Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Using Mouse Models of Insulin Resistance

Garrett Heinrich
Tags: Metabolism

What is insulin resistance?

Insulin is a hormone secreted by the pancreas that can stimulate cells to take up glucose. In the muscle and adipose tissues, insulin acts by binding to insulin receptors on the cell surface and initiating a signaling cascade that results in blood glucose uptake by glucose transporters. In the liver, insulin acts to regulate the switch between gluconeogenesis and lipogenesis. When insulin fails to activate the insulin receptors found on the cell surface, the cells of the target tissues are considered resistant to insulin. Insulin resistance is the hallmark of Type 2 Diabetes because when cells become resistant, the pancreas responds by secreting even more insulin to compensate and keep systemic glucose levels normal. Unfortunately, this increased insulin secretion can cause even further insulin resistance, requiring more compensation. Thus a vicious cycle begins that can end with the pancreas being unable to secrete enough insulin for the glucose load, resulting in Type 2 Diabetes.

Ways to make models of insulin resistance

A number of different stressors can cause insulin resistance, and many debates have occurred over what sequence of events happens first. Mouse models have been developed to study insulin resistance due to the similarity of the mouse genome to humans, the ability to perform genetic manipulations within mice, and the cost/time advantages compared to other animal models. One of the early mouse models that attempted to create insulin resistance involved genetically deleting part of the insulin receptor throughout the mouse, leading to what is known as the Insulin Receptor Knockout (or IRKO) mouse. Unfortunately, these mice die of ketoacidosis not long after birth. The introduction of Cre-lox technology allowed for the inactivation of the insulin receptor in specific tissues of interest, such as muscle (MIRKO), adipose (FIRKO), liver (LIRKO), and beta cell (BIRKO), just to name a few. Cre-lox crosses one mouse with expression of Cre recombinase driven by a tissue-specific promoter with another mouse expressing alleles with a portion of the gene of interest surrounded by loxP sites that delete the surrounded gene sequence upon recombination (Fig. 1). The progeny of these mice then have an allele of tissue-specific gene deletion which can then be bred to homozygosity. Unexpectedly, single tissue-specific deletions of the insulin receptor often did not produce systemic insulin resistance and hyperinsulinemia because non-targeted tissues could compensate and increase glucose transport elsewhere.

Cre-lox system for tissue-specific gene knockout

Figure 1. Cre-lox system for tissue-specific gene knockout


Another downside of these Cre-lox models is that they have lost the gene while developing in utero, while insulin resistance develops in people later in life. To delete a gene in adult mice, researchers have developed inducible models that can be recombined at a time of the researcher’s choosing. In one system, the cre-recombinase is fused to a binding domain from the estrogen receptor (ER), and thus can be turned on by giving mice an ER activator such as tamoxifen. Another type of system uses a reverse tetracycline transactivator protein (rtTA), Cre under inducible regulation by a tet operator (tetO), and a floxed target gene. In this system, genes can be turned off in specific tissues either by treating with doxycycline when the gene is to be recombined away (Tet-ON), or mice can be treated with doxycycline to prevent recombination until treatment is stopped (Tet-OFF). In yet another system, mice can be engineered to have specific promoter-expressing cell types such as specific neurons or brown adipose tissue ablated by the administration of diphtheria toxin. Each of these strategies has successfully been used to make insulin-resistant mice or mice protected from insulin resistance.

While knocking out the insulin receptor may not produce insulin resistance, targeting other parts of the insulin signaling pathway that lead to glucose uptake has been successful. Knocking out the insulin receptor substrate 1 (Irs1) or the facilitated glucose transporter 4 (Glut4) are sufficient to lead to insulin resistance. Reducing insulin clearance in the liver, a process where insulin is removed from the circulation and the insulin receptor is returned to the cell surface to detect more insulin, can also induce insulin resistance and cause hyperinsulinemia.

