Supplementary Methodologies:
Materials: Normal pellet diet (NPD; 10 % calories from fat) was procured from Aashirwad
industries, Mohali, Punjab, India. Lard was procured from local slaughter house. High fat diet
(HFD; 60% calories from fat) was prepared using a standard composition described
elsewhere.1 Energy output of NPD is ~ 4.75 kcal/gm whereas HFD had caloric values of 5.25
kcal/gm. The diets fed to animals (HFD to HFD + IMOs/ Lycopene/ combination of both)
were of isocaloric in nature. LycoVit®10 CWD/S containing 10 % Lycopene as was a kind
gift from BASF-the chemical company (BASF India Ltd, Mumbai, India). Isomaltooligosaccharides (IMOs) as VitaFiberTM Basic (Powder), was a kind gift from BioNeutra
North America Inc. It is freely soluble in water, sweetness ~ 60 % of sucrose, low calories,
non-carcinogenic, ≥ 90% non digestible/ resistant oligos, ≤3% free glucose, free of glutan and
other allergen, stable to wide range of pH and heat condition, least flatulent, pleasant mouth
feel and humectants. All the reagents used were of highest commercial grades available.
Doses and anthropometric paramteres: Lycopene and IMOs were dissolved in distilled water
and administered par orally and the control groups received distilled water (five days in week
i.e. except Sunday and Wednesday). Doses of lycopene and IMOs selected for the study were
based on available literature.2-6 For the dose preparation of lycopene 10% active lycopene in
the powder was considered for calculation. The study was conducted for a total of 12 weeks.
Body weights were measured weekly. Naso-anal length and waist circumference was
measured in the last week of the study. The anthropometric parameters e.g. BMI (body
weight in g/ naso-anal length2 in cm), Lee’s index (body weight in g0.33×1000)/ naso-anal
length in cm).
In the last week total locomotor activity was measured using photoactometer (IMCORP),
Ambala, India. At the end of the experiment, from the fasted mice, blood was collected retroorbitally and mice were sacrificed by cervical dislocation, except the animals were kept for
histological examinations. Visceral white adipose tissue (vWAT), subcutaneous white
adipose tissue (sWAT), brown adipose tissue (BAT), hypothalamus, pancreas, liver were
dissected, snap frozen and stored at -80 °C. Entire gut from the base of oesophagus to rectum
was isolated and weighed. The cecum was dissected and its content was isolated asceptically,
weighed and snap frozen for extraction of bacterial DNA. At the same time content of
alimentary canal was removed and disembowelled body weight was calculated by subtracting
weight of gut to total body weight.
Blood and tissue biochemistry: The OGTT test was performed during the 12th week (4th and 5th)
day before the sacrifice of the mice. Mice were fasted overnight (12 h), and basal blood
glucose concentration (0 min) was determined using Glucocard
TM
strips (Arkray Factory
Inc., Japan). Then 2 g/kg body weight of glucose was administered orally and blood glucose
(mg/dL) was determined at 15, 30, 60, 120 minutes. Using the basal blood glucose level
(mM/L) and fasted serum insulin level, the % beta cell activity (%B) and insulin resistance
(IR) was calculated using updated Homeostasis Model Assessment (HOMA) calculator
7
(HOMA2 calculator v2.2.3 developed by Diabetes Trial Unit, University of Oxford). Another
insulin sensitivity index quantitative insulin sensitivity check index (QUICKI) [QUICKI =
1/(log glucose in mg/dL + log insulin in µIU/ mL)] and McAuley’s index in terms of fat-free
mass (Mffm/I) for insulin resistance [Mffm/I = e(2.63 – 0.28 ln (Insulin in µIU/mL) – 0.31 ln (TG in mM/L)]
using fasted blood glucose and serum insulin and serum TG were calculated.8 Serum
collected at the end of experiment was used for determining serum lipid profile i.e. total
cholesterol, low density cholesterol (LDL-c), high density cholesterol (HDL-c), triglyceride
using commercially available kits (Accurex biomedical pvt. ltd., Maharashtra, India) and free
fatty acid (Sigma-aldrich Inc., St. Louise, MO, USA). Liver lipids were extracted using
method of Folch et al., 1957.9 The extracted lipids were resuspended in 2% Triton X-100
10
and similarly assayed for total cholesterol, LDL-c, HDL-c and triglyceride (TG) using
commercial kits as for serum. Atherogenic fraction in liver was calculated as TC minus HDLc.
Different biochemical parameters e.g. lipid peroxidation (LPO) in terms of malondialdehyde
(MDA), reduced glutathione (GSH), total nitrite, superoxide dismutase (SOD) and catalase
were assayed in supernatant of tissue homogenate (10 % w/v) of sWAT, liver, pancreas,
vWAT. Only total nitrite was assayed in BAT tissue. All these estimation were performed
using previously described methods
11
and the values were expressed in terms of per mg
protein, which is quantified using Bradford’s reagent.
