Name: evaluation of hepatic glucose production in a polycystic ovary syndrome mouse model
Uploaded: 2022-03-05T13:30:00
Description: Watch this Scientific Journal Video about Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model at JoVE.com

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.

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
<ol>
	<li>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 a...

<ol>
	<li>March, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+polycystic+ovary+syndrome+in+a+community+sample+assessed+under+contrasting+diagnostic+criteria.">The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria.</a> Human Reproduction. 25 (2), 544-551 (2009).</li><li>Yildiz, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+phenotype+and+cardiometabolic+risk+of+polycystic+ovary+syndrome+under+different+diagnostic+criteria.">Prevalence, phenotype and cardiometabolic risk of polycystic ovary syndrome under different diagnostic criteria.</a> Human Reproduction. 27 (10), 3067-3073 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+2003+consensus+on+diagnostic+criteria+and+long-term+health+risks+related+to+polycystic+ovary+syndrome.">Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome.</a> Fertility and Sterility. 81 (1), 19-25 (2004).</li><li>Goodarzi, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome:+etiology,+pathogenesis+and+diagnosis.">Polycystic ovary syndrome: etiology, pathogenesis and diagnosis.</a> Nature Reviews. Endocrinology. 7 (4), 219-231 (2011).</li><li>Azziz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction:+Determinants+of+polycystic+ovary+syndrome.">Introduction: Determinants of polycystic ovary syndrome.</a> Fertility and Sterility. 106 (1), 4-5 (2016).</li><li>Baskind, N. E., Balen, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic-pituitary,+ovarian+and+adrenal+contributions+to+polycystic+ovary+syndrome.">Hypothalamic-pituitary, ovarian and adrenal contributions to polycystic ovary syndrome.</a> Best Practice and Research. Clinical Obstetrics &amp; Gynaecology. 37, 80-97 (2016).</li><li>Burghen, G. A., Givens, J. R., Kitabchi, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+hyperandrogenism+with+hyperinsulinism+in+polycystic+ovarian+disease.">Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease.</a> The Journal of Clinical Endocrinology and Metabolism. 50 (1), 113-116 (1980).</li><li>Bremer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome+in+the+pediatric+population.">Polycystic ovary syndrome in the pediatric population.</a> Metabolic Syndrome and Related Disorders. 8 (5), 375-394 (2010).</li><li>Dunaif, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+insulin+receptor+serine+phosphorylation+in+cultured+fibroblasts+and+in+skeletal+muscle.+A+potential+mechanism+for+insulin+resistance+in+the+polycystic+ovary+syndrome.">Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome.</a> The Journal of Clinical Investigation. 96 (2), 801-810 (1995).</li><li>H&#248;jlund, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin-stimulated+phosphorylation+of+Akt+and+AS160+in+skeletal+muscle+of+women+with+polycystic+ovary+syndrome+is+reversed+by+pioglitazone+treatment.">Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment.</a> Diabetes. 57 (2), 357-366 (2008).</li><li>Moghetti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergences+in+insulin+resistance+between+the+different+phenotypes+of+the+polycystic+ovary+syndrome.">Divergences in insulin resistance between the different phenotypes of the polycystic ovary syndrome.</a> The Journal of Clinical Endocrinology and Metabolism. 98 (4), 628-637 (2013).</li><li>Ovalle, F., Azziz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance,+polycystic+ovary+syndrome,+and+type+2+diabetes+mellitus.">Insulin resistance, polycystic ovary syndrome, and type 2 diabetes mellitus.</a> Fertility and Sterility. 77 (6), 1095-1105 (2002).</li><li>Dunaif, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+peripheral+insulin+resistance,+independent+of+obesity,+in+polycystic+ovary+syndrome.">Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome.