New, improved models of human obesity aim to improve understanding of leptin’s impact on CNS regulation of metabolism.

Mind Over FatterFour hundred years ago in Europe being obese was a sign of wealth. In other words, if a person were obese, it meant that they were wealthy enough to afford to obtain more than the bare minimum of food to eat. Photos of obese royalty feasting on food are the dominant images of renaissance culture. Today, obesity is a growing problem in the United States, with dieting being the main topic of many health providers and of our popular culture, in general. And of course, obesity has become a major therapeutic area for drug development.

It is also not news that the human central nervous system (CNS) regulates metabolism, and consequently, body weight. Research into the cause of obesity has all pointed to the brain as the master regulator of metabolism. And for about sixty years now, biotechnology companies, and academic researchers alike, have been trying to develop better animal models to figure out how this regulation works. Along the way, a protein hormone called leptin was discovered to be a major player in CNS-mediated regulation of body weight and metabolism. And, after years of research that still continues today, the story of leptin is just beginning to unfold.

Leptin on the brain
Satya P. Kalra, PhD, distinguished professor emeritus, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, Fla., is a leptin enthusiast. Kalra’s research involves the use of three different rodent models of obesity (the ob/ob mouse, the high fat diet-induced obesity model, as well as insulinopenic Akita mice, and streptozotocin-treated mice). Collectively, these models enable Kalra and other obesity researchers to examine a multitude of leptin functions.

“All of these models provide a unique substrate to gain deeper insight into leptin neurobiology by elucidating various brain mechanisms concerned with the dysregulation underlying obesity...,” says Kalra, who adds that  by introducing the leptin gene into the hypothalamus of ob/ob mouse to restore leptin signaling with the help of gene therapy approaches, his lab has successfully identified the neural pathways and mechanism of action of leptin in controlling appetite, obesity, energy expenditure, insulin secretion, glucose disposal related to diabetes and systemic inflammation-dependent cardiovascular ailments and bone growth.

The ob/ob mouse, which contains a complete loss of leptin, enables the analysis of “the effects of leptin on brain (hypothalamic) signaling in regulating energy homeostasis, independent of a multitude of effects of leptin in the periphery,” says Kalra. Also making use of the ob/ob mouse and diet-induced obesity models is the contract research organization VivoPath (Worcester, Mass.), who employs these models to perform tasks required for early-stage obesity and diabetes drug discovery.

“When these animals lack leptin, they become obese,” says Philip D. Lambert, PhD, chief scientific officer of VivoPath. “The ob/ob model does not produce any leptin protein, so they don’t have any way of signaling back to the CNS to indicate the peripheral adiposity of the animal. And when signaling no longer occurs, the animals become obese,” says Lambert.
The ob/ob model does not accurately model human obesity because, ironically, obese humans have high levels of leptin, whereas the absence of leptin causes obesity in the mouse model. Moreover, obesity in humans is a polygenic trait, controlled by multiple organ systems including the leptin-CNS feedback loop.

In addition to the ob/ob mouse, VivoPath also uses a diet-induced obesity (DIO) model, which it obtains through Taconic, Hudson, N.Y., to perform drug screening. “I think that the [diet-induced obesity] model is more predictive of the human situation than the ob/ob mouse, so we tend to use the former as a primary model for drug screening,” says Lambert. “Compound efficacy testing in type II diabetes as well as obesity is what we use the two models for. So we use those models to look at how compounds can affect glucose, body weight and food intake. Compounds that work in these models tend to work in human obesity and human type II diabetes.”

Compared to the genetic models of obesity, developing a DIO model seems a lot simpler by comparison. “The dietary-induced obesity models are just normal animals [capable of producing leptin] that you expose to a very high fat diet,” says Lambert. “So if you take half of the mice and put them on a high-fat diet and then leave the other half on their regular chow, over a 10-week period you’ll see the high fat fed group become significantly obese and show signs of type II diabetes such as insulin resistance and high blood glucose.” Kalra, who also uses rat and mouse-based DIO models capable of producing leptin says that “the high fat diet model attempts to reproduce the energy-enriched, diet-induced obesity observed in humans.

