Douglas Coleman’s Parabiosis Studies in Obese and Diabetic Mice
DOUGLAS L. COLEMAN and KATHERINE HUMMEL
Effects of Parabiosis of Normal with Genetically Diabetic Mice
American Journal of Physiology 217, 1298-1304
DOUGLAS L. COLEMAN (1973)
Effects of Parabiosis of Obese with Diabetes and Normal Mice
Diabetologia 9, 294-298
Comments by Ruth Harris.
In the late 1960s and early 1970s Douglas Coleman and his collaborator Katherine Hummel at The Jackson Laboratories published two papers [1, 2] describing parabiosis between two strains of mice that were obese due to different single autosomal recessive gene mutations, obese (ob/ob) and diabetic (db/db) mice. Even though the mutations were on different chromosomes, the mice had identical obese and diabetic phenotypes when they were expressed on the same background strain . Despite these similar phenotypes the two strains of mice showed very different responses when joined in parabiosis, leading to the conclusion that the diabetes mice were insensitive to a hypothesized circulating satiety factor whereas obese mice did not produce the factor. These observations laid the groundwork for the eventual identification of leptin as the mutant protein in ob/ob mice and purported negative-feedback signal in the regulation of energy balance .
Parabiosis is a surgical procedure that produces a chronic common blood supply between two animals. This preparation can be used to demonstrate the involvement of a circulating factor, or hormone, in a physiologic response by making an intervention in one member of a pair and looking for a response in the partner. The rate of blood exchange between parabionts is relatively slow, with total blood volume exchanging approximately 10 times a day . This means that any factor that is biologically active in the untreated partner must have a relatively long half-life in the circulation and survive degradation by two livers.
The first parabiosis study, specifically designed to test for the presence of a circulating signal in the control of energy balance, was reported by Romaine Hervey in 1959 . He demonstrated that when one member of a parabiosed pair of rats became hyperphagic and obese due to a lesion of the ventromedial hypothalamus, the non-lesioned partner appeared to reduce its food intake, lost weight and had a greatly reduced fat mass. Hervey concluded that the non-lesioned partner reduced its food intake due to a hypothalamic response to a circulating signal of increased adiposity originating in the lesioned rat.
Douglas Coleman was a biochemist studying the genetics of spontaneous mutations in mice with a particular focus on db/db and ob/ob mice. Both db/db and ob/ob mice are insulin resistant and develop diabetes with a syndrome similar to that of type 2 diabetes mellitus that develops in the setting of obesity in humans. Coleman and Hummel  first paired db/db mice with wild-type controls in order to determine whether the diabetic mouse produced a circulating factor that promoted insulin release or the wild-type mouse produced a factor that inhibited insulin release. However, they summarily discarded this aspect of the experiment as there was no evidence for a change in insulin production in either of the partners and the relatively slow rate of exchange between parabionts meant that neither insulin nor glucose exchanged effectively between partners. The majority of the discussion focuses on observations that the wild-type partners appeared to have little food in their stomachs and that most of them died within weeks of the surgery. Although it was not possible to measure the intake of individual members of a pair, the total intake of the db/db-wild-type pairs was not significantly greater than that of a single db/db mouse, suggesting that the wild-type mouse had a suppressed food intake. These measures may have been somewhat compromised by the db/db mice being food restricted for 6 weeks before surgery and then allowed to eat ad libitum after surgery; however, consistent with Hervey’s hypothesis , Coleman and Hummel  concluded that the wild-type partners of db/db mice died due to starvation caused by a circulating satiety factor produced by the obese db/db partner. They also concluded that the results were consistent with the interpretation that the db/db partner was unable to respond to the signal due to a defective hypothalamus, but they were reluctant to exclude the possibility that the diabetes syndrome was the result of a defect in the endocrine pancreas.
