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 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&#183;min) and 2 mg/(kg&#183;min), respectively. These concentrations were optimized by several prior pilot studies.</li>
	<li>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.</li>
	<li>Prepare the second infusate by dissolving both [6,6-2H2]glucose tracer along with non-isotopic glucose (~20 mg/kg&#183;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&#183;min) (fasting condition) and (~) 1.9 mg/kg&#183;min (glucose rich condition) was used. In order to mimic the &#39;glucose rich condition&#39;, D-glucose infusion rate was set at (~) 18.8 mg/(kg&#183;min).</li>
	<li>Once the solutions are prepared, sterile filter the solutions with a 0.22 &#181;m filter and store at 4 &#176;C. Warm the solutions to room temperature prior to the infusion. Load the solutions on prelabeled 1 mL syringes.</li>
	<li>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 &#181;L/h for 3 h.</li>
	<li>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 &#181;L/h. Set the pump to a constant rate infusion at 150 &#181;L/h for the additional 3 h.</li>
</ol>
2. Set-up of infusion experiments
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Assemble the 1&#160;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.</li>
	<li>Prepare the infusion pump by setting the rate to 150 &#181;L/h, which is the basal rate.</li>
	<li>Heat a water bath to 48 &#176;C.</li>
	<li>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.</li>
	<li>After 3 h of fasting, begin the catheter insertion process, which is detailed below.</li>
</ol>
3. Catheter insertion
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wrap a piece of 1 inch transpore tape around the insertion site to secure the catheter. Remove the small tourniquet from the tail.</li>
</ol>
4. Infusion set-up and first infusion
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Disconnect the flush from the tail vein catheter and place a small clamp on the catheter&#39;s silastic tubing to prevent backflow while connecting the infusate line from the pump.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Once infusion lines are noted to be functioning properly, remove the cover from the mouse. Place standard bedding around the mouse.</li>
	<li>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.</li>
</ol>
5. Blood sampling
<ol>
	<li>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.</li>
	<li>Collect ~75 &#181;L of blood in the desired vial. To deproteinize samples in preparation for mass spectrometry, add approximately 15 &#181;L of blood to 500 &#181;L 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.</li>
</ol>
6. Second infusion
<ol>
	<li>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.
	<ol>
		<li>To reach a steady state, run a bolus of the second infusate at 600 &#181;L/h for 15 min. Note the starting time for each group of cages. Decrease the infusion rate to 150 &#181;L/h to complete the 3 h of total infusion time.</li>
	</ol>
	</li>
	<li>Repeat steps 5.1 and 5.2.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Mass spectrometry
<ol>
	<li>Send out the samples for mass spectrometry.</li>
	<li>Analyses
	<ol>
		<li>Measure the isotopic enrichment of [6,6-2H2]glucose by gaschromatography - mass spectrometry (GCMS) using the pentaacetate derivative21,22. Briefly, this method involves preparation of the pentaacetate derivative of glucose, followed by sample analysis using GCMS 20,22.</li>
	</ol>
	</li>
	<li>Calculations
	<ol>
		<li>Perform all kinetic measurements under steady state conditions. Total plasma glucose appearance rate (glucose Ra) 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&#183;min)) (GPR) = glucoseRa - exogenous glucose.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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><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 &gt;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...

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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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Research

JoVE Journal

Medicine

2.9K Views.  Baylor College of Medicine. 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.

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

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Developmental Biology

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Biology

Polycystic Ovary Syndrome Induction in Mice Through Controlled Letrozole Release 

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of PCOS and for related drug development. Letrozole-induced mouse models replicate the metabolic and reproductive phenotypes of patients with PCOS. The traditional method of letrozole treatment in PCOS mice requires daily dosing over a certain period, which can be labor-intensive and cause significant stress to the mice. This study describes a simple and effective method for inducing PCOS in mice by implanting a controlled letrozole-releasing mini-pump. A mini-pump capable of stable, continuous release of a quantitative amount of letrozole was fabricated and implanted subcutaneously in mice under anesthesia. This study demonstrated that the mouse model successfully mimicked PCOS features after letrozole mini-pump implantation. The materials and equipment used in this study are readily available to most laboratories, requiring no special customization. Collectively, this article provides a unique, easy-to-perform method for inducing PCOS in mice.

Author Spotlight: Investigating the Mechanisms and Inducing Models of Polycystic Ovary Syndrome

This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of ...

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice

Source: Ajoy, R. and Chou, S. Y. <a href="https://www.jove.com/video/56410/studying-hypothalamic-insulin-signal-to-peripheral-glucose">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain</a>.&#160;J. Vis. Exp. (2018).
This video introduces how to perform an oral glucose tolerance test on a laboratory mouse.

Source: Ajoy, R. and Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into ...

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

Summary

Explore More Videos

Chapters in this video

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice