Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Summary

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Abstract

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-²H₂]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-²H₂]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

Introduction

Polycystic ovary syndrome (PCOS) is a common disorder occurring in 12%-20% of reproductive-aged women^1,2. It is a complex disease resulting in variable phenotypes involving polycystic ovaries, irregular menses and clinical or laboratory evidence of hyperandrogenemia, and is typically diagnosed when a woman meets two of the three criteria³. A predominant aspect of PCOS, and a key factor in its pathogenesis, is metabolic derangements that are found in women who have the disease. Women with PCOS have higher incidences of insulin resistance, glucose intolerance, obesity, and metabolic syndrome^3,4,5,6. Insulin resistance is not only a manifestation of the disease, but it is thought to contribute to its pathogenesis by potentiating the action of luteinizing hormone in the ovary thereby leading to increased androgen production^7,8. Insulin resistance is thought to have several possible origins but studies suggest it may be due to abnormal patterns of insulin receptor signaling^9,10. Studies have evaluated insulin resistance in PCOS patients using the gold standard technique of hyperinsulinemic-euglycemic clamp^{11,12,13,14,15}. Women with PCOS, irrespective of BMI, have higher levels of insulin resistance compared with controls. Insulin control over glucose production is impaired in disorders of insulin resistance leading to excess glucose production. For example, diabetic patients have increased rates of gluconeogenesis and impaired suppression of glycogenolysis¹⁶. Furthermore, impaired suppression of glucose production has been observed in diabetic rats¹⁷. Although clamp studies can give a measurement of insulin resistance, few studies in PCOS focus on direct measurement of glucose production in fasting and fed states. This requires the use of a non-radioactive isotopic glucose tracer infusion and measurement via mass spectrometry.

Animal models have been extensively used in PCOS research. Both lean and obese-type PCOS murine models have been created by administering androgens prenatally, prepubertally, or post-pubertally¹⁸. Rodent PCOS models also demonstrate metabolic differences compared with their respective controls. Previous data from our lab demonstrated abnormal glucose tolerance tests (GTT) in PCOS mouse models (lean and obese), consistent with human PCOS literature¹⁹. Use of a lean and obese animal model allows further investigation into metabolic differences. Specifically, this model allows evaluation of the rate of glucose production directly using isotopic glucose tracers. One of the most commonly used stable isotopic glucose tracer is [6,6-²H₂]glucose. The [6,6-²H₂]glucose enrichment can be measured using a pentaacetate derivative as previously described²⁰.

In this study, our aim was to measure the rate of hepatic glucose production in fasting and glucose-rich state in PCOS mice using isotopic glucose infusion. These techniques can be applied to a wide range of experiments involving glucose kinetics.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Baylor College of Medicine.

1. Preparation of [6,6-²H₂]glucose

1. One day before the procedure, prepare the stable isotope glucose tracer in normal saline. For this experiment, [6,6-²H₂]glucose was used as a tracer to measure plasma glucose appearance rate.
   NOTE: In this experiment, glucose production during fasting and glucose-rich conditions were measured, so the glucose isotope was prepared in two different preparations. Prepare the solutions in such a way that the final infusing dose will be close to 1 mg/(kg·min) and 2 mg/(kg·min), respectively. These concentrations were optimized by several prior pilot studies.
2. Prepare the first infusate with only [6,6-²H₂]glucose as a tracer to represent the fasting condition by dissolving sterile and pyrogen free [6,6-²H₂]glucose in sterile 0.9% sodium chloride solution.
3. Prepare the second infusate by dissolving both [6,6-²H₂]glucose tracer along with non-isotopic glucose (~20 mg/kg·min) in sterile 0.9% sodium chloride solution to simulate the fed condition.
   NOTE: In the present experiment, a primed constant rate infusion of [6,6-²H₂]glucose at (~) 1.08 mg/(kg·min) (fasting condition) and (~) 1.9 mg/kg·min (glucose rich condition) was used. In order to mimic the 'glucose rich condition', D-glucose infusion rate was set at (~) 18.8 mg/(kg·min).
4. Once the solutions are prepared, sterile filter the solutions with a 0.22 µm filter and store at 4 °C. Warm the solutions to room temperature prior to the infusion. Load the solutions on prelabeled 1 mL syringes.
5. Set up the pump to facilitate a primed constant rate infusion of the first infusate (for basal condition) containing only the tracer, and program the pump for a constant rate infusion of the isotope at 150 µL/h for 3 h.
6. At the end of the 3 h fasting set up, replace the first infusate syringe with the second infusate syringe containing the isotopic tracer and D-glucose (for the simulated fed state), and prime the infusion for the intial 15 min at 600 µL/h. Set the pump to a constant rate infusion at 150 µL/h for the additional 3 h.