Knowing the correlations between insulin resistance and obesity, researchers have given mice various diets to induce weight gain and hyperinsulinemia. Feeding mice diets enriched in fats and carbohydrates produces obesity and insulin resistance while the usual regular chow they are fed does not, just like people with unhealthy eating habits. Typical high fat diet (HFD) fed to mice to induce obesity (called diet-induced obesity, or DIO) and insulin resistance contains 40-60% fat. Some researchers use what they call a Western diet (WD) to simulate an unhealthy human diet, though the exact composition of this named diet can vary. Sometimes, this WD contains ~40% fat with fructose or sucrose supplement. Sometimes, the WD is enriched with cholesterol and salt to also induce atherosclerosis. All versions induce insulin resistance in the mice. Two factors to consider in using mouse models of insulin resistance are the genetic background of the mice, and the temperature in which the mice are housed. Mice used in research have been bred over many generations, with strains made from different mouse types and sources. These differences make each strain have a signature level of gene expression, known as the genetic background. Some strains are more prone to insulin resistance than others. The C57BL/6 strain is commonly used in metabolic studies due to its propensity to become obese and insulin-resistant when fed a high fat diet. Thermoneutrality is the temperature at which energy is expended to maintain basal metabolism. Mice are actually thermoneutral at a higher temperature than humans (30⁰C vs. 22⁰C). The room temperature at which many experiments are performed and mice are housed makes the mice expend more energy and be more insulin sensitive than if they were at their thermoneutral temperature, complicating making comparisons between the mouse model and the human state.

Phenotyping Insulin-Resistant Mouse Models

While obesity is easily seen visually and measured by body weight, insulin resistance is harder to determine. To determine if a mouse model is insulin-resistant, it must be metabolically phenotyped. The simplest way to measure signs of insulin resistance is to fast the mouse overnight until insulin secretion reaches its basal steady state and perform an insulin assay to detect basal hyperinsulinemia. Insulin is produced by the pancreas as a pro-peptide called proinsulin that is cleaved into fragments before it is released into the bloodstream. These fragments are known as C-peptide and insulin, and can be measured by the Mouse C-peptide ELISA kit and Insulin (Mouse) ELISA kit. Since insulin is secreted from the pancreas in the same ratio as C-peptide but cleared from the blood at a much faster rate, one measure of the insulin clearance is to calculate the C-peptide to insulin ratio (C/I ratio). When insulin clearance is impaired, leaving more insulin in the circulation and contributing to insulin resistance, the C/I ratio is lower.

Another way to phenotype mice for insulin resistance is to remove the target tissues of insulin such as the muscle, fat, and liver, and measure insulin receptor activation. This can be done in vivo by fasting mice and giving an insulin or glucose injection, or by refeeding and dissecting at a given time after stimulation. The insulin receptor is found on the plasma membrane with an extracellular alpha-subunit region and transmembrane beta-subunit region. When insulin binds the receptor on the extracellular alpha-subunit, the receptor auto-phosphorylates tyrosine residues in the intracellular part of the beta-subunit. This activation can be measured by Western blot for these phosphorylated tyrosine residues (Table 1). Further down the insulin signaling pathway, protein kinase B (Akt) is phosphorylated at residues Ser473 and Thr308. These are also commonly measured by Western blot by comparing phosphorylation to the total amount of Akt protein (Table 1). Both phosphorylated Akt and total Akt can be measured on the same Western blot membrane when using primary antibodies of different host species.

Anti-Mouse Antibodies to Measure Insulin Signaling
pTyr-1158/1162/1163 Insulin Receptor and pTyr-1131/1135/1136 IGF- Receptor (rabbit)
Insulin receptor monoclonal antibody (9H4) (mouse)
Insulin receptor beta monoclonal antibody (C18C4) (mouse)
[pSer473] Akt monoclonal antibody (11E6) (mouse)
[pSer473] Akt polyclonal antibody (rabbit)
Akt (phosho Thr308) polyclonal antibody (rabbit)
Akt (phosphorylated) (pThr308), pAb (rabbit)
Akt monoclonal antibody (5C10) (mouse)
Akt polyclonal antibody (rabbit)

Table 1. Anti-mouse antibodies to measure insulin signaling.

Mice compensate for insulin resistance by producing and secreting more insulin from the pancreas. Immunohistochemistry for mouse insulin measures how much insulin and beta-cell area is present in the pancreas. The pancreas is removed from the mouse, fixed, embedded in paraffin, cut into thin slices and placed on a slide. An antigen retrieval step allows the proper epitope to be recognized by the primary antibody, such as Insulin (D3E7) antibody. Enzo has a number of streptavidin-based and biotin-free solutions to label the insulin antibody location with color producing reactions using horseradish peroxidase (HRP) or alkaline phosphatase (AP). The slide can be mounted with coverslip and visualized by microscope.

