TRANSGENIC COMPLEMENTATION OF LEPTIN RECEPTOR ...

Articles in PresS. Am J Physiol Endocrinol Metab (November 4, 2003). 10.1152/ajpendo.00349.2003

TRANSGENIC COMPLEMENTATION OF LEPTIN RECEPTOR DEFICIENCY II. Increased Leptin Receptor Transgene Dose Effects on Obesity/Diabetes and Fertility/Lactation in Lepr-db/db Mice

Streamson C. Chua, Jr.1*, Shun Mei Liu1, Qiong Li1, Aijun Sun2, Walter F. DeNino2, Steven B. Heymsfield2, and X. Edward Guo3

1. Department of Pediatrics, Columbia University, New York, NY 2. New York Obesity Research Center, St. Luke’s-Roosevelt Hospital, New York, NY 3. Department of Biomedical Engineering, Columbia University, New York, NY

* To whom all correspondences should be directed

Running title: Leptin receptor transgene dose effects

Copyright (c) 2003 by the American Physiological Society.

1 ABSTRACT We have generated mice that are homozygous for a leptin receptor transgene that is expressed exclusively in neurons (NSE-LEPR-B). We had previously shown that this transgene in the hemizygous state is effective in ameliorating almost all aspects of leptin receptor deficiency. Now, in the homozygous state, we show that the transgene almost fully corrects the excess adiposity of LEPR-deficient (db/db) mice. Body composition analyses indicate that the transgene is able to restrain the massive increase in adiposity observed in LEPR-deficient mice. Examination of hypothalamic AGRP and POMC mRNA shows normalization of these leptinregulated transcripts. Interestingly, despite normalization of circulating leptin concentrations by the transgene in the fed state, transgenic db3J/db mice did not show fasting-induced reductions of circulating leptin. Increased adiposity of the transgenic db/db mice at 4 weeks of age, immediately post-weaning, suggests the transgene is less effective in correcting the preferential fat deposition caused by LEPR deficiency. We noted that the morphology of brown adipose tissue is nearly normal, concordant with the cold tolerance conferred by the transgene. Aspects of the diabetes phenotype are also corrected: glucose and insulin concentrations are nearly normal; and islet hyperplasia is greatly diminished. The transgene also corrects the infertility of db/db females and confers the ability to lactate sufficiently to nurse normal sized litters. Finally, the slightly increased adiposity and mild insulin resistance of transgenic db/db dams was not a contributory factor to the increased fat content of transgenic db/db male progeny.

Keywords: monogenic model of obesity, hypothalamus

2 INTRODUCTION The adipocyte-derived hormone leptin acts on multiple organs and its effects are readily examined in animal models of leptin (ob/ob) and leptin receptor (db/db) deficiency (18). Upon initial observation of these animal models, one is immediately struck by the massive obesity that is caused by hyperphagia, decreased thermogenesis, and preferential deposition of triglycerides in white fat. These phenotypes are mediated by leptin’s actions (4) on hypothalamic neurons bearing the STAT (Signal Transduction And Transactivator)-signalling competent isoform of leptin receptor, LEPR-B (7). All of the phenotypes relevant to obesity and diabetes are probably mediated by these LEPR-B-bearing hypothalamic neurons (6, 17). However, direct effects of leptin are also manifest in the T cells of the immune system (20, 21), platelets (15, 23), and vascular endothelium (3, 30). Signal transduction pathways independent of STATs may mediate some of these actions. We have previously reported the effects of a transgene, NSE-LEPR-B, which expresses solely in the mouse central nervous system due to the specificity conferred by the neuron specific enolase (ENO2) promoter (17). This transgene in the hemizygous state ameliorates, but does not fully normalize, all of the phenotypes of diabetes mice related to energy balance, namely hyperphagia, defective nonshivering thermogenesis, and obesity, along with correcting the infertility of males. However, the hemizygous transgenic db/db mice still exhibited increased adiposity, insulin resistance, and female infertility. Leptin treatment of ob/ob females restores fertility but the females fail to lactate (6). A similar phenotype of restoration of fertility with failure of lactation is observed in ob/ob Y4receptor double mutant females (28). As ob/ob females without Neuropeptide Y (NPY) are partially fertile and successfully raise their pups (10), it is quite likely that overexpression of