Another widely used rodent model of obesity, the ZDF (Zucker Diabetic Fatty) rat, is part of the animal model portfolio of Charles River Laboratories, Wilmington, Mass. The ZDF rat model has a spontaneous point mutation in the leptin gene that allows researchers to study the entire cascade associated with metabolic syndrome and type II diabetes.

“We also have other models that we’ve developed in the last couple of years which are polygenic obesity models. These were developed by selection within a breed colony for either large animals or smaller animals, and subsequently populations have been characterized that are either obesity-prone or obesity-resistant based on normal diet. We have expanded populations of those animals,” says Terrence Fisher, MBA, general manager, North American Research Models, Charles River. “A lot of these animals are basic research tools (use of disease models) but some are being used in drug research, such as to perform pharmacokinetics and ADME testing.”

Obesity’s complications
In a recent issue of the journal Obesity, King et al. described the development of a hyperlipidemic murine model to study obesity and its effect on cardiovascular disease (King et al. Obesity (Silver Spring) 2009 Jun 4), using the apoE knockout mouse fed a high fat diet enriched in lard.

“Our initial interest was in determining if obesity per se had an effect on the development of atherosclerosis,” says Victoria King, PhD, scientist, Department of Internal Medicine, Division of Cardiovascular Medicine, University of Kentucky, Lexington, Ky. “So we were hoping that the mice would just get fat and have more atherosclerosis, but the mice fed the high fat diet also had alterations in glucose tolerance ...I think it is going to be very difficult to actually dissect out the effects of these different mediators on the development of atherosclerosis.” In other words, for reasons unclear to King and her colleagues, hyperlipidemic mouse models that develop atherosclerosis also develop glucose tolerance regardless of the type of diet they are given, but they are investigating the reasons for this phenomenon. King et al. also demonstrated that atherosclerosis observed in these mice correlated with an increase in body weight and a marked (albeit non-statistically significant) increase in leptin levels.

In addition to an increase in leptin, King et al. also noticed an increase in the acute phase protein, serum amyloid A (SAA), a pro-inflammatory mediator. “We are interested in SAA because it is an acute phase protein and its expression is up-regulated in both obesity and cardiovascular disease,” says King. It was observed that mice with elevated levels of SAA had a greater degree of atherosclerosis. “One of the interesting thing that we found was that SAA, which is generally carried on HDL, was carried on both the antiatherogenic HDL [high density lipoprotein] and the proatherogenic lipoproteins, very low density lipoprotein and low density lipoprotein; that’s why we associated SAA with atherosclerosis.” King adds that while liver is the primary source of SAA, adipose tissue is also a source of SAA, but that they are still trying to determine if the SAA is coming from adipocytes or from some of the inflammatory cells in the adipose tissue.

“We know that during the development of obesity we see a lot more inflammatory cells moving into the fat … Our hypothesis is that the increase in SAA that occurs with diet-induced obesity is in fact coming from the adipose tissue and that may account for why we are just having such a chronic low-level elevation versus a marked acute phase response.”
The current goal of the lab is to use a triple knockout mouse, i.e. a mouse deficient in apoE, SAA-1, and SAA-2, to determine if the observed increase in atherosclerosis is due to SAA or if there are other factors involved such as leptin.

In summary, obesity is a complex polygenic disorder characterized, in humans at least, by elevated levels of the adipose-associated protein hormone leptin. Researchers aimed at characterizing leptin function have employed a number of rodent models whose development dates back to the 1950s. Continued efforts by biotechnology companies and academic researchers to build better models of human obesity have led to a better understanding of the biology of obesity and its complications as well as an improvement in screening methods for drugs to combat them.

This article was published in Bioscience Technology magazine: Vol. 32, No. 9, September, 2009, pp. 1, 10-12.