Marathon mice: researcher view on potential roles in preclinical research.

Introduction

Physical inactivity is one of the most important causative factors for the development of obesity, and it is known that resistance training programs such as high endurance performance are helpful in reducing fat mass.[1] High endurance performance can be acquired by physical training, special diet programs as well as genetic predisposition. Researchers from the Howard Hughes Medical Institute, Maryland, United States have developed a genetically modified “marathon mouse,” a noninbred model with modified muscle composition using transgenic techniques that doubled the running capacity by augmenting the expression of peroxisome proliferator-activated receptor-delta (PPAR-delta).[2] These PPAR-delta-enhanced marathon mice could run 1800 m before exhaustion, unlike the normal mice that run only about 900 m. Marathon mice, when fed with the high-fat diet, that usually causes obesity in normal mice, enhanced the PPAR-delta protein, are also resistant to weight gain. Therefore, marathon mice models will provide an intricate link between the running capacity and hepatic lipid metabolism, thereby providing useful information on the metabolic flux studies.

The prime insight on the use of marathon mice models is to understand the physiological molecular changes that occur as a result of physical activity. This further helps the researchers to envisage the drug development that enhances muscle strength which mimics the benefits of exercise. These drugs can be used as a medicament in various disease conditions with a caveat about these drugs abuse as muscle strength-enhancing drugs by athletes to enhance their sports performances.

The genetically modified marathon mice are known to have altered muscle composition and enough physical endurance as compared to normal mice. Usually, exposure to lower temperatures and physical training regulate the white to beige adipose tissue conversion (browning). Table 1 shows the differentiating features between the marathon mice and normal mice. The peroxisome proliferator-activated receptor gamma coactivator-1-alpha present in the muscle and fat plays a vital role in the thermogenesis of beige fat regulating the body weight. Therefore, the main differences between the marathon mice and normal mice include browning markers, genes related to oxidative phosphorylation pathway, and genes regulating mitochondrial fission and fusion, especially in response to voluntary exercise.

Table 1

Differentiating Features of Marathon Mice and Normal Mice[3]

Factor	Normal mice	Marathon mice
Beige fat formation	Exercise-induced	Inbuilt
Effect of voluntary physical activity	Increased expression of a gene that is involving in the PGC1α related pathways	Increases the expression of brown fat gene markers such as
	Increased transcription of mitochondrial genes	TBX1
		CIDEA
	Increasing mitochondrial biogenesis forming brown adipose tissue	UCP1
PGC1α in muscle and fat	Increased expression with physical exercise	Unaffected by exercise
Electron transport chain	Normal expression of individual complex subunits of electron transport chain in subcutaneous fat	Increased expression of individual complex subunits of electron transport chain in subcutaneous fat
Mitochondrial Fission and Fusion in response to physical activity	Normal mitochondrial fission and fusion gene expression	Fis1 (mitochondrial fission gene) overexpression
		Mitochondrial fragmentation and simultaneous fusion blocking
Hepatic genetic analysis	Normal glycolytic pathway, fatty acid production, and normal levels of cholesterol production. Normal progesterone and sex steroid levels	Increased glycolytic pathway
		Increased fatty acid production
		Increased cholesterol production (all the above effects are regulated by sterol regulatory element-binding protein)
		Increased progesterone levels
		Low levels of sex steroids

TBX1=T-box protein 1, CIDEA=Cell death activator, UCP1=Uncoupling protein 1, PGC1α=Proliferator-activated receptor-gamma-coactivator-1-alpha

Marathon mice already have beige subcutaneous tissue indicating large mitochondrial mass and elevated mitochondrial biogenesis which can be further enhanced by training. Due to the mitochondrial biogenesis further oxidative phosphorylation in subcutaneous fat and increased expression of genetic markers of browning might lead to the superior running ability.[1]

How to Validate Marathon Mice Animal Model

In 1984, Wilner proposed three external validations for an animal model which include construct, face, and predictive validity. Among these, construct validity is very important to validate an animal model in preclinical research. Marathon mice model provides good predictive validity at the transgenic level and can be easily extrapolated to humans as they mimic the human adipose tissue metabolism.

Marathon Mice Has Therapeutic Potential

In obesity

The current epidemiological status of obesity has reached at least 2.8 million people dying each year as a result of complications associated with being overweight or obese. Obesity is also prevalent in low- and middle-income countries as well. So much biomedical research is on the track to discover medicament for this health ailment. The treatment strategy for obesity is the physical activity mainly through voluntary exercises as well as antiobesity drugs. Most antiobesity drugs aim at reducing appetite which is the hardest thing for the human population since our appetite is genetically determined, thereby posing an important challenge in front of us in designing antiobesity drugs.