3 NPY is affecting prolactin release, perhaps via effects on hypothalamic dopaminergic neurons. Hemizygous transgenic db/db females are infertile and we were unable to determine their ability to lactate. As the partial correction of the phenotypes by the NSE-LEPR-B transgene was probably due to mosaic expression of the transgene within the brain, we reasoned that it would be possible to improve the normalization of the obesity/diabetes phenotypes of db/db mice by increased expression of the transgene in the homozygous state. There are instances where a tyrosinase transgene in the homozygous state conferred increased and more uniform pigmentation over the hemizygous condition in albino, tyrosinase-deficient mice (22, 31). In this report, we describe the effects of the NSE-LEPR-B transgene in the homozygous condition on the body composition of db/db mice along with its effects on female fertility. As homozygous transgenic db/db females are fertile, we are also able to assess their ability to lactate and suckle their pups. Since our model is based on correcting the LEPR-deficient phenotypes on a transgene expressed in the brain, we should be able to evaluate the neurally mediated actions of the leptin-leptin receptor system on peripheral organ systems.

4 METHODS Animals Growth data were obtained on a cohort of NSE-LEPR-B mice (12-20 mice per group). Other data have group sizes specified for each data set. Animals were fed a standard laboratory diet (Picolab Mouse Diet 20: 55% carbohydrate, 20% protein, 9% fat; PMI Nutrition International) and water ad libitum. All animals were housed under barrier conditions at 22° C with a 14h:10h light/dark cycle. Pathogen testing was done on a quarterly basis and all serologies were negative throughout the study. Genotyping was done as previously described (17). For simplicity, we refer to the Leprdb allele as db and the Leprdb3J allele as db3J. We have performed parallel analyses between transgenic db/db and transgenic db3J/db3J mice and we have not observed difference in growth or body composition between the two genotypes (unpublished data, SC). For the purposes of analyses, mice of these two genotypes have been grouped together. Similarly, non-transgenic db/db and db3J/db3J mice have also been grouped together when data was available. The genotypes of mice are specified in the legends when relevant. Live mice were weighed on an Ohaus balance with a precision to 0.1g. Animals were euthanized by asphyxiation with carbon dioxide. Euthanized animals were weighed using a Mettler precision balance (AC 100, Mettler) and naso-anal length measurements were taken. Blood for leptin, glucose, and insulin concentration determinations was collected from live, awake mice via a nicked tail vein. Fed serum samples were collected between 0900 and 1200. Fasted serum samples were collected between 0900 and 1200 after an overnight fast (chow removed at 1800) or after a 5 hour fast with serum collected at 1400 with chow removed

5 at 0900. All procedures were described in protocols that were approved by the Columbia University IACUC. The procedures were designed to minimize distress and pain. Production of Homozygous Transgenic Animals Transgenic hemizygotes were mated to produce putative transgene homozygous animals. Homozygosity was verified by test matings to wild type animals and subsequent genotyping of the progeny. Mice that produced all transgenic progeny (at least 10 pups tested) when mated to wild type animals were considered transgenic homozygotes. Progeny from subsequent matings between transgenic homozygotes were not tested. Colonies were maintained by matings between db3J/db3J Tg/Tg pairs or db3J/+ Tg/Tg mice mated to db3J/db3J Tg/Tg mates. Compound db/db3J heterozygotes were produced by matings between db/db Tg/Tg or db/+ Tg/Tg mice to db3J/+ Tg/Tg mates. Tissue Histology Pancreata were fixed in Bouin’s solution whereas other tissues were fixed in buffered formalin. Tissues were embedded in paraffin and 5 micron sections were cut. Pancreatic sections were immunostained for insulin content and counterstained with hematoxylin. Other tissues were stained with hematoxylin and eosin. Digital images were obtained on a Nikon Eclipse 400 microscope and a SPOT Insight digital camera. Assessment of Female Fertility Transgenic db/db females were mated at 6-8 weeks to transgenic db/db males. The number of days from mating to the delivery of the first litter was recorded. The number of pups born and the number of pups weaned 21 days after birth were noted. Ovaries of virgin females were removed at sacrifice and processed for histology.