An overload of free fatty acids (FFA), as seen with HFD, can induce mitochondrial dysfunction, reducing the amount of ATP produced by the cell and increasing reactive oxygen species (ROS). These abnormalities can be monitored in live cells (such as isolated hepatocytes) with the Enzo MITO-ID® Membrane potential detection kit. To label the mitochondria for assessing total mass or co-localizing with proteins of interest, the MITO-ID® Green detection kit or MITO-ID® Red detection kit (GFP-CERTIFIED) can be utilized, depending on the fluorescent detection color of need.

The gold standard for measuring insulin resistance in mouse models is still the hyperinsulinemic-euglycemic clamp. In this system, continuous infusion raises the insulin level, and glycaemia is kept constant throughout the clamp by monitoring at regular time intervals. The amount of glucose needed to maintain euglycemia as it is taken up by cells and how effectively hepatic gluconeogenesis is shut-off determines how sensitive the mice are to insulin. Radioactive tracer glucose is also infused during the clamp, which can later be used to measure glucose uptake in individual tissues. This procedure is usually performed by a specialized core facility.


How can Enzo help guide your metabolic research?

Do you have more questions on mouse models of insulin resistance and how to find the best assays and molecules for your research? Reach out to our Technical support team. We will be happy to assist!

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

 

Did you enjoy the reading?
Share it on your favorite social media

 
comments powered by Disqus

Related Products

Insulin receptor monoclonal antibody (9H4) 

ELISA, IHC, WB | Print as PDF
 
ADI-905-647-100 100 µg 467.00 USD
Do you need bulk/larger quantities?
 

Insulin receptor β monoclonal antibody (C18C4) 

Purified from ascites., WB | Print as PDF
 
ADI-905-683-100 100 µg 314.00 USD
Do you need bulk/larger quantities?
 

[pSer473]Akt polyclonal antibody 

Purified from rabbit serum., WB | Print as PDF
 
ADI-905-773-100 100 µg 313.00 USD
Do you need bulk/larger quantities?
 

Akt polyclonal antibody 

Purified from rabbit serum., IHC (PS), IP, WB | Print as PDF
 
ADI-KAP-PK004-D 50 µg 175.00 USD
 
ADI-KAP-PK004-F 200 µg 384.00 USD
Do you need bulk/larger quantities?
 

[pSer473]Akt monoclonal antibody (11E6) 

ELISA, Flow Cytometry, IHC, IP, WB | Print as PDF
 
ALX-804-401-C100 100 µg 562.00 USD
Do you need bulk/larger quantities?
 

Akt monoclonal antibody (5C10) 

ELISA, WB | Print as PDF
 
ALX-804-553-C100 100 µg 456.00 USD
Do you need bulk/larger quantities?
 

Diphtheria toxin (Corynebacterium diphtheriae) 

From Corynebacterium diphtheriae. | Print as PDF
 
BML-G135-0001 1 mg 846.00 USD
Do you need bulk/larger quantities?
 

[pTyr1158/1162/1163]Insulin receptor and [pTyr1131/1135/1136]IGF-1 Receptor polyclonal antibody 

WB | Print as PDF
 
BML-SA392-0020 20 µl 148.00 USD
 
BML-SA392-0100 100 µl 575.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Red detection kit (GFP-CERTIFIED®) 

Photostable,  non-toxic and selective mitochondrial dye that stains regardless of membrane  potential
Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51007-0100 100 tests 89.00 USD
 
ENZ-51007-500 500 tests 262.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Membrane potential detection kit  

The only assay that monitors energetic status using a simple mix-and-read, no-wash protocol
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51018-0025 25 tests 177.00 USD
 
ENZ-51018-K100 100 tests 489.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Green detection kit  

Photostable,  non-toxic and selective mitochondrial dye that stains regardless of membrane potential
Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51022-0100 100 tests 93.00 USD
 
ENZ-51022-K500 500 tests 261.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
Keep in touch

©2021 Enzo Life Sciences, Inc.,