MOUSE :: HMDP :: Diet induced obesity

E.         Hybrid Mouse Diversity Panel:  Diet induced obesity

1.         Mice (B. Parks)
2.         Diets (B. Parks)
3.         Body composition, NMR (B. Parks)
4.         Plasma lipids, insulin & glucose (L. Castellani)
5.         Tissues, weights (B. Parks)
6.         Gut flora composition (B. Parks)
7.         Expression arrays, adipose (B. Parks)
8.         Expression arrays, liver (B. Parks)
9.         Liver lipids (S. Hui)
10.        Expression arrays, muscle (B. Parks)

 

1.         Mice (B. Parks)

Male and female mice from the HMDP were obtained from the Jackson Laboratories and bred at UCLA.  At 8 to 10 weeks of age mice were switched from a chow diet to a high fat diet for 8 weeks. After 8 weeks on high fat diet mice were fasted for 4 hours (beginning at 6 am) and processed for plasma and tissues (as described below).

 

2.         Diets (B. Parks)

High fat diet was obtained from Research Diets, Inc. (#D12266B) and contains 31.8 % Kcal from fat. 

 


3.         Body composition, NMR (B. Parks)

NMR (Bruker) scanning of live mice to determine fat and lean mass was performed at 0, 2, 4, 6, and 8 weeks after the start of high fat diet. 

 

4.         Plasma lipids, insulin & glucose assays (L. Castellani)

Following 4 hour fast mice were bled retro-orbitally under isoflurane into EDTA-coated microtubes and Heparin-coated microtubes and kept on ice.  Blood was centrifuged for 10 minutes at 12,000 RPM and the plasma was collected and stored at -80°C. 

General Overview: The assays that we routinely perform are total cholesterol, HDL cholesterol, unesterified cholesterol (UC), unesterified (free) fatty acids (FFA), triglycerides (TG) and glucose. Total plasma cholesterol is assayed after treating the samples with cholesterol esterase to hydrolyze cholesterol esters and then performing the cholesterol assay, which will now encompass all of the cholesterol that was in both the cholesterol ester and unesterfied cholesterol pools. Hence, this is the “total” cholesterol assay. Unesterified cholesterol is assayed by performing the cholesterol assay without treating the plasma sample with cholesterol esterase, so that only the free or unesterified cholesterol is determined. This value can then be subtracted from the total cholesterol to calculate the esterfied or cholesterol esters (CE). The HDL cholesterol is determined by performing the total cholesterol assay on the supernatant after precipitation of the apoB containing lipoproteins with Heparin/Manganese Chloride. The triglyceride assay is a typical assay that actually determines the mass of glycerol released after the hydrolysis of fatty acids from triglycerides. However, we also do a triglyceride (glycerol) “blank” assay to measure the endogenous levels of glycerol present in the mouse plasma prior to hydrolysis of triglycerides. This endogenous “blank” value is then subtracted from the total glycerol determined after the hydrolysis of triglycerides in order to correct for the endogenous plasma free glycerol concentrations, which are considerably higher in mice than in humans. Most of the pipetting is performed using a Beckman Biomek 2000 Automated Workstation with the assays read in a 96 well format using a Molecular Devices Spectramax-Plus microplate reader.

Quality Control:
Internal quality control: All assays are run in triplicate determinations on a 96 well plate. We also run standards on each plate as well as control samples with known analyte concentrations on each plate in order to validate the accuracy of the assay. This allows us to run 26 unknown samples (in triplicate) on each plate, with the remainder of the wells used by the standards, control samples, and sample blanks, all of which are also run in triplicate.

External quality control: We participate in the Centers for Disease Control and Prevention Lipid Standarization program. Our laboratory ID # is LSP-251. Each quarter we receive 12 test samples from the CDC, and one of the CDC test samples is included with an actual run of our unknown samples each week. The values we obtain from the test samples are submitted to the CDC at the end of each quarter, and we are notified if we have met their criteria for accuracy and precision. We have passed each quarter for the past 18 years.