</a> Diabetes. 38 (9), 1165-1174 (1989).</li><li>Hutchison, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+on+insulin+resistance+and+body+composition+in+overweight+and+obese+women+with+and+without+polycystic+ovary+syndrome.">Effects of exercise on insulin resistance and body composition in overweight and obese women with and without polycystic ovary syndrome.</a> The Journal of Clinical Endocrinology Metabolism. 96 (1), 48-56 (2011).</li><li>Stepto, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Women+with+polycystic+ovary+syndrome+have+intrinsic+insulin+resistance+on+euglycaemic-hyperinsulaemic+clamp.">Women with polycystic ovary syndrome have intrinsic insulin resistance on euglycaemic-hyperinsulaemic clamp.</a> Human Reproduction. 28 (3), 777-784 (2013).</li><li>Basu, R., Schwenk, W. F., Rizza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+fasting+glucose+production+and+disappearance+are+abnormal+in+people+with+&amp;#34;mild&amp;#34;+and+&amp;#34;severe&amp;#34;+type+2+diabetes.">Both fasting glucose production and disappearance are abnormal in people with &amp;#34;mild&amp;#34; and &amp;#34;severe&amp;#34; type 2 diabetes.</a> American Journal of Physiology, Endocrinology and Metabolism. 287 (1), 55-62 (2004).</li><li>Blesson, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+dependent+dysregulation+of+hepatic+glucose+production+in+lean+Type+2+diabetic+rats.">Sex dependent dysregulation of hepatic glucose production in lean Type 2 diabetic rats.</a> Frontiers in Endocrinology. 10, 538 (2019).</li><li>Caldwell, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+reproductive,+metabolic,+and+endocrine+features+of+polycystic+ovary+syndrome+in+female+hyperandrogenic+mouse+models.">Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models.</a> Endocrinology. 155 (8), 3146-3159 (2014).</li><li>Chappell, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+androgen+induced+lean+PCOS+impairs+mitochondria+and+mRNA+profiles+in+oocytes.">Prenatal androgen induced lean PCOS impairs mitochondria and mRNA profiles in oocytes.</a> Endocrine Connections. 9 (3), 261-270 (2020).</li><li>Chacko, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+gluconeogenesis+using+glucose+fragments+and+mass+spectrometry+after+ingestion+of+deuterium+oxide.">Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide.</a> Journal of Applied Physiology. 104 (4), 944-951 (2008).</li><li>Bier, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+&amp;#34;true&amp;#34;+glucose+production+rates+in+infancy+and+childhood+with+6,6-dideuteroglucose.">Measurement of &amp;#34;true&amp;#34; glucose production rates in infancy and childhood with 6,6-dideuteroglucose.</a> Diabetes. 26 (11), 1016-1023 (1977).</li><li>Chacko, S. K., Sunehag, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconeogenesis+continues+in+premature+infants+receiving+total+parenteral+nutrition.">Gluconeogenesis continues in premature infants receiving total parenteral nutrition.</a> Archives of Disease in Childhood. Fetal and Neonatal Edition. 95 (6), 413-418 (2010).</li><li>Chacko, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ghrelin+on+glucose+regulation+in+mice.">Effect of ghrelin on glucose regulation in mice.</a> American Journal of Physiology, Endocrinology and Metabolism. 302 (9), 1055-1062 (2012).</li><li>Marini, J. C., Lee, B., Garlick, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-surgical+alternatives+to+invasive+procedures+in+mice.">Non-surgical alternatives to invasive procedures in mice.</a> Laboratory Animals. 40 (3), 275-281 (2006).</li><li>Jacobs, J. D., Hopper-Borge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+infusions+for+pharmacokinetic+and+pharmacodynamic+analysis+of+taxanes+in+mice.">Carotid artery infusions for pharmacokinetic and pharmacodynamic analysis of taxanes in mice.</a> Journal of Visualized Experiments: JoVE. (92), e51917 (2014).</li><li>Ayala, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperinsulinemic-euglycemic+clamps+in+conscious,+unrestrained+mice.">Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice.</a> Journal of Visualized Experiments: JoVE. (57), e3188 (2011).</li><li>Kmiotek, E. K., Baimel, C., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Intravenous+Self+Administration+in+a+Mouse+Model.">Methods for Intravenous Self Administration in a Mouse Model.</a> Journal of Visualized Experiments: JoVE. (70), e3739 (2012).</li><li>Marini, J. C., Lee, B., Garlick, P. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride solution</td>