The second study reported by Coleman  was specifically designed to address the role of circulating factors in mediating obesity in db/db and ob/ob mice. The results of the previous study with db/db parabionts was compared with reports from other investigators that ob/ob mice paired with wild-type mice stopped gaining weight, apparently due to a factor that was transmitted from the wild type partner [7, 8]. Coleman therefore tested pairs in which db/db mice were parabiosed to ob/ob mice, pairs in which ob/ob mice were parabiosed to wild-type mice and pairs containing two ob/ob mice. Additional single ob/ob mice were also monitored. Parabiosis with either wild-type or db/db mice caused a significant reduction in blood glucose and insulin and prevented the normal rate of weight gain in ob/ob mice. Partners of wild type mice gained weight at approximately half the rate as was found in single mice, but ob/ob partners of db/db mice experienced a dramatic loss of weight and the mice died within weeks of surgery. Post mortem analysis indicated a significant loss of body fat and little food in the GI tract of these ob/ob mice. By contrast, the db/db parabionts remained hyperphagic, hyperglycemic and obese. Coleman concluded that db/db mice produced, but did not respond to a satiety factor that normally regulated food consumption. By contrast, ob/ob mice had normal satiety centers that were capable of responding to a satiety factor produced by wild-type or db/db mice, but were either unable to produce the satiety factor, or did not produce enough to influence the food intake of a wild-type parabiotic partner. Thus, mutations known to be located on two different chromosomes [2, 3] produced an identical phenotype because the defects in db/db and ob/ob mice occurred in the same metabolic pathway.
It is worth noting that a study that has proven pivotal to our understanding of the control of energy balance included very few data. As mentioned above, it was not possible to measure the food intake of individual parabionts and 24-hour intake, uncorrected for spillage, was measured only three times for pairs and single ob/ob mice. Data on body weight and blood glucose of pairs are also reported for only three time points during the 4-month study. Twelve ob/ob-db/db pairs survived between 13 and 90 days with the ob/ob partner dying first. Body fat and gut content were observed, but not measured, during autopsy, and blood exchange between partners was not confirmed. Thus, evidence for a circulating feedback signal in the control of energy balance was derived from an experiment in which neither food intake nor body fat were directly measured in the responsive partner. Following the discovery of leptin, a repetition of the experiment confirmed that ob/ob partners of db/db mice had a gut content that was only 25% that of members of ob/ob pairs and that body fat was reduced by 60% after only 18 days .
The two parabiosis studies reported by Coleman [1, 2] are identified as foundational evidence for the involvement of a circulating feedback signal in the control of energy balance and, more importantly, led to the search for the circulating factor that we now know as leptin. The identity of the satiety factor remained unknown for more than 20 years. Coleman pursued the idea that it was a pancreatic factor  whereas others tested the efficacy of CCK  or adipose-derived factors, such as glycerol . Ultimately, evidence that the ob/ob mouse was deficient in the satiety factor led to the initiation of an eight-year collaboration between Rudolph Leibel and Jeffrey Friedman at The Rockefeller University using newly developed positional-cloning techniques to successfully identify the mutant protein in ob/ob mice in 1994 . Other groups were also trying to identify the mutations and the db/db mutation was identified in 1995 by a group at Millenium Pharmaceuticals . ob/ob and db/db mice are now also referred to as lepob/ob and leprdb/db mice, respectively.
Although Coleman is not a co-author on papers that document progress in identifying the diabetes  or obese gene , his contribution to discussion of the projects is acknowledged . His critical contribution to the discovery of leptin was also appropriately recognized when he and Jeffrey Friedman were named as co-recipients of several prestigious awards, including the Lasker Basic Medical Research Award in 2010 for a fundamental discovery that opens up a new area of biomedical science. The discovery of leptin  can be credited for a resurgence in investigation of the control of food intake and energy balance, but Coleman’s parabiosis studies clearly were a major stimulus leading to the search for a satiety signal.
Both Hervey  and Coleman  concluded that the primary function of the circulating factor in parabionts is to inhibit food intake. Subsequent studies in which one partner of a pair was made obese by over-feeding suggest that loss of body fat from the normal partner of an overfed rat can be achieved with only a minimal inhibition of intake . Similarly, studies in which leptin is infused chronically, demonstrate that an initial decline in food intake is followed by a return to control levels that maintain body weight at a new stable, lean level , suggesting that the primary response to leptin administration is a metabolic adjustment to reduce the amount of fat present and that hypophagia is a means to achieve this goal. In addition, as more became known of the multiple physiologic systems that are modulated by leptin, it was proposed that rather than an increase in leptin functioning as a satiety signal, a fall in leptin serves a protective function during periods of food scarcity by promoting hunger, but inhibiting energy expensive functions such a reproduction . Thus, although it is clear that leptin plays an essential role in the control of energy balance, it may not be a satiety factor per se, opening the door for new investigations searching for a factor that inhibits appetite in response to overfeeding .
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