6 Dual Energy X-ray Absorptiometry Whole body and tibia DXA measurements were done on a Lunar PIXIMUS scanner (Lunar Piximus, GE Medical Systems, Waukesha, Wisconsin) functioning in the pencil beam mode. Before each series of scans, a tissue calibration scan was performed using the manufacturer’s provided phantom. Live mice were anesthetized with a ketamine/xylazine mixture. Each mouse was placed on the scanner dish in a prone position with fore and hind legs outspread. Scans provided determinations of fractional body fat content, total body fat mass and total fat-free mass. Hormone and Glucose Measurements Glucose was determined on whole blood with a Bayer Glucometer using immobilized glucose oxidase. Insulin and leptin concentrations in serum were measured with ELISA kits from Alpco (insulin) and Crystal Chem (leptin) using mouse insulin and leptin as standards (15). Serum levels of hormones were quantified using commercial radioimmunoassay kits (Diagnostic Systems Laboratories). Quantification of Neuropeptide mRNA Hypothalamic RNA was isolated and used for cDNA synthesis and amplification as described previously (17). AGRP and POMC mRNA were analyzed using HPRT mRNA as an internal control for each sample. Real time quantification was performed on an Opticon 2 (MJ Research, MA). The data were analyzed to correct for differences in amplification efficiencies and normalized to HPRT mRNA content (25). Statistical Analysis All values are expressed as mean + SD unless otherwise noted. Group differences were tested for significance using the Student’s unpaired t test after initial tests to show a significant

7 effect of genotype. Data from the hypothalamic neuropeptide mRNA quantification were analyzed by a non-parametric test, the Wilcoxon rank-sum test.

8 RESULTS Near Normal Adiposity in db/db Mice Homozygous for the NSE-LEPR-B Transgene We have generated a line of mice, backcrossed to the C57BL/6J strain for 4 generations, that is homozygous for the NSE-LEPR-B transgene by standard mating procedures. We have found no evidence of deleterious effects of the homozygous condition, suggesting that the transgene does not disrupt any essential genes. The transgenic homozygous mice (db/+ and +/+) are fully fertile and we have not observed any phenotypic differences from wild type animals. The growth curves of transgenic db/db and db/+ mice are presented in Figure 1. The growth curves of transgenic db3J/db3J and transgenic db/db mice are not significantly different and the data are pooled in Figure 1. Between 1 and 3 months of age, transgenic db/db mice of both sexes are heavier than transgenic db/+ mice. At 4 months of age, the male db/+ transgenic and db/db transgenic mice have equivalent weights. At 6 months of age, the male db/+ transgenic males are actually heavier than the db/db transgenic males while the db/+ and db/db transgenic females are equivalent in weight. We performed body composition analysis by DXA on mice at 1 and 6 months of age, comparing transgenic and non-transgenic mice (Figure 2). The db/db mice of both sexes have the highest adipose tissue mass and fractional body fat at both ages, as expected. At 1 month of age, transgenic db/db mice of both sexes have adipose mass that is intermediate between genetically lean (db/+ and Tg db/+) and genetically obese (db/db) mice. At 6 months of age, the transgenic db/db male mice have body compositions that are very similar to transgenic db/+ males. However, the non-transgenic db/+ mice had a lower mean fat mass than either of the transgenic male groups, suggesting that either the transgene itself has an effect or that residual CBA alleles from the transgenic founder may be contributing to increased adiposity. The adult

9 transgenic db/db female mice still have slightly increased fat-free mass relative to db/+ and transgenic db/+ mice. There are some minor effects on fat-free mass, such as the increased fatfree mass in 1 month old male db/db mice, while significant, represent a small fraction of the total body mass. It is interesting to note that the 6 month old transgenic db/db males have a slightly lower fat-free mass than any of the other male genotypes. In summary, the data demonstrate that a major effect of the transgene in the homozygous state is the restraint on the massive increase in fat mass (>20 grams over 5 months) in post-weaning db/db mice.

Leptin Concentrations In Transgenic db3J/db Mice Are Not Reduced By Fasting We measured fed leptin concentrations in transgenic db3J/db and transgenic db3J/+ mice at 5 months of age (Figure 3). Fed leptin concentrations in transgenic db3J/db mice (males and females) are not statistically different from transgenic db3J/+ mice. However, leptin concentrations of fed db3J/db3J males was greatly elevated. Fasting reduces the leptin concentrations in transgenic db3J/+ mice of both sexes but does not reduce the leptin concentrations of transgenic db3J/db mice of either sex, suggesting that the transgene does not completely restore modulation of leptin secretion.

Correction of Brown Adipose Tissue Morphology by the Transgene A component of the obesity syndrome is defective nonshivering thermogenesis due to atrophy of brown adipose tissue. We had previously shown that the NSE-LEPR-B transgene normalizes cold tolerance in the hemizygous state. With the transgene in the homozygous condition, we observe normal brown adipose tissue in 5-8 month old transgenic db3J/db3J mice (Figure 4) whereas we were unable to observe any brown adipose tissue in the interscapular area

10 of db3J/db3J mice of the C57BL/6J strain (12 mice dissected). Histological examination shows the presence of adipocytes with the multiloculated fat droplets typical of brown adipocytes in the interscapular brown fat pad of transgenic db3J/db3J mice. The brown adipocytes have a slightly larger degree of lipid accumulation in transgenic female db3J/db3J mice than in lean mice or transgenic db3J/+ mice.

