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 syndr....

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 ....

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