Supplies/Reagents needed:

A.  0.9 % sodium chloride solution
B.  0.65ml minitubes (Phenix catalog# M-931B)
C. 96 well round bottom plates (USA Scientific catalog #5665-0101)
D.  96 well flat bottom plates (USA Scientific catalog #5665-    5101)
E.   Reagents for triglyceride and triglyceride blank assays

Triglyceride Assay Reagents: Triglyceride reagent (Sigma catalog # F6428 triglyceride [free glycerol] reagent) reconstituted as per manufacturers recommendations and 8000U lipase (EMD Biosciences catalog #437707). Reagent for triglyceride ‘blank’ assay is made exactly the same way but the lipase is omitted.

F.  Glucose assay reagent (Fisher catalog # SB1070-125)
G.  Glucose standard (Sigma catalog # G6152)
H.  Triglyceride standard (Sigma catalog #G556-100ml)
I.    Cholesterol standard (Fisher catalog # SB1012-030)
J.  FFA assay reagents (reconstituted per manufacturers recommendations)
NEFA Color reagent A (Wako catalog# 999-34691)
NEFA Solvent A: (Wako catalog# 995-34791)
NEFA Color reagent B: (Wako catalog# 991-34891)
NEFA Solvent B: (Wako catalog# 995-35191)
NEFA Standard solution (1mEq/L) (Wako catalog # 276-76491)

K.  Control samples
Control 1- SER-T-FY-1 Level 1 human control serum (Stanbio cat # G427-86)
Control 2 is SER-T-FY-2 Level 2 human control serum (Stanbio cat # G428-86).
L.  Reagents for cholesterol assays

Cholesterol Assay Reagent:
4-amino antipyrine (Aldrich catalog # A3,930-0), KCl (Fisher catalog # P217-500), sulfonic acid (Research Organics catalog # 6062H-3), sodium cholate (USB catalog # 13630), pipes (Sigma catalog # P-3768), Triton X-100 (Sigma catalog # T-6878), horseradish peroxidase (Amresco catalog # 0417), cholesterol oxidase (EMD Biosciences catalog # 228250) and cholesterol esterase (EMD Biosciences catalog # 228180) [Omit the cholesterol esterase from the reagent for ‘UC’ assay]

Heparin/MnCl2 for precipitation reagents for HDL assay     
MnCl2 (Fisher catalog # M87-100), Heparin solution (EMD Biosciences catalog # 375095)

 

Preparation of Plasma Samples, Standards, Controls, and Blanks:

General Overview: The enzymatic colorimetric assays are read on a 96 well plate in a Molecular Devices SpectraMax plus plate reader. This allows the analysis of 26 samples per plate when run in triplicate determinations, with the remainder of the wells used for the 3 standards, 2 control plasmas, and 1 assay blank, all of which are also run in triplicate. Therefore, a total of 32 minitubes should be labeled for each assay being run (26 for the samples, 3 for standards, 2 for control plasmas and 1 for the assay “blank”). After the tubes are labeled proceed with the preparation of the plasma samples, standards, controls and blanks for each assay as described below.

Initial Dilution of Plasma Samples: The frozen mouse plasma samples, which should have at least 100ul per tube, are thawed on ice. Once thawed they are vortexed and centrifuged in a table top microcentrifuge to recover all of the sample at the bottom of the tube. Depending on the strains and diets, the plasma is diluted from 4 to 16 fold with 0.9% NaCl, since very lipemic samples can occur with genetically modified mouse strains on a high fat/high cholesterol diet. For assays of common inbred strains on a normal low fat chow diet, we take 75 ul of plasma and add 225 ul of 0.9% saline for a 4 fold dilution. This will give enough total diluted sample to run all of the assays in triplicate. Obviously, for lipemic samples that will be diluted more, a smaller volume of plasma is sufficient to run all assays. The goal of the dilutions is have the lipid concentrations high enough to give reliable OD readings significantly above background, without exceeding or approaching the maximal OD reading of the plate reader. Thus, different batches of samples have to be diluted differently to meet these requirements.

Preparing Standards for each assay: Standards for the various assays are initially prepared at the concentrations listed below for each assay. Each standard is then diluted with 0.9% NaCl to the same fold dilution as the unknown plasma samples, with the following exceptions. The most concentrated cholesterol standard (standard 4; 400 mg/dl) is diluted only half as much as the unknown plasma samples, and none of the FFA standards undergo further dilution after they are prepared.

