Lasker basic medica l research award commentary

A historical perspective on

Douglas L Coleman

As the only child of a self-employed and largely that the db mutation is on chromosome 4 and, self-taught Canadian radio-and-refrigeration like the ob/ob mutant mouse, the db/db mouse repairman, I spent much of my childhood develops marked and hyperphagia, but, investigating how things worked by taking unlike the ob/ob mutant, it develops severe, them apart. As I grew older, I developed a life-shortening diabetes2. Figure 1 illustrates keen interest in science, and, while I was an the striking obesity of the db/db mutant undergraduate at McMaster University, my mouse compared with a normal littermate at scientific interests converged on biology, geol- eight weeks of age. The db/db mouse is much ogy and chemistry. During this time, a very larger than the normal mouse (30 grams com- dynamic biochemistry professor encouraged pared with 20 grams), and the extra weight is me to attend graduate school at the University all . Because both ob/ob and db/ of Wisconsin and study biochemistry. Upon db mice become morbidly obese with similar Figure 1 An obese db/db mouse (right) next to a receiving my PhD from the University of speed, the db/db mutant shown in this figure normal mouse (left). Wisconsin in 1958, I knew that the prospects looks just like an ob/ob mouse; to the unaided of returning to employment in Canada were eye, the two mouse types are phenotypically poor, and I was offered a position at the Jackson identical2. the db/db mutant to a normal mouse3 (Fig. 2a). Laboratory in Bar Harbor, Maine. When I When I began to characterize this new After what seemed to be good wound healing accepted this position, my plan was to remain mutation, I wondered whether some circu- and a healthy-looking parabiotic pair, I was at the Jackson Laboratory for one or two years lating factor might control the obese pheno- surprised to see that the normal mouse died. to further my education in biology, especially type. For example, could a factor produced My immediate thought was that I was a pretty genetics and immunology. As things turned in a normal mouse prevent or remediate the poor surgeon, but repeated attempts con- out, however, the Laboratory provided a very metabolic abnormalities? Conversely, might a sistently yielded the same outcome: only the fertile environment—with excellent colleagues factor produced by the db/db mutant mouse normal mouse in the pair died. Encouraged and world-class mouse models of disease—and cause obesity in a normal mouse? If this hypo- by this stereotypic pattern, I initiated a more I spent my entire career in Bar Harbor. I never thetical factor were carried through the blood, detailed characterization of the normal part- dreamed that I would work on obesity and I reasoned, I could test for its presence by link- ners and found that, after about one week, or that my research would one day be ing the blood supplies of the various mouse their blood sugar concentrations had declined deemed important enough to be considered for strains. Fortunately, two of my colleagues at to starvation levels. Moreover, at necropsy, the a major scientific award. the Jackson Laboratory were using parabio- normal mice not only consistently lacked food In 1958, only one obese mutant mouse—the sis to characterize anemia mutant mice and in their stomachs and food remnants in their ob/ob mouse—was known1. It had been discov- were pleased to instruct me in this technique. intestines but also had no detectable glycogen ered in 1950 and characterized as a model for Parabiosis requires the surgical joining of two in their livers. In marked contrast, the diabetes mild diabetes, but it was not the subject of any mice by skin-to-skin anastomosis from the partners consistently retained elevated blood ongoing studies by other investigators at the shoulder to the pelvic girdle. Wound healing sugar concentrations, and their stomachs and Jackson Laboratory. The ob mutation is located and cross circulation is established in two to intestines were distended with food and food on chromosome 6, and the mouse is character- three days. To avoid a vigorous immune-medi- residues. This was my ‘Eureka’ moment! These ized by massive obesity, marked hyperphagia ated rejection, this technique requires that the results led me to conclude that the db/db mouse and mild transient diabetes. In 1965, a new mice be on the same genetic background, an produced a blood-borne satiety factor so pow- obesity mutant—the db/db mouse—was dis- issue that would delay some desired pairings erful that it could induce the normal partner to covered. I was asked to assist in characteriz- (see below). Most parabiosis experiments fail, starve to death, even in the face of the limited ing this new mutant mouse and, in particular, either because there is no factor or because the cross-circulation between parabiotic pairs. comparing it to the ob/ob mutant. We found factor exchanged is insufficient, owing to the Despite my excitement, my colleagues and lack of major blood vessels in the skin to medi- most of the scientific community remained ate the cross-circulation. largely unconvinced. Douglas L. Coleman is at the Jackson Laboratory, Because the ob/ob and db/db mutant mice At about the same time as these first para- Bar Harbor, Maine, USA. were on different genetic backgrounds, my first biosis studies, a new obese mutant mouse e-mail: [email protected] parabiosis experiment involved the ­joining of spontaneously emerged4. This mouse did not