The enhancement of PPAR-delta leads to the enhancement of slow-twitch fibers which are having a large number of mitochondria converting fat to energy than fast-twitch fibers. The presence of more mitochondria in slow-twitch fibers of marathon mice possibly results in more fat burning and releasing energy in the form of heat. Marathon mice have less chance of gaining weight when compared to normal mice despite consuming the same amount of food and engaging in the same level of exercise. Intramuscular triglycerides, which are related to insulin resistance and diabetes in obese people, were found to be lower in these marathon mice.[4]

Previously, vigorous physical training is the only known way to increase endurance until the generation of the genetically modified mouse. Therefore, the development of such models provides an avenue of opportunities to design the drugs which enhance the PPAR-delta pathway that would let the human population, like our marathon mice, have the same diet quantity, but would increase their metabolism to burn more energy which can act as a potential anti-obesity agents.

Debilitating conditions

The debilitating ill patients need muscle power, but they are unable to do voluntary physical activity because of their health ailment. The researchers discovered that the PPAR delta agonist could improve the muscle power of debilitating patients. Muscle transformation of PPAR-delta activity has confirmed by administering the GW501516, a PPAR-delta receptor agonist, which produced muscle and metabolic benefits similar to that of transgenic mice. Hence, such drugs enhancing the PPAR-delta activity can be a boon for patients suffering from debilitating conditions.

Marathon Mice in Gene Doping

Gene doping can stretch the physical limits of human strength and endurance. Gene doping is a protrusion of gene therapy by inserting DNA to enhance athletic performance. The World Anti-Doping Agency (WADA), an international organization formed in 1999 to “promote, coordinate, and monitor the fight against doping in sport in all its forms,” defines gene doping as the “nontherapeutic use of cells, genes, genetic elements, or modulation of gene expression, having the capacity to enhance performance” (WADA, 2008). In marathon mice, athletic training not only changes muscle fiber quality but also enhances the circulation and motor neuron innervation. The heart is also redesigned to pump a larger amount of blood in these marathon mice. In certain ways, they have created a metabolic shield that prevents them from gaining weight.

Gene Doping Example

An athlete could be genetically modified to express a gene that increases their performance. Repoxygen, developed by Oxford Biomedica pharmaceuticals, is a gene therapy method of producing the hormone erythropoietin (EPO), that is, important for the production of oxygen-carrying red blood cells (RBCs). Repoxygen boosts up the number of RBCs in muscles, thereby increasing oxygen delivery. It was abandoned in the preclinical stage because of its mention in a criminal trial by a German athlete coach Thomas Springstein. The athletes were given Repoxygen as a performance-enhancing drug without their consent. The prosecution was done in open court with the evidence of e-mail requisition of repoxygen by the coach. This event in 2006 got media coverage at the global level and it was the first confession in public regarding the athlete's involvement in gene doping.

Identification of Gene Doping

There is an evolution in analytical techniques over years to identify gene doping. Researchers could detect the banned drug metabolites in the urine of gene dopers only for a few weeks in the past.

Currently, the duration of detection of such metabolites has been expanded to a couple of months.

The athletes are more likely to inject EPO DNA which is synthesized from bacteria. Since bacterial EPO DNA codes differently from that of humans, the dopers can be easily identified by transgenic DNA identification techniques. The mass spectrometers are used for the detection of the metabolites of the banned compounds such as metandienone, oxymetholone, and stanozolol in the urine of the gene dopers.

PROS and CONS of Gene Doping

PROS

Gene doping enables athletes to enhance their physical performance by serving as a valuable supplement to their training regimens without affecting the mental discipline, practice, and skills of the athletes

The muscle tissue samples are needed for the detection of gene doping which is an invasive process currently under athlete's privacy rules and regulations

Gene doping could help athletes in avoiding debilitating permanent and expensive sport-related injuries.

CONS

Gene doping is a violation under sports law. If the result of marathon mice when replicated in humans, it would be an unfair advantage to the athlete by making mental practice, skill factor, and practice insignificant

Hence, many efforts globally are underway in the development of noninvasive methods for the detection of gene doping. Still, there are ongoing efforts for the detection of appropriate noninvasive methods for many gene doping agents

The introduction of gene doping technology would mislead the young budding athletes as well as their parents to seek the technology to improve performance.

Conclusion and Future Perspectives

Researchers have already found that genetically modified mice can run long distances with enough endurance when compared to normal mice. With the discovery of marathon mice, we can understand that modifying a single gene can make a big change in physiology and metabolism. Such genetic model disposition provides new avenues for numerous preclinical researches for the screening of drugs under different clinical conditions. Drugs that stimulate the PPAR-delta pathway have the potential to improve muscle strength, reduce obesity, and protect against diabetes. Marathon mice may be a promising animal model in future for testing the impact of voluntary exercise on adipose tissues. Hence, the marathon mice model can be extrapolated to other significant preclinical research for various clinical conditions under strict guidelines and surveillance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.