Triglyceride (glycerol) standards: Standard 1- 4.81 mg/dl glycerol (equivalent to 46.25mg/dl TG), Std 2- 9.62 mg/dl glycerol (equivalent to 92.5mg/dl TG), Standard 3- 19.24 mg/dl glycerol (equivalent to 185mg/dl TG) and Standard 4- 38.48 mg/dl glycerol (equivalent to 370mg/dl TG).

Cholesterol Standards: Standards 5 and 4 are prepared by taking the cholesterol standard (Fisher catalog # SB1012-030; 200mg/dl) directly as provided. Standards 3 through 1 are then prepared by serial twofold dilutions from standard 4. Thus, the actual initial concentrations of your standards are Standard 1- 25 mg/dl, Standard 2- 50 mg/dl, Standard 3- 100 mg/dl, Standard 4- 200 mg/dl, and Standard 5- also 200 mg/dl. When performing the assay, standards 4 through 1 are diluted with 0.9% NaCl to the same fold dilution as the unknown samples, while Std 5 undergoes only half the dilution of the unknowns.

FFA (Nonesterified fatty acid) standards: The FFA concentration in the standard solution we order (Wako catalog # 276-76491) is 28.25 mg/dl. Standard 1 is the stock solution diluted 1.5 x of your sample dilution and is labeled 18.83 mg/dl. Standard 2 is the stock at the same dilution as your samples and is labeled 28.25 mg/dl. Standard 3 is the stock diluted 0.5x your sample dilution and is labeled 56.5 mg/dl.

Glucose standards: The glucose (Sigma catalog #G6152) standards are made at the following conentrations. Standard 1; 100 mg/dl, Standard 2; 200 mg/dl, and standard 3; 400 mg/dl. Once the three standards have been prepared, they are each then diluted further with 0.9% NaCl to the same extent as the unknown samples.

Preparing Control Samples: We run two control samples, a low value (Control 1) and a high value (Control 2), for each assay on each 96-well plate. The controls are purchased from Stanbio (Boerne, Tx, USA). Control 1 is SER-T-FY-1 Level 1 human control serum (cat # G427-86) and Control 2 is SER-T-FY-2 Level 2 human control serum (cat # G428-86). The exact concentration of each analyte varies slightly by lot# and the specific values for a lot are included on the lot specification sheet with each shipment. The values for each analyte for each control are generally in the following ranges:
Glucose- Control 1; 95mg/dl  Control 2; 300mg/dl
Cholesterol- Control 1; 95mg/dl  Control 2; 300mg/dl
Free fatty acids- Control 1; 9mg/dl  Control 2; 40mg/dl
HDL chol- Control 1; 95mg/dl  Control 2; 300mg/dl

Preparing Assay blanks: Sample “blanks” are prepared for each assay by adding 75 ul of saline, rather than plasma, to make the initial dilutions from which aliquots are taken for all of the assays. This “blank” is then run exactly the same way as the unknown plasma samples for all of the assays (except HDL, see below), and the “blank” OD reading (which should be essentially zero) is subtracted from all other values. In addition to the saline “blank” described above, the HDL assay also includes a heparin-MnCl2 blank, since there is a heparin-MnCl2 precipitation step in the HDL assay. In the case of the heparin-MnCl2 blank, you do not actually have a precipitate (since it has saline instead of plasma), but take 30uls of the “supernatant” just as you would for the samples which contain plasma. This “blank” from the precipitation is used to subtract from the HDL cholesterol values obtained for the unknown and control samples, while the saline “blank”, prepared as described above, is used for the standards on the HDL assay.