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a b c d Further studies with db/db mutant mice that were lesioned in the ventromedial nucleus or the arcuate nucleus of the (or both) suggested that the receptor for this satiety db/db +/++db/db ob/ob ob/ob /++/++/+ factor is found in these brain areas7. Additional support for the idea that a satiety factor acts on a receptor in the brain came from a study in which rats with lesions in the ventromedial Diabetes Diabetes Obese nucleus of the hypothalamus became hyper- ↑Body weight ↑Body weight ↓Food intake phagic and obese8. When lesioned rats were ↑Adipose tissue mass ↑Adipose tissue mass ↓Insulinemia Lean ↓Blood sugar parabiosed with normal rats, the normal rat Lean Obese Normal insulin ↓Food intake ↓Food intake Normal blood sugar lost weight and failed to grow but did not die. ↓Insulinemia ↓Adipose tissue mass ↓Fat pad size ↓Blood sugar ↓Insulinemia Lean Despite these clear results, many of my col- Death by starvation ↓Blood sugar No change leagues and many in the obesity field main- Death by starvation tained the dogma that obesity is entirely Figure 2 Summary of the parabiosis experiments. (a–d) Four different parabiotic combinations and the behavioral, not physiological. On the basis of main phenotypes observed in each mouse in the pair. these experiments, however, some investigators did accept a physiological basis as an underly- ing cause that contributes to obesity, and the have diabetes and therefore appeared identical to eat? Parabiosis of mutant mice of the same hunt for the satiety factor became a race. None to the ob/ob mutant, but, like the db/db mutant, weight would address this issue. These addi- of the early satiety factor candidates (cholecys- the mutation mapped to chromosome 4. This tional parabiosis experiments revealed that tokinin, somatostatin, pancreatic polypeptide new mutation arose in an unrelated strain, but the ob/ob and db/db mutant mice responded and others) stood up to rigorous experimen- when crossed into either the C57BL/Ks inbred similarly to the procedure regardless of inbred tation, and I continued my own attempts to background (in which the db/db mutation was background. After collateral circulation devel- identify the factor. In control parabiosis studies maintained) or the related C57BL/6 inbred oped, blood sugar concentration in the ob/ob using two normal mice (Fig. 2d), I had found background (in which the ob/ob mutation mutant mouse declined, eventually reaching that the pairs remained active and healthy for was maintained) it caused markedly different starvation levels. Survival time ranged from four months (until they were culled). But, at disease syndromes—leading me to wonder 20 to 30 days. At necropsy, it was clear that the necropsy, they did show one abnormality: the whether the inbred background could medi- adipose tissue mass in the ob/ob parabiont had fat pads were smaller in size than those iso- ate the severity of the disease. To answer this decreased and that neither food nor food resi- lated from unparabiosed normal mice. This question, I placed the diabetes mutation on the due could be found in the stomach or intestinal suggested to me that the factor might be a C57BL/6 background and the obese mutation tract of the ob/ob partner. By contrast, the db/ component of adipose tissue. Although the fac- on the C57BL/Ks background by five cycles of db mutant was gorged with food and gaining tor was produced by adipose tissue, my time- cross-intercross breeding, thereby producing weight. Consistent with the lack of food in the consuming attempts to isolate it proved futile, two new congenic lines5. I found that both the ob/ob partner, the food consumption of the as I concentrated my efforts on individual fatty ob/ob and the db/db mutations, when main- pair was decreased to about that typical of a acids or lipid extracts of adipose tissue. tained on the C57BL/Ks inbred background, single db/db mutant mouse. These striking Following a tour-de-force positional cloning produced identical syndromes: marked obesity results clearly indicated that the ob/ob mutant exercise carried out over many years, the long- with severe life-shortening diabetes. In con- mouse, like the normal mouse, recognized and sought satiety factor was definitively identified trast, when maintained on the C57BL/6 back- responded to the factor provided by the db/db by Jeffrey Friedman9. This satiety factor was ground, both mutations produced obesity but partner6. named leptin and, with the subsequent clon- with only mild and transient diabetes. These Several additional parabiosis and lesion ing of the leptin receptor, the field exploded. studies clearly established that the severity of experiments tied up loose ends. Parabiosis of Essentially all of the predictions made from the diabetes depended on modifying genes ob/ob mutant mice with normal mice (Fig. 2c) the parabiosis experiments were verified: ob/ob in the inbred background. Interestingly, the not only slowed the weight gain in the ob/ob encodes a blood-borne (leptin) that modifying genes remain unknown to this day, mutant mouse and decreased food consump- functions in a negative feedback loop to control although a great deal of effort has gone into tion of the pair to that seen in normal-with- adipose tissue mass by modulating appetite; the identifying them. normal parabionts but also resulted in a pairs db/db gene encodes the leptin receptor; leptin Knowing that two genes on separate chromo- of parabionts that survived for months, until is produced in adipose tissue; and the leptin somes produce identical syndromes strongly the end of the experiment. This study showed receptor is expressed primarily in the hypo- suggested that these genes mediate a com- that the factor produced by the normal mouse thalamus. These discoveries not only changed mon metabolic pathway. Having both types was probably the same as that produced by the prevailing theory about obesity, from being of mutant mice on both inbred backgrounds the db/db mutant, but in insufficient amounts caused by a lack of willpower to being caused permitted the parabiosis of the ob/ob mutants to be lethal. My overall conclusions from the by an imbalance of hormone signaling, but also with the db/db mutants without fear of rejec- parabiosis studies were that the db/db mutant showed that adipose tissue is not just a use- tion (Fig. 2b). This experiment would allow me mouse overproduced a satiety factor but could less and unwanted fat storage site but rather an to answer a question that had always nagged at not respond to it—perhaps owing to a defec- important and essential endocrine organ. me: was the normal parabiont starving because tive receptor—whereas the ob/ob mutant rec- In humans, many leptin deficiency syn- it was continually being dragged around by the ognized and responded to the factor but could dromes are responsive to leptin replacement larger db/db partner and never had a chance not produce it. therapy. As leptin is involved in numerous