Normalization of Carbohydrate Metabolism and Pancreatic Islet Morphology Major concomitant phenotypes associated with obesity of LEPR deficiency are glucose intolerance and pancreatic islet hyperplasia. We measured fed and fasting glucose (Figure 5A) and insulin (Figure 5B) concentrations in transgenic db3J/db mice at 5 months of age. Fed glucose and insulin concentrations were indistinguishable between the two genotypes. In the fasting condition, glucose tended to be higher in the transgenic db3J/db mice but did not reach statistical significance. Fasting insulin concentrations tended to be higher in the transgenic db3J/db mice but only the difference between the females reached statistical significance. Concordant with these findings, we observed near-normalization of islet size in the pancreata of transgenic db3J/db and transgenic db3J/db3J mice (Figure 5C) although there were islets in the transgenic db3J/db3J pancreata that were larger than the largest islets observed in the pancreata of genetically lean mice.

Restoration of Female Fertility and Lactation by the Transgene In the hemizygous state, the transgene did not correct the infertility of females. However, the transgene in the homozygous state restores fertility to transgenic db/db females. The data in Table 1 show that female transgenic db/db mice, when mated to male transgenic db/db or db/+

11 mice, become pregnant and deliver normal sized litters. The ovaries of db/db females (Figure 6) histologically have many large empty follicles with few primary or secondary follicles and no evidence of proper corpora lutea. The ovaries of transgenic db/db females show numerous primary and secondary follicles with the presence of corpora lutea. Moreover, the females suckle their pups and are successful in raising all of their progeny to weaning, even in their first litters (Table I).

Excess Adiposity in Trangenic db/db Dams Does Not Increase Adiposity of Progeny Due to the increased adiposity and mild insulin resistance of the female transgenic db/db females, we were concerned that our results might be affected by potential confounding maternal effects. We generated progeny from two types of mating pairs: 1) transgenic db3J/+ females mated to transgenic db3J/db3J males; and 2) transgenic db3J/db3J females mated to transgenic db3J/+ males. We determined the body compositions (Table II) of transgenic db3J/db3J males at 4 weeks of age that were produced by the two types of matings. We found no significant effect of maternal genotoype on fractional body fat content in the male progeny.

Transgene Modulation of Hypothalamic Neuropeptide Gene Expression We had previously shown that the transgene in the hemizygous condition normalized POMC mRNA and reduced but did not normalize AGRP mRNA. We analyzed the effect of the transgene in the homozygous state on AGRP and POMC mRNA by real-time RT-PCR (Table III). As expected, db3J/db3J male mice have elevated AGRP mRNA and reduced POMC mRNA, relative to db3J/+ mice. The transgene in the homozygous state reduced AGRP and increased POMC mRNA to concentrations that were indistinguishable from db3J/+ males.

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Dose Effects of the Trangene on Obesity and Diabetes of db/db Mice We have compared some obesity and diabetes parameters of 5 month old hemizygous and homozygous transgenic db3J/db3J mice (Table IV) to be able to directly show a transgene dose effect. The body weights and BMI of db3J/db3J Tg/Tg mice, both males and females, are not significantly elevated above db3J/+ mice. The db3J/db3J Tg (transgene in hemizygosity) mice have elevated weights and BMI that are significantly different from the db3J/+ groups. There is also a similar gradation in fasting leptin concentrations due to the transgene dose with the db3J/db3J Tg/Tg animals having lower leptin concentrations than the db3J/db3J Tg animals. The fasting glucose values for db3J/+ and transgenic mice are not statistically different, whereas the db3J/db3J animals have elevated glucose values. There are slight differences in the glucose and leptin data since the data in Table IV are collected after a 5 hour fast whereas the data in Figure 5 are from an overnight fast.