2. Set-up of infusion experiments

1. Remove the mice from their home cages and place them in their fasting cages 3 h prior to the start of the experiment. For this experiment, a 4-month-old female mice from the C57BL/6J strain was used.
   NOTE: This procedure does not require any analgesia prior to or during the procedure given its minimally invasive nature.
2. Assemble the caging equipment by grouping the mice into a set number of mice per insulin pump. Place partitions on top of a flat, stable base to make individual stalls for the mice. It is important to have access to the tail vein catheter for the infusion to run, so ensure that there is a notch in the bottom of the cage door.
   NOTE: Cages shown in the video were made specifically for this experiment. Cages are clear and made out of plexiglass. There is a flat, stable base (standard caging equipment) upon which the partitions sit. This setup consists of several pieces to allow ease of setup in several different environments depending on the height of the table and the needs of the experimenter. The door to the cage slides to close and has a notch at the bottom to allow the tail to fit through.
3. Assemble the 1 mL syringes containing 1 mL of basal infusate, and then connect to the infusion pump tubing using the polyethylene tubing 0.28 mm ID x 0.61 mm.
4. Prepare the infusion pump by setting the rate to 150 µL/h, which is the basal rate.
5. Heat a water bath to 48 °C.
6. Prepare the catheter insertion station adjacent to the water bath containing the 30 G 0.5 inch needles, 0.3 mm ID x 0.64 mm silastic tubing, and 1 inch clear transpore tape.
7. After 3 h of fasting, begin the catheter insertion process, which is detailed below.

3. Catheter insertion

1. Select one mouse and place it in a secure holder with access to the tail. An example of what to use is a bottle cut in half with a notch for the tail. Place the holder on a flat base. Place a piece tape over the proximal portion of the tail to allow space for catheter insertion more distally.
   NOTE: The type of tape chosen for this task must have moderate strength and adhesiveness. Multipurpose, paper-based labeling tape was used in this experiment as it is mild and easy to peel off.
2. Prepare the catheter (30 G needle attached to 0.3 mm silastic tubing and PE- 10 were gas sterilized) by attaching it to a 1 mL syringe containing sterile heparinized saline flush. Flush the catheter gently.
3. Bring the mouse to the water bath and insert the tail in the water bath for approximately 30-45 s. This helps to dilate the tail vasculature for catheter placement.
4. Perform the catheter insertion under sterile conditions. Once the tail is warmed, clean the tail with benzalkonium wipes and place a small copper, toothless alligator clamp, that was previously contoured to the shape of the tail, as a tourniquet at the proximal end of the tail. Visualize the lateral tail vein under a magnifying glass, and then carefully insert the catheter into the tail vein and withdraw the needle. Flush the solution gently to ensure patency of the catheter.
5. Wrap a piece of 1 inch transpore tape around the insertion site to secure the catheter. Remove the small tourniquet from the tail.

4. Infusion set-up and first infusion

1. Place the mouse in its individual cage and close the sliding door, ensuring the tail is protruding through the notch and remains outside of the cage.
2. Place an additional piece of tape over the whole catheter and the tail to secure it to the base plate of the cage. Multipurpose colored, label tape was used for this step.
3. Disconnect the flush from the tail vein catheter and place a small clamp on the catheter's silastic tubing to prevent backflow while connecting the infusate line from the pump.
4. Once securely connected, remove the clamp and flush with the priming solution, which consists of the infusate. Ensure that the solution is clear in the tubing and not blood-stained.
5. Make note of the time of initiation of the infusion to ensure that it runs for the total time of approximately 3 h. If multiple cages are used simultaneously, they should be staggered to manage infusion times effectively.
6. Once infusion lines are noted to be functioning properly, remove the cover from the mouse. Place standard bedding around the mouse.
7. Start the infusion with the first infusate containing the tracer and run it for 3 h continuously. For the duration of the infusion, continue to check mice wellbeing as well as infusion lines. Ensure that the infusion tubing is properly secured and that there are no leaks from the line connection points.

5. Blood sampling

1. After the first infusion has completed, stop the infusion, place a clamp on the sylastic tubing on the catheter to prevent back flow. Gently remove the mice from their enclosures without disturbing the catheter in order to collect blood. Place them at a spot near the cages for blood draw. For this experiment, the mice underwent cheek venipuncture using a 4 mm lancet.
2. Collect ~75 µL of blood in the desired vial. To deproteinize samples in preparation for mass spectrometry, add approximately 15 µL of blood to 500 µL of acetone. The remaining blood can be used to check the blood glucose level via the glucometer and/or centrifuged to separate plasma for future hormone assays.