			<td>McKesson</td>
			<td>275595</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL BD Luer-Lok tip syringe</td>
			<td>VWR</td>
			<td>75846-756</td>
			<td>Two syringes per animal (one for isotopic glucose solution, one for glucose-rich isotopic solution)</td>
		</tr>
		<tr>
			<td>1-inch clear transpore tape</td>
			<td>3M</td>
			<td>70200400169</td>
			<td></td>
		</tr>
		<tr>
			<td>1-inch Labeling tape</td>
			<td>Fisher</td>
			<td>GS07F161BA</td>
			<td>Brand is example</td>
		</tr>
		<tr>
			<td>5 mL syringe containing heparanized saline flush</td>
			<td>McKesson</td>
			<td>191-MIH-2235</td>
			<td>One can also prepare a heparin flush solution (10 units/mL heparin in 0.9% sodium chloride)</td>
		</tr>
		<tr>
			<td>5 mm Medipoint Goldenrod animal lancets</td>
			<td>Fisher Scientific</td>
			<td>NC9891620</td>
			<td>5 mm if animal is between 2 and 6 months</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced hot plate stirrer</td>
			<td>VWR</td>
			<td>97042-602</td>
			<td>Brand is example</td>
		</tr>
		<tr>
			<td>BD 27 gauge 0.5 inch needles</td>
			<td>Health Warehouse</td>
			<td>A283952</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 30 gauge 0.5 inch needles</td>
			<td>Medvet</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm</td>
			<td>VWR</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm</td>
			<td>VWR</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker, 1000 mL</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Caging pellets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clear VOA glass vials with closed-top cap</td>
			<td>Fisher Scientific</td>
			<td>05-719-120</td>
			<td>For storage of acetone and blood draw samples</td>
		</tr>
		<tr>
			<td>Copper toothless alligator clamp for tourniquet</td>
			<td>Amazon</td>
			<td></td>
			<td>Any Brand; smooth toothless alligator clips made of solid copper</td>
		</tr>
		<tr>
			<td>D-(+)-glucose &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose (6,6-D2, 99%)</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-349-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning silastic tubing 0.3 mm ID x 0.64 mm OD</td>
			<td>VWR</td>
			<td>62999-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying glass</td>
			<td>Amazon</td>
			<td></td>
			<td>Any brand; similar to LANCOSC Magnifying Glass with Light and Stand</td>
		</tr>
		<tr>
			<td>Microbalance</td>
			<td>Ohaus Adventurer Pro</td>
			<td>AV264C</td>
			<td>Any similar model with 0.0001g accuracy can be used</td>
		</tr>
		<tr>
			<td>Nalgene bottle, 500 mL</td>
			<td>Sigma-Aldrich</td>
			<td>B0158-12EA</td>
			<td>Or any Similar brand; saw in half (including lid) and cut tail-sized notch in the bottom</td>
		</tr>
		<tr>
			<td>PHD Ultra multi-syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3024A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglass sheet</td>
			<td></td>
			<td></td>
			<td>Any brand; to stabalize mouse during catheter insertion</td>
		</tr>
		<tr>
			<td>Plexiglass sheets and dividers</td>
			<td></td>
			<td></td>
			<td>Any brand; used to cage mice during infusion</td>
		</tr>
	</tbody>
</table>

evaluation of hepatic glucose production in a polycystic ovary syndrome mouse model

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.

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 &#177; 2.53 mg/(kg&#183;min) after 6 h fasting a...

Watch this Scientific Journal Video about Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model at JoVE.com

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

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. ...

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