13 DISCUSSION We have previously shown that the NSE-LEPR-B transgene has effects on all aspects of energy regulation relevant to leptin signalling (17). Mice hemizygous for the transgene and homozygous for Lepr mutations (db and db3J) were leaner, less insulin resistant, and had normal cold tolerance and lower corticosterone concentrations. Moreover, male db/db mice carrying the transgene were fertile although female transgenic db/db mice remained infertile. In the current report, we describe the phenotypes of db/db mice with the transgene in the homozygous state. We reasoned that the increasing transgene dosage would increase LEPR expression and improve the normalization of the phenotypes related to LEPR deficiency. Our data support this proposition since adiposity is normalized in adult male transgenic db/db mice when compared to transgenic db/+ males and nearly normalized in adult female transgenic db/db mice. The data show that the transgene in the homozygous state prevents the acquisition of over 20 grams (in the course of 5 months) of body fat due to LEPR deficiency. Our conclusion is tempered by the slight differences in adiposity observed in db/+ and transgenic db/+ male mice. It is possible that the difference could be due to a transgene effect, with increased transgenic leptin receptor expression leading to increased body fat content. Alternatively, the differences might be due to residual CBA alleles derived from the founder B6xCBA F2 mouse that increase body fat content. The normalization of hypothalamic AGRP and POMC mRNA in adult transgenic db3J/db3J males is also consistent with the supposition that transgene homozygosity reduces mosaicism and increases transgene expression. Previously, the transgene in the hemizygous state normalized POMC mRNA but only reduced AGRP mRNA without bringing AGRP mRNA to concentrations similar to lean mice.

14 The body composition data at one month of age indicated that the transgene is less effective, compared to its efficacy in adult db/db mice, in normalizing body composition. Thus, there may be components of the leptin-leptin receptor system that have differential effects in the preweanling mouse pup and the adult mouse. Further studies will be needed to define the contribution of leptin to the regulation of body composition of precocial organisms that undergo extremely rapid postnatal growth, such as the mouse pup. Measures of leptin concentrations in the blood showed that the transgene effectively reduced leptin concentrations to those found in transgenic db3J/+ mice. However, fasting did not reduce leptin in transgenic db3J/db mice, suggesting that there is still a defect in the regulation of leptin secretion that is not corrected by the transgene. We had previously remarked upon this in our analysis of transgenic hemizygous db3J/db3J animals although the elevated circulating insulin concentrations confounded our ability to make firm conclusions (1, 2, 16, 27). With the present animals, we show that fasting insulin concentrations in transgenic db3J/db male mice is significantly reduced. In addition, the fasting insulin concentrations of transgenic db3J/db females was well below 1ng/ml. Thus, we can exclude elevated insulin concentrations in the fasting state as a stimulus to elevated fasting leptin concentrations in the transgenic db3J/db mice. The data are highly suggestive of an autoregulatory loop of adipocyte secretion of leptin (26, 32, 35-37). The growth curves of the db3J/db compound mutants were not different from the db and db3J homozygotes, with or without the transgene (unpublished data, SC). We went to the effort of using db3J/db compound heterozygotes to measure circulating leptin concentrations to avoid potential confounding effects from elevations of a circulating leptin binding protein (11) produced by the db3J mutant allele (19). The db allele provides LEPR isoforms that could contribute to leptin metabolism, perhaps for catabolism after binding to

15 leptin receptors in the kidneys and the lungs (9, 29). This would avoid confounding results from potential leptin clearance issues that might be caused by the loss of non-B LEPR isoforms. One component of the obesity syndrome is defective nonshivering thermogenesis (8). This defect is most likely the combined result of decreased sympathetic activation of BAT and mild central hypothyroidism that has been documented to be due to indirect and direct actions of leptin on hypothalamic TRH neurons (12, 24). We had previously shown that the cold intolerance of db/db mice was corrected by the NSE-LEPR-B transgene. In this report, we show that the BAT of transgenic db3J/db3J mice has near-normal morphology, suggesting that trophic effects on BAT have been normalized by the transgene. The glucose intolerance of LEPR-deficient mice is due to insulin resistance and straindependent responses to hyperglycemia (8). The NSE-LEPR transgene normalizes nearly all parameters of glucose metabolism, although female transgenic db3J/db mice did not show a fasting-induced decline in circulating insulin concentrations. There are indications of very mild insulin resistance in the transgenic db3J/db animals although we can not determine whether this is due to long-term effects of the excess adiposity immediately post-weaning or to the absence of LEPR-B expression in some crucial neuronal types. Nevertheless, the expression of LEPR-B in neurons is sufficient to nearly normalize pancreatic islet mass. However, the current model does not allow the separation of potential trophic effects of insulin resistance/hyperglycemia on beta cell mass from potential direct modulation of beta cell mass from the hypothalamus. Another aspect of leptin receptor biology relates to fertility and lactation. We note that LEP-null females treated with leptin become fertile and have normal sized litters (5, 6). However, these leptin-treated females are unable to lactate. Similarly, LEP-null Y4-null females are fertile but fail to lactate (28). Interestingly, LEP-null NPY-null females are fertile and