6. Second infusion

1. Remove the infusates from the syringe pumps by disconnecting the tubing from the syringes and replace it with the second infusate containing the tracer along with the glucose. Repeat steps 4.2 through 4.7 using the glucose-rich isotopic glucose infusion.
   1. To reach a steady state, run a bolus of the second infusate at 600 µL/h for 15 min. Note the starting time for each group of cages. Decrease the infusion rate to 150 µL/h to complete the 3 h of total infusion time.
2. Repeat steps 5.1 and 5.2.
3. Stop the infusion pump and remove the tail vein catheter gently, apply pressure at the catheter sites until the bleeding stops, and return mice to their home cages.
4. Santize and disinfect the caging setup thoroughly with standard soap and water.
   NOTE: This is a survival procedure. Mice can be returned to cages and kept for further experiements if needed. It is recommended that no further experiementation is performed for at least a week following this procedure to ensure adequate animal welfare.

7. Mass spectrometry

1. Send out the samples for mass spectrometry.
2. Analyses
   1. Measure the isotopic enrichment of [6,6-²H2]glucose by gaschromatography - mass spectrometry (GCMS) using the pentaacetate derivative^21,22. Briefly, this method involves preparation of the pentaacetate derivative of glucose, followed by sample analysis using GCMS ^20,22.
3. Calculations
   1. Perform all kinetic measurements under steady state conditions. Total plasma glucose appearance rate (glucose R_a) was calculated from the M+2 enrichment of [6,6-²H2]glucose in plasma using established isotope dilution equations²¹. Under steady state conditions, it is assumed that the rate of appearance of glucose is equal to the rate of disappearance of glucose. Rate of endogenous glucose production (mg/(kg·min)) (GPR) = glucoseR_a - exogenous glucose.

Results

Using previously described isotope dilution equations, the total plasma glucose rate (glucoseR_a) was calculated from M+2 enrichment of [6,6-²H₂]glucose in fasting and glucose-rich conditions using the pentaacetate derivative²¹. Under steady-state conditions, it is assumed that rate of appearance of glucose is equal to the rate of disappearance of glucose. In the control group, the total glucoseR_a was 19.98 ± 2.53 mg/(kg·min) after 6 h fasting a...

Discussion

Hyperglycemia and abnormal glucose metabolism/homeostasis are features of PCOS. Blood glucose level is maintained by a combination of glucose from diet and glucose production via glycogenolysis and gluconeogenesis and glycogenesis, under the control of hormone and enzymes. Hepatic glucose production is suppressed by the presence of increased circulating glucose levels. In disorders of abnormal glucose metabolism, regulation of the suppression of glucose production is compromised leading to hyperglycemia. While m...

Materials

Name	Company	Catalog Number	Comments
0.9% sodium chloride solution	McKesson	275595
10 mL BD Luer-Lok tip syringe	VWR	75846-756	Two syringes per animal (one for isotopic glucose solution, one for glucose-rich isotopic solution)
1-inch clear transpore tape	3M	70200400169
1-inch Labeling tape	Fisher	GS07F161BA	Brand is example
5 mL syringe containing heparanized saline flush	McKesson	191-MIH-2235	One can also prepare a heparin flush solution (10 units/mL heparin in 0.9% sodium chloride)
5 mm Medipoint Goldenrod animal lancets	Fisher Scientific	NC9891620	5 mm if animal is between 2 and 6 months
Acetone	Sigma-Aldrich	650501
Advanced hot plate stirrer	VWR	97042-602	Brand is example
BD 27 gauge 0.5 inch needles	Health Warehouse	A283952
BD 30 gauge 0.5 inch needles	Medvet	305106
BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm	VWR	63019-004
BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm	VWR	63019-004
Beaker, 1000 mL			Any brand
Caging pellets
Clear VOA glass vials with closed-top cap	Fisher Scientific	05-719-120	For storage of acetone and blood draw samples
Copper toothless alligator clamp for tourniquet	Amazon		Any Brand; smooth toothless alligator clips made of solid copper
D-(+)-glucose >99.5%	Sigma-Aldrich	G8270
D-glucose (6,6-D2, 99%)	Cambridge Isotope Laboratories, Inc.	DLM-349-PK
Dow Corning silastic tubing 0.3 mm ID x 0.64 mm OD	VWR	62999-042
Magnifying glass	Amazon		Any brand; similar to LANCOSC Magnifying Glass with Light and Stand
Microbalance	Ohaus Adventurer Pro	AV264C	Any similar model with 0.0001g accuracy can be used
Nalgene bottle, 500 mL	Sigma-Aldrich	B0158-12EA	Or any Similar brand; saw in half (including lid) and cut tail-sized notch in the bottom
PHD Ultra multi-syringe pump	Harvard Apparatus	70-3024A
Plexiglass sheet			Any brand; to stabalize mouse during catheter insertion
Plexiglass sheets and dividers			Any brand; used to cage mice during infusion