 

Setting up for the Individual Assays:

Total cholesterol, unesterified cholesterol, and HDL cholesterol assays:

Additional Step for HDL Assay Only: The cholesterol assays for total cholesterol and unesterified cholesterol are done directly on aliquots of the diluted plasma samples. For the analysis of HDL cholesterol, prior to running the cholesterol assay the HDL has to be isolated from the other lipoproteins by precipitation. The apoB containing lipoproteins are precipitated from 100ul of the diluted plasma in the 96 well U-bottom plates using heparin-MnCl2. The plates are then centrifuged for 30min at 40c at 2500rpm, in a Beckman TJ-6 (or comparable) centrifuge.  30ul of the supernatants are then taken for the cholesterol assay. Since the plasma sample is diluted further in the heparin-MnCl2 precipitation step, the HDL cholesterol values have to be multiplied by 1.2.

Cholesterol Assay: The total cholesterol assay is done on 20ul of the diluted plasma sample, the UC assay on 25ul of the diluted plasma sample, and the HDL assay on 30ul of the supernatant after heparin-MnCl2 precipitation. The samples, controls and standards are added to 0.65 ml minitubes to which 600 ul of the cholesterol reagent is added. The reagent with esterase is used for the total cholesterol and HDL assays, and the reagent without esterase is used for the UC assay. The samples are then incubated at 37degrees C in a water bath. After the incubation 170ul aliquots in triplicates are loaded into the 96 well flat bottom plates. Read the plates at 515nm, subtracting the “blank” values from all readings.. Because the total cholesterol is higher than unesterified or HDL cholesterol, use standards 100 mg/dl, 200 mg/dl, and 400 mg/dl. For the HDL assays, use standards 50 mg/dl, 100 mg/dl, and 200 mg/dl. For the UC assay use standards 25 mg/dl, 50 mg/dl, and 100 mg/dl.

Triglyceride and Triglyceride blank assays: For the triglyceride and triglyceride blank assays aliquot 30ul of the diluted sample, controls, and standards into 0.65ml minitubes. Use Glycerol standards 46.25 mg/dl, 92.5 mg/dl, and 185 mg/dl for the triglyceride blank assay and glycerol standards 92.5 mg/dl, 185 mg/dl, and 370 mg/dl for the triglyceride assay. Add 600uls of the triglyceride assay reagent with lipase to each tube for the triglyceride assays, and add 600uls of the triglyceride reagent without lipase to each tube for the triglycride blank assay. Incubate the tubes for 10 min at 37 degrees C in a water bath.  After the incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 540nm. After running the triglyceride and triglyceride blank assays for each sample, the value of the triglyceride blank is subtracted from the triglyceride value to correct for endogenous levels of free glycerol in the plasma.

FFA assay: For the free fatty acid assay aliquot 30ul of the diluted plasma sample, controls, and standards into 0.65ml minitubes. Add 400uls of reagent A and incubate for 5 min at 37 degrees C in a water bath. Then add 200uls of reagent B and incubate for 5 min at 37 degrees C in a water bath.  After incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 550nm and subtract “blank” values from the reading.

Glucose assay:
For the glucose assay, aliquot 15ul of the diluted plasma, controls, and standards into 0.65ml minitubes. Add 600uls of the glucose reagent directly as supplied by the manufacturer, (Fisher cat # SB1070-125 manufactured by Stanbio) to each tube and incubate for 5 min at 37 degrees C in a water bath. After the incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 505nm. Subtract the assay “blank” from the values.

Insulin assay:
Insulin levels in plasma were measured using the ALPCO Mouse Ultrasensitive Insulin ELISA according to manufacturer’s instructions as described : http://www.alpco.com/products/Insulin_Ultrasensitive_Mouse_ELISA.aspx

0. We defined an eQTL as local if the peak association signal was within a 10Mb sliding window of the physical location of the gene(s). We then calculated the average distance between these local eQTL and the transcription start site of the corresponding gene(s) transcription start site.

Estimation of power and mapping resolution: We evaluated the statistical power of the HMDP through simulation studies, with various parameters including the variance explained by SNP, variance explained by genetic background, and variance explained by random errors, and the number of repeated measurements per strain. For the comparison of power with single RI set or classical inbred (CI) only studies, we selected a subset of the simulated phenotypes for each RI or CI set and evaluated the power in the same way. Since there are 8 possibilities of SNPs being polymorphic among three sets of RI strains, the putative causal SNPs are categorized into 8 classes and power is evaluated for each class. The significance threshold per each RI set is determined separately using parametric bootstrapping described below.  See Supplemental Methods for comparison of BXD RI set to the full HMDP and simulations.