16 successfully raise litters (10). In our transgenic db/db females, reproduction has been restored and the ability to lactate has also been restored. These observations suggest that there may be a critical period for the action of leptin on mammary gland development that remains to be explored. As neither overexpression of NPY (14, 33) nor MC4R deficiency (13) prevent mammary gland development sufficiently to affect lactation, the mechanism and neural pathways involved in leptin’s regulation of the mammary gland remains to be determined. There have been studies that indicate the db/+ genotype has an effect on body composition via a maternal effect. Female db/+ mice of the C57BLKS/J strain become glucose intolerant during pregnancy and their progeny are macrosomic (34). We were concerned that the slightly increased adiposity of transgenic db/db dams might affect the body compositions of their progeny, potentially confounding the effects of maternal environment and the genotype of the progeny. Our data suggest that neither the increased adiposity nor the mild insulin resistance of transgenic db/db females affects the fractional body fat content of their progeny. Thus, the increased adiposity of the transgenic db/db mice at 4 weeks is probably due to deficient LEPR signaling during development. The NSE-LEPR transgene has limitations in terms of addresssing the components of the neural network that mediate leptin’s effects on ingestion, body composition, energy expenditure, and fertility. The transgene is unable to direct expression to neurons that are defined by their neurotransmitter content, as might be obtained by a transgene that is driven by the promoters of the Pomc or the Npy genes. However, the current model would be interesting to use to dissect the central and peripheral actions of leptin on immune cells and the skeletal system. Since the transgene effectively reduces insulin resistance and glucocorticoid concentrations, the transgene effectively simplifies the model and removes confounding metabolic conditions that may mask

17 the direct actions of leptin on peripheral tissues. The current model does not address questions that arise from examination of the phenotype of the transgenic db/db mice – 1) Which neurons contribute to the insulin resistance of db/db mice?; 2) Which neurons are responsible for the elevated glucocorticoids of db/db mice?; 3) What is the contribution of the development of insulin resistance and elevated glucocorticoids to the obesity/diabetes syndrome; 4) What are the mechanisms that cause the early onset adiposity of preweaning db/db pups? In summary, we have described the effects of a leptin receptor B isoform neuron-specific transgene on the phenotype of db/db mice. The transgene in the homozygous state causes near normalization of adiposity in adult db/db animals. The transgene appears to be less effective in reducing the adiposity during the pre-weaning phases of development. Furthermore, we have data to suggest that the maternal environment is not a factor in the increased adiposity of young transgenic db/db mice. The transgene corrects glucose intolerance and the beta cell hyperplasia accompanying the insulin resistance of LEPR deficiency. Finally, the fertility of female db/db mice is restored by the transgene along with the ability to lactate.

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ACKNOWLEDGEMENTS This work was funded by NIH grants DK42618 (SBH), DK57621 (SCC), and DK26687 (SBH, SCC).

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24 FIGURE LEGENDS Figure 1. Growth curves of db/db and transgenic db/db mice. Growth curves (A and B) of male and female mice are presented. The data points for db/+ Tg and db/db Tg mice represent weights of groups of 10-18 mice of each genotype/sex combination. The points for the db/db mice are means of 4-5 mice for each sex and are presented for comparison purposes. The asterisks signify a difference from the db/+ Tg/Tg groups.

Figure 2. Fat mass and fat-free mass of transgenic db/db mice. Fat mass (A and C) and fat-free mass (B and D) of males and females at 1 and 6 months of age as determined by DXA are presented in absolute weights for the four genotypes. Group sizes for transgenic mice ranged from 8 to 16 while groups sizes for nontransgenic mice were 37. An asterisk represents a statistically significant difference from the db/+ group, a caret (^) represents a difference from the db/+ Tg/Tg group (db/db and db/db Tg/Tg groups only), and a @ represents a difference between the db/db and the db/dbTg/Tg group. The fat-free mass of male transgenic db/db mice at 6 months was lower than all other groups and is represented by a pound sign (#).

Figure 3 Leptin concentrations in transgenic db3J/db and db3J/+ mice. Leptin concentrations were measured by ELISA and presented as group means (N=4-7 per group). The mice were 5 months of age. An asterisk signifies a difference due to fasting (p