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pathways, the effects of too little leptin are that govern this crucial hormone, it is likely 2. Hummel, K.P., Dickie, M.M. & Coleman, D.L. Diabetes, a new mutation in the mouse. Science 153, 1127– widespread (including infertility, impaired that combination therapies involving lep- 1128 (1966). immune function, decreased insulin response tin will prove efficacious in other diseases. 3. Coleman, D.L. & Hummel, K.P. Effects of parabiosis of and altered homeostasis). Although leptin Indeed, exciting preclinical work from Roger normal with genetically diabetic mice. Am. J. Physiol. 217, 1298–1304 (1969). mutations are not common in humans, leptin Unger’s laboratory raises the possibility that 4. Hummel, K.P., Coleman, D.L. & Lane, P.W. The influ- therapy in the few people who lack an active leptin supplementation therapy might be ben- ence of genetic background on the expression of muta- form of this hormone is a godsend, transform- eficial in the treatment of type 1 diabetes10. tions at the diabetes locus in the mouse 1. C57BL/ KsJ and C57BL/6J strains. Biochem. Genet. 7, 1–13 ing them from morbidly obese individuals (1972). ACKNOWLEDGMENTS living in a state of perceived starvation into 5. Coleman, D.L. & Hummel, K.P. The influence of genetic I thank my many mentors, especially A. Renold for background on the expression of the obese (ob) gene in much leaner individuals with a more normal continued support and guidance from the beginning of the mouse. Diabetologia 9, 287–293 (1973). lifestyle. Leptin therapy has also successfully my career in the diabetes-obesity studies up to his death. 6. Coleman, D.L. Effects of parabiosis of obese with treated patients with reduced adipose tissue I also thank the Jackson Laboratory staff in the 1960s, diabetes and normal mice. Diabetologia 9, 294–298 1970s and 1980s, especially E. Leiter, for continued help (1973). mass resulting from, for example, lipodystro- and encouragement. Special thanks go to my family, 7. Coleman, D.L. & Hummel, K.P. The effects of hypotha- lamic lesions in genetically diabetic mice. phy or amenorrhea. However, leptin therapy especially my son, Tom, for editing this manuscript. My Diabetologia 6, 263–267 (1970). is not a panacea for curing obesity, because wife, Beverly, was my strongest supporter for nearly 55 8. Hervey, G.R. The effects of lesions in the hypothalamus most obese humans, who have normal genes years. She would have been pleased. in parabiotic rats. J. Physiol. (Lond.) 145, 336–352 for leptin and its receptor, are leptin resistant. (1959). COMPETING FINANCIAL INTERESTS 9. Zhang, Y. et al. Positional cloning of the mouse obese Despite extensive efforts in many laboratories, The author declares no competing financial interests. gene and its human homologue. Nature 372, 425–432 it remains unclear why ordinary obese people (1994). 1. Ingalls, A.M., Dickie, M.M. & Snell, G.D. Obese, a new 10. Wang, M.Y. et al. Leptin therapy in insulin-deficient become resistant to their own leptin. As we mutation in the house mouse. J. Hered. 41, 317–318 type 1 diabetes. Proc. Natl. Acad. Sci. USA 107, learn more about the molecular mechanisms (1950). 4813–4819 (2010).

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