Departments of 1Pediatrics and 3Biomedical Engineering, Columbia University, New York 10032; and 2New York Obesity Research Center, St. Luke's-Roosevelt Hospital, New York, New York 10025
Submitted 31 July 2003 ; accepted in final form 28 October 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
monogenic model of obesity; hypothalamus
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 Y4-receptor double-mutant females (28). Because ob/ob females without neuropeptide Y (NPY) are partially fertile and successfully raise their pups (10), it is quite likely that overexpression of 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 NSELEPR-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 in which 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. Because homozygous transgenic db/db females are fertile, we are also able to assess their ability to lactate and suckle their pups. Because 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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Live mice were weighed on an Ohaus balance with a precision to 0.1 g. Animals were euthanized by asphyxiation with carbon dioxide. Euthanized animals were weighed using a precision balance (AC 100, Mettler), and nasoanal length measurements were taken.
Blood for leptin, glucose, and insulin concentration determinations was collected from live, awake mice via a nicked tail vein. Serum samples from fed animals were collected between 0900 and 1200, and those from fasted animals were collected between 0900 and 1200 after an overnight fast (chow removed at 1800) or after a 5-h fast with serum collected at 1400 after chow removal at 0900. All procedures were described in protocols that were approved by the Columbia University Institutional Animal Care and Use Committee. These 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 (10 pups tested) when mated to wild-type animals were considered transgenic (Tr) 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 and 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-µm 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 wk 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 euthanized virgin females were removed and processed for histology.
Dual-energy X-ray absorptiometry. Whole body and tibia dual-energy X-ray absorptiometry (DEXA) measurements were made 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 with immobilized glucose oxidase. Insulin and leptin concentrations in serum were measured with ELISA kits from Alpco (insulin) and Crystal Chem (leptin), with 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). Agouti gene-related peptide (AGRP) and proopiomelanocortin (POMC) mRNAs were analyzed using hypoxanthine-phosphoribosyltransferase (HPRT) mRNA as an internal control for each sample. Real-time quantification was performed on an Opticon 2 (MJ Research, Waltham, 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 means ± SD unless otherwise noted. Group differences were tested for significance using the Student's unpaired t-test after initial tests to show a significant effect of genotype. Data from the hypothalamic neuropeptide mRNA quantification were analyzed by a nonparametric test, the Wilcoxon rank-sum test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The growth curves of transgenic db/db and db/+ mice are presented in Fig. 1. The growth curves of transgenic db3J/db3J and transgenic db/db mice are not significantly different, and the data are pooled in Fig. 1. Between 1 and 3 mo of age, transgenic db/db mice of both sexes are heavier than transgenic db/+ mice. At 4 mo of age, the male db/+ transgenic and db/db transgenic mice have equivalent weights. At 6 mo of age, the male db/+ transgenic males are actually heavier than the db/db transgenic males, whereas the db/+ and db/db transgenic females are equivalent in weight.
|
We performed body composition analysis by DEXA on mice at 1 and 6 mo of age, comparing transgenic and nontransgenic mice (Fig. 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 mo 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 mo of age, the transgenic db/db male mice have body compositions that are very similar to transgenic db/+ males. However, the nontransgenic 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, derived from the transgenic founder B6xCBA F2 mouse, may be contributing to increased adiposity. The adult 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 fat-free mass in 1-mo-old male db/db mice, which, although significant, represent a small fraction of the total body mass. It is interesting to note that the 6-mo-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 g over 5 mo) in postweaning db/db mice.
|
Leptin concentrations in transgenic db3J/db mice are not reduced by fasting. We measured leptin concentrations in fed transgenic db3J/db and transgenic db3J/+ mice at 5 mo of age (Fig. 3). Leptin concentrations in fed transgenic db3J/db mice (males and females) are not statistically different from those of fed transgenic db3J/+ mice. However, leptin concentrations of fed db3J/db3J males were 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 (BAT). 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 BAT in 5- to 8-mo-old transgenic db3J/db3J mice (Fig. 4), whereas we were unable to observe any BAT in the interscapular area 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 (Fig. 5A) and insulin (Fig. 5B) concentrations in transgenic db3J/db mice at 5 mo 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 (Fig. 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/+ mice, become pregnant and deliver normal-sized litters. The ovaries of db/db females (Fig. 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 1).
|
|
Excess adiposity in trangenic db/db dams does not increase adiposity of progeny. Because of the increased adiposity and mild insulin resistance of the 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 2) of transgenic db3J/db3J males at 4 wk 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 3). 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.
|
Dose effects of the transgene on obesity and diabetes of db/db mice. We have compared some obesity and diabetes parameters of 5-mo-old hemizygous and homozygous transgenic db3J/db3J mice (Table 4) to be able to show directly a transgene dose effect. The body weights and body mass indexes (BMIs) of db3J/db3J Tg/Tg mice, both males and females, are not significantly elevated above those of db3J/+ mice. The db3J/db3J Tg (transgene in hemizygosity) mice have elevated weights and BMIs 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, because the data in Table 4 are collected after a 5-h fast, whereas the data in Fig. 5 are from an overnight fast.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The body composition data at 1 mo of age indicated that the transgene is less effective, compared with 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 the level of 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 are significantly reduced. In addition, the fasting insulin concentrations of transgenic db3J/db females were well below 1 ng/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 those of the db and db3J homozygotes, with or without the transgene (unpublished data, S. C. Chua). 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 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 non-shivering 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 previously showed 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 strain-dependent 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 cannot determine whether this is due to long-term effects of the excess adiposity immediately postweaning 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 -cell mass from potential direct modulation of
-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 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 melanocortin-4 receptor deficiency (13) prevents mammary gland development sufficiently to affect lactation, the mechanism and neural pathways involved in leptin's regulation of the mammary gland remain to be determined.
There have been studies indicating that 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 wk 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. Because the transgene effectively reduces insulin resistance and glucocorticoid concentrations, it effectively simplifies the model and removes confounding metabolic conditions that may mask 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 preweaning 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 -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.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|