MOUSE :: HMDP :: Akita Diabetes Study, Pilot

D.         Hybrid Mouse Diversity Panel:  Akita Diabetes Study, Pilot

1.         Mice (R. Davis)
2.         Plasma lipids, insulin & glucose (L. Castellani)
3.         Body composition, NMR (R. Davis)
4.         Nephritis, kidney (R. LeBoeuf)
5.         Heart functions (R. Davis)
6.         Atherosclerosis (R. Davis)
7.         Urine metabolites (R. Davis)
8.         Neuropathy (R. Davis)


1.         Mice (R. Davis)

The Akita mutation in the mouse insulin2 gene causes a misfolding of the insulin protein that leads to unfolded protein response and apoptosis in the pancreatic islets.  Thus, the Akita mutation is dominant and leads to deficiency of islet cells resulting in a type 1 diabetes phenotype.  The diabetes is much more severe in males than in females but, males show relatively uniform hyperglycemia by 8 weeks of age, independent of genetic background.  By contrast, diabetic complications such as nephropathy are strongly dependent on genetic background.  We hypothesized that we could map the determinants of diabetic complication susceptibility by mating the Akita mutation to mice of the HMDP.   In our pilot experiments, we had several objectives.

  1. Monitor a range of phenotypes for diabetic complications that would establish the most robust and efficient assays for mapping studies with the full HMDP
  2. Determine if a high fat diet would induce atherosclerosis in the Akita F1 hybrids so that susceptibility to diabetic atherosclerosis could be mapped.
  3. Determine if a high fat diet would drive insulin production in females sufficient to induce comparable islet depletion as seen in males.


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

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: Glucose levels in blood were measured using the AlphaTRAK blood glucose monitoring system (Abbott Laboratories).  This unit has the advantage that it is calibrated for use with mice, taking into account the difference in blood cell density that normally causes erroneous results in units calibrated for humans.  Also, the AlphaTRAK meter has a maximum reading of 750 mg/DL so that more measurements are within the instrument’s range compared to other devices such as the OneTouch Ultra that we had previously used.

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 :


3.         Body composition, NMR (R. Davis)

Animals were measured for total body fat mass and lean mass by nuclear magnetic resonance (NMR) using the Bruker Minispec with software from Echo Medical Systems (Houston, TX) as described by Taicher GZ, Tinsley FC, Reiderman A, Heiman ML. Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis. Anal Bioanal Chem 377: 990–1002, 2003.


4.         Nephritis, kidney (R. LeBoeuf)

In development.


5.         Heart functions (R. Davis)

Cardiomyopathy is an important complication of human diabetes.  Cardiomyopathy in the Akita mouse on a C57BL/6 background had previously been characterized by Basu, et al. Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function. 

They found echocardiographic evidence of cardiomyopathy and Christoph Rau, in our lab was able to see similar changes in our Akita mice using the Visualsonics Vevo 2100 in the lab of Yibin Wang.  The Vevo family of instruments provides functionality similar to that found on a clinical ultrasound instrument, but at much higher resolution. Imaging a murine heart with a clinical instrument provides a low resolution picture of the organ, whereas with the high frequency capabilities of the Vevo systems, the murine heart is visible in great detail, with enough clarity to make important measurements of cardiopulmonary function.  However, as a screening measurement for cardiomyopathy in the proposed Akita F1 HMDP, we chose to monitor ANP (A-type natriuretic peptide) and BNP (B-type natriuretic peptide)  mRNA levels. Basu, et al., showed that BNP mRNA levels in Akita hearts were one of the earliest and most robust indicators of cardiomyopathy in this model.  Quantitative PCR for the natriuretic peptides was carried out using the method described by Ellmers, et al.

Basu R, Oudit GY, Wang X, Zhang L, Ussher JR, Lopaschuk GD, Kassiri Z. Am J Physiol Heart Circ Physiol. 2009 Dec; 297(6): H2096-108.

L. J. Ellmers, J. W. Knowles, H.-S. Kim, O. Smithies, N. Maeda and V. A. Cameron ( Am J Physiol Heart Circ Physiol 283:707-714, 2002) modified to use non-fluorescent primers.


