers Grove, IL, USA), performed in duplicate, as per the manufacturer’s guidelines and expressed in ng mL−1. Alpha-amylase and lipase activities were determined by kinetic method using the commercial kits of Labtest, Minas Gerais, Brazil and Bioclin, Minas Gerais, Brazil, respectively .
Estimation of tissue lipids
Tissue (Liver, kidney) lipids were extracted from the experimental animals as per Floch et al.  using a chloroform–methanol mixture (2:1, v/v). The tissues were rinsed with ice-cold physiological saline, dried, homogenized in cold chloroform-methanol (2:1, v/v) and the contents were extracted after 24 h. The extraction was repeated four times. The combined filtrate was washed with 0.7% KCl and the aqueous layer was discarded. The organic layer was made up to a known volume with chloroform and used for tissue lipid analysis.
Adiposity index (AI), a measure of the total weight of the visceral fat depots (epididymal, retroperitoneal and mesenteric) in the body, was determined according to Taylor and Phillips method  using the formula: AI = (sum of the weights of the visceral fat depots/body weight) × 100.
At the end of the experiment, rats were fasted overnight and euthanized by CO2 inhalation and subjected to necropsy. Major vital organs like heart, lungs, liver, kidneys, mesenteric fat pads, epididymal and retroperitoneal fat pads were collected. After detailed necropsy examination, these organs were weighed and organ-to-body weight ratio was measured.
Liver antioxidants analysis
After the completion of the experimental period, rats were anesthetized, sacrificed; the liver was excised, rinsed in ice-cold normal saline followed by ice-cold 10% KCl solution, blotted, dried and weighed. A 10% w/v homogenate was prepared in ice-cold KCl solution and centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant thus obtained was used for the estimation of thiobarbituric acid substances (TBARS) , assay of catalase  (CAT), reduced glutathione (GSH) , superoxide dismutase (SOD)  and glutathione peroxidase (GpX) .
RT- PCR analysis
Total RNA was isolated from adipose tissue by using tri-reagent (Sigma-Aldrich, USA) according to manufacturer’s protocol and reverse-transcribed to obtain cDNA using DNA synthesis kit (Applied Biosystems, Foster City, USA).
Twenty ng of cDNA was used for semi-quantitative PCR analysis with specific primers such as PPAR-γ, TNF-α, SREBP-1c, HMG-CoA reductase, acetyl-CoA carboxylase (ACC), fatty-acid synthase (FAS), fatty-acid-binding protein-4 (Fab-4), and uncoupling protein-2 (UPC-2). The sequences of primers are mentioned in Table 1. PCR was performed for 38 cycles using the following cycling conditions: 30 s of denaturation at 94 °C, 30 s of annealing at 59 °C and 1 min of extension at 72 °C.
Histopathology of adipose and liver tissues
Liver and adipose tissues were collected from experimental rats, cut into pieces and kept in 10% formalin solution. A small piece of the tissue was sectioned with microtome, fixed on slides, stained using haematoxylin and eosin (H&E) staining procedures and observed under an optical microscope with 40X magnification and photographed.
Body fat distribution was determined by 7 Tesla MRI. The rats were placed in the magnet and fixed in a nonmagnetic device. For the detection of pure fat images, respiration-triggered water- suppressed Spin Echo sequences (repetition time: 744 ms, echo time: 12.24 ms, four averages, field view: 70 mm, matrix: 192–192, water suppression: Gauss pulse 3.9 ms, 701 HZ bandwidth) were used to obtain 30 slices (slice thickness: 2 mm) in the abdominal region. For the data analysis, 20 slices at a reproducible position covering the area from the nose to anus were considered. The area of retraperitoneal fat was visually identified in the selected slice package. Tissue volumes were calculated by multiplying the corresponding number of segmented pixels by in-plane pixel dimensions and the slice thickness.
Results are expressed as the Mean ± S.D. All the grouped data were statistically evaluated with SPSS\19.0 software. Hypothesis testing methods included one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test. Significance level at p < 0.05, 0.01, and 0.001 was considered for various parameters to indicate statistical significance.