Gene expression analysis: Total RNA was isolated from Liver, vWAT and hypothalamus
using the Trizol® based RiboPure RNA extraction kit (InvitrogenTM) according to
manufacturers’ instructions. Total RNA was quantified using Infinite® M200 Pro NanoQuant
(Tecan) and the integrity and purity was determined using 1% agarose gel. Intact RNA
samples [1µg (vWAT), 2 µg (liver) and 3 µg (hypothalamus)] were used for c-DNA synthesis
using ReverTra Ace® qPCR RT master mix with gDNA remover kit (Toyobo c. ltd., Japan),
High capacity RNA-to-cDNA kit (Applied Biosystems) and SuperScript III First-strand
synthesis supermix kit (InvitrogenTM) respectively according to manufacturer’s instructions.
Quantitative PCR: The relative expression of different genes in Liver and vWAT (suppl.
table 1 - for the list of genes and primers) were determined using SYBR® based dye using
quantitative PCR (Applied Biosystem 7500 Fast Real-Time PCR machine). Anorectic and
orexigenic gene expression in hypothalamus was determined using a customized mouse PCR
array plate (CAPM12379C, SABiosciences, Qiagen). RT-PCR was carried out under the
following conditions: 95ºC for 10 min, followed by 45 cycles of 95ºC and 60ºC for 1 min.
Data were analysed using the ∆∆Ct method, and values are expressed as fold change relative
to the NPD group.
Histological Examinations: Animals were perfusion fixed by 10% buffered formalin and the
liver, vWAT, ileum and colon were isolated and stored in 10 times volume of 10% buffered
formalin for further processing using haematoxylin and eosin staining (H&E) and all the
images were captured using 20X objective (Final camera magnification 14X). Only frozen
tissues were used for neutral lipid specific oil red O (ORO) staining. For H&E staining
paraffin embedded tissues were microtomized (3 – 5 µm) and stained. The mean adipocyte
size in adipose tissue sections were estimated in 20 images of each tissue (2 animals per
group), using Imagescope (Version 12.1.0.5029, Aperio Technologies Inc.) by manual
counting. The cell size were categorised on a 10 point arbitrary scale: Category 1 = 240 – 499
µm2; 2 = 500 – 999 µm2; 3 = 1000 – 1499 µm2; 4 = 1500 – 1999 µm2; 5 = 2000 – 2499 µm2;
6 = 2500 – 2999 µm2; 7 = 3000 – 3499 µm2; 8 = 3500 – 3999 µm2; 9 = 4000 – 4499 µm2; 10
= 4500 µm2 <. Ileal sections were also analysed by same software for villus and crypts
length.
Frozen liver tissues (0.8 cm × 0.8 cm) were directly embedded in tissue freezing medium
(Leica Biosystem Nussloch GmbH, Germany) and 12 µm sections were made for staining
with ORO dye. Images (2 tissues per group, 10 representative images per tissue, 40X
objective) were analyzed using ImageJ software as described elsewhere.12 For quantification
of entrapped ORO, sections were washed with 200 µL of isopropyl alcohol and washed liquid
were collected and absorbance was determined at 510 nm. The relative absorbance of
extracted ORO was calculated as % of NPD group.
Microbial analysis of cecal content: Approximately 100 mg of cecal content was taken and
DNA was isolated using QIAmp® DNA stool mini kit (Qiagen) according to manufacturer’s
instruction. Quantification was performed using Infinite® M200 Pro NanoQuant (Tecan),
integrity was evaluated on 0.8% agarose gel. qPCR was performed for Lactobacillus spp.
(LAB), Bifidobacteria (BIF), Akkermensia muciniphilia (AKK), Roseburia spp. (ROS),
Enterobacteriaceae (ENTB), Bacteroidetes (BACT), Firmicutes (FIRM), taking total bacteria
(TB) as internal control was performed as described above (suppl. table 1 – For list of genes
and primers).
Short chain fatty acids (SCFAs) analysis in cecal content: The system was equipped with
Agilent’s controller pump (model no. G1311C), an auto sampler unit (model no. G1329B)
and a diode array detector (DAD; model no. G1315D) set at 210 nm (peak width 2.5 Hz,
band width 2nm). Chromatographic separation of acetate, propionate and butyrate was
achieved at 60ºC using an anion exchange column (300 × 7.7 mm; 8 µm particle size) along
with a guard column made with the same material. The mobile phase a mixture of 0.1%
formic acid in Milli-Q water (Merck Millipore, 0.22 µm filtered, resistivity 18.1 – 18.3 MΩ
cm) was used. The column was equilibrated and eluted with isocratic flow rate of 0.6 mL/min
at 60ºC for 30 min. Data was acquired and processed with EZchrom elite software with the
system suitability option installed. The method was optimized and validated in house
(unpublished data). Weighed samples were suspended in sufficient volume of acidified water
(pH 1 – 2) by rigorous vortexing. Then keeping at room temperature (10 min) followed by
centrifugation (6000 rpm, 20 min, 4ºC) and filtration with 0.2 µm nylon filter (Millipore
Millex-GN). Isobutyric acid was used as an internal standard (5µM). Injection volume was
set at 20 µL and minimum two technical and three biological replicates were processed in
each group analyzed. Data was represented as µM concentration of these SCFAs per mg
sample used.
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