6.         Atherosclerosis (R. Davis)

For the assessment of aortic lesions, mice were euthanized and the upper portion of the heart and proximal aorta were removed, embedded in OCT compound (Miles Laboratories), and stored at −70°C. Serial 10 μm–thick cryosections from the middle portion of the left ventricle of the aortic arch were collected and mounted on poly-d-lysine–coated plates. Sections were stained with oil red-O and hematoxylin, and the lipid staining areas were counted in a blinded fashion by light microscopy.


7.         Urine metabolites (R. Davis)

As a measure of nephropathy, we measured albumin to creatinine ratios (ACR) in urine collected overnight in metabolic cages designed for the purpose.  Albumin and creatine were measured using the Exocell kits for Albumin (Albuwell M™.(#1011) and the Creatinine Companion Protocol (#1012).
Albuwell M is an indirect competitive ELISA designed to monitor kidney function in the mouse by measurement of urinary albumin. To complete the assay, sample and rabbit anti-murine albumin antibody are added to albumin coated wells. The antibody interacts and binds with the albumin immobilized to the stationary phase or with albumin in the fluid phase, hence the notion of competitive binding.

A subsequent reaction with anti-rabbit -HRP conjugate labels the probe with enzyme. After washing, only the antibody-conjugate bound to the stationary phase remains in the well, and this is detected using a chromogenic reaction. Color intensity is inversely proportional to the logarithm of albumin in the fluid phase. The assay may be completed in less than 2.5 hours.
Specimen Required:    Urine, 10ul
Assay Range: 0.3-10 ug/ml
Precision: Intraassay and interassay precision for samples within the useful range of the assay have a C.V.<10% of the mean.

The Creatinine Companion, a chemical assay designed to monitor urinary creatinine,
is based upon the Jaffe' reaction of alkaline picrate with creatinine. Standards or samples are added to a microplate, and alkaline picrate reagent is added. Absorbance at 500 nm is determined after a 10 minute incubation on the benchtop. Subsequently, an acid solution is added, and absorbance is again determined after a 5 minute incubation. The difference between these absorbance values is directly proportional to the the creatinine concentration, and a standard curve is generated from the response to the standards. Unknown samples are evaluated by comparing response to the standard curve. The assay may be completed in less than 30 minutes.

The assay serves as a normalization procedure for other analytes in urine samples, particularly for urinary albumin (1,2). It allows for a measure of kidney function based on a spot urine sample rather than the usual 24 hour pool.
Specimen Required:    Urine, 20 ul
Assay Range: 10-100 ug/ml or 1-10 mg/dL
Precision: Intra- and interassay precision of samples within the useful range have a C.V.<10% of the mean.

Bennet, P. H. et. al. Screening and management of microalbuminuria in patients with diabetis mellitus. Am. J. Kidney Dis. 25:107, 1995.

Warram, J. H. et. al. Effect of duration of type 1 diabeties on the prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine ratio. J. Am. Soc. Nephrol. 7:930, 1996.


8.         Neuropathy (R. Davis)

As measures of neuropathy, we measured foot-pad sensitivity to mechanical (Von Frey’s test) and heat (Hargreaves test) stimulation.

Von Frey Hair Test: This test quantitatively evaluates for the presence or absence of allodynia. A hand hair digital Von Frey Hair touch stimulator is be applied to each hind-limb. The digital device records the maximum force applied at the time of the paw withdrawal reflex. The test is performed five times per testing occasion on each animal. The animals are non-restrained during the testing situation.

Hargreaves Test: Mice are placed in a 4x4x7 inch (WxDxH) plexiglass box on an elevated heated glass plate and after 15min of habituation an infrared beam, of adjustable intensity, is shone onto the rear of the footpad. At an intensity setting of 5, the response, a definitive paw flick, occurs within 5-10s under basal conditions, the test is terminated at 15s if the animal has not responded. This test does require 2 days of habituation in the test chamber, 15 min each so that the mouse is not moving but not asleep.

These tests were carried out with the equipment and assistance of Wendy Walwyn (