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.
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.
Polycystic ovary syndrome (PCOS) is a common disorder occurring in 12%-20% of reproductive-aged women1,2. 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 syndrome3,4,5,6. 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 production7,8. Insulin resistance is thought to have several possible origins but studies suggest it may be due to abnormal patterns of insulin receptor signaling9,10. Studies have evaluated insulin resistance in PCOS patients using the gold standard technique of hyperinsulinemic-euglycemic clamp11,12,13,14,15. 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.
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
2. Set-up of infusion experiments
3. Catheter insertion
4. Infusion set-up and first infusion
5. Blood sampling
6. Second infusion
7. Mass spectrometry
Using previously described isotope dilution equations, the total plasma glucose rate (glucoseRa) 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 glucoseRa was 19.98 ± 2.53 mg/(kg·min) after 6 h fasting and 25.80 ± 1.76 mg/(kg·min) during glucose-rich conditions. Using the calculation listed above, GPR was 19.08 ± 2.53 mg/(kg·min) after 6 h fasting and 8.56 ± 1.40 mg/(kg·min)) in glucose rich conditions (Table 1 and Figure 1).
Figure 1: Glucose production rate in fasting and glucose-rich conditions Please click here to view a larger version of this figure.
Fasting | Glucose-rich | |
(mean ± SD) | (mean ± SD) | |
Glucose Ra, mg/(kg.min) | 19.98 ± 2.53 | 25.80 ± 1.76 |
GPR, mg/ (kg.min) | 19.08 ± 2.53 | 8.56 ± 1.40 |
Ra, rate of glucose appearance; GPR, glucose production rate |
Table 1: Ra and GPR in fasting and glucose-rich conditions
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 many studies have demonstrated indirect measurements of hepatic glucose production in a PCOS animal model, few have measured hepatic glucose production directly. In this study we describe a straightforward way to measure the rate of hepatic glucose production in multiple mice at the same time. This technique can be applied to many studies involving glucose metabolism in various mouse models. Furthermore, this technique can serve as the foundation upon which additional measurements can be applied, such as measurement of gluconeogenesis and glycogenolysis20,23.
The critical components of this experiment are the catheter insertion and defining the exact measurements of [6,6-2H2]glucose and natural glucose when creating infusion solutions in order to adequately perform GCMS. The catheter insertion technique used is described by Marini et al. 200624. Although there are invasive methods of administering infusions and sampling via carotid artery and jugular vein catheters25,26,27, insertion of a minimally invasive tail vein catheter accomplishes the same goals in a less invasive and less time-intensive fashion. Although two catheters may be placed in each tail vein, one for infusing and one for sampling, we used one catheter for infusion and then performed sampling via cheek venipuncture since there were only two time points for sampling. However, if a study included multiple time points for sampling, insertion of a second tail vein catheter can help facilitate this28.
Exact measurements are crucial for calculations involving mass spectrometry to ensure accurate results. We used the glucose tracer to measure the appearance of glucose is [6,6-2H2]glucose21. Although other non-radioactive tracers have been employed in other studies, this a stable isotope that is commonly used in our lab20. Stable glucose tracers are preferred over radioactive glucose tracers due the improved safety profile, natural presence, lack of half-life affecting study time, and ability to combine different tracers29. The choice of tracer is up to the discretion and expertise of the researchers performing the study.
There are some limitations of this study, including the technical demand of the procedure. However, compared to more invasive techniques for catheterization (i.e., catherization of carotid artery and external jugular vein), this technique is straightforward, efficient, and less morbid. If utilizing two tail vein catheters, there have been reports of erroneous enrichment values from the infusion interfering with the sampling catheter24. However, in most studies the need for multiple samples is not warranted because steady-state is known. Lastly, although mass spectrometry gives precise measurements, it requires additional skills, expertise, and cost.
In this study, we describe a straightforward, accurate way to measure the rate of total hepatic glucose production in a PCOS mouse model. This technique should serve as the foundation for multiple studies involving glucose metabolism of mouse models.
The authors have nothing to disclose.
This work was supported by training grants by the Department of Obstetrics and Gynecology, Baylor College of Medicine (ALG) and R-01 research grant (Grant # DK114689) for CSB, SC and JM from National Institutes of Health.
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 |