Genome-wide Significance Threshold: Genome-wide significance threshold in genome-wide association mapping is determined by the family-wise error rate (FWER) as the probability of observing one or more false positives across all SNPs per phenotype. We ran 100 different sets of permutation tests and parametric bootstrapping of size 1000, and observed that the mean and standard error of the genome-wide significance threshold at family-wise error rate (FWER) of 0.05 were 3.9 x 10-6 ± 0.3 x 10-6, and 4.0 x 10-6 ± 0.3 x 10-6, respectively. This is approximately an order of magnitude larger than the significance threshold obtained by Bonferroni correction (4.6 x 10-7). We also performed parametric bootstrapping under simulated the genetic background effect from population structure using EMMA. With 50% and 100% of variance explained by genetic background, the thresholds were determined to be 1.6 x 10-6 ± 0.2 x10-6 and 1.7 x 10-6 ± 0.2 x 10-6. The reduction in the significance threshold compared to no genetic background effect is due to the fact that inter-SNP correlation due to long-range LD reduces when conditioning on the population structure.  A detailed explanation of these analyses is provided in the Supplemental Methods.

 

5.         Tissues, weights (B. Parks)

At sacrifice (following 8 weeks high fat diet intervention) mice were dissected and the following tissues were flash frozen in liquid nitrogen:  liver, spleen, right kidney, heart, gonadal fat pads, retroperitoneal fat pads, mesenteric fat pad, subcutaneous fat pads, segment of intestine (duodenum), skeletal muscle (quadriceps), and cecum. The following tissues were weighed:  liver, spleen, right kidney, heart, lungs, gonadal fat pads, retroperitoneal fat pads, mesenteric fat pad, and subcutaneous fat pads.

 


6.         Gut flora composition (B. Parks)

Flash frozen cecum samples were excised open to expose feces and total DNA was isolated using MOBIO power soil kit.  Isolated DNA was then used to amplify 16s bacterial ribosomal DNA using custom barcoded primers developed by Rob Knights group (U. of Colorado).  Amplified samples were then cleaned and processed for 454 pyrosequencing.  Sequences were analyzed using QIIME.

 


7.         Expression arrays, adipose (B. Parks)

Flash frozen samples were weighed and homogenized in Qiazol (Qiagen) according to manufacturer’s protocol.  Following homogenization livers were isolated in RNeasy 96 columns (Qiagen) using the manufacturers protocol.

 


8.         Expression arrays, liver (B. Parks)

Flash frozen samples were weighed and homogenized in Qiazol (Qiagen) according to manufacturer’s protocol.  Following homogenization livers were isolated in RNeasy 96 columns (Qiagen) using the manufacturers protocol.

 


9.         Liver lipids (S. Hui)

Lipids were extracted from livers by modified Folch’s extraction protocol (JBC, 226: 497).  Briefly, liver samples (~0.1g) were homogenized in ice-cold PBS (1:10, w/v).  Homogenate (0.5 ml) was transferred to a glass tube for lipid extraction with 2.5 ml chloroform/methanol (2:1, v/v).  Tubes were shaken vigorously to ensure adequate mixing of the aqueous and organic phases.  Tubes were then centrifuged at 2000 rpm for 15 min at 4ºC.  The bottom organic phase was transferred to a new glass tube.  Extraction of lipids from the aqueous phase was repeated with 2.5 ml chloroform.  After phase separation by centrifugation, the bottom organic phase from the second extraction was combined with the one from the first extraction.  The combined organic extract was then dried completely under nitrogen.  Dried lipids were re-solubilized in 0.5 ml 0.5% Triton X-100/PBS at 55 ºC. 

For triglyceride assay, lipids were further diluted 4x in 0.5% Triton X-100/PBS and sonicated in an ultrasonic water bath for 2 minutes at 55 ºC.  Amounts of triglyceride in each sample were determined by a colorimetic assay kit from Wako Diagnostics.

 


10.        Expression arrays, muscle (B. Parks)

In development.