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1 The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA
2 New York Obesity Research Center, St Luke’s-Roosevelt Hospital Center, 1111 Amsterdam Avenue WH 1020, New York, New York 10025, USA
(Requests for offprints should be addressed to Simon Klebanov at New York Obesity Research Center; Email: seklebanov{at}hotmail.com)
Olga DeSimone’s present address is Department of Pediatrics, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, USA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leptin-deficient patients can also be successfully treated with recombinant leptin (Farooqi et al. 2002, Oral et al. 2002). However, as with insulin injections in diabetes, leptin injections in leptin deficiency are a psychological burden and may result in a suboptimal pattern of the hormone throughout the day. Thus, similarly to replacing insulin via islet transplantation, replacing leptin via WAT transplantation may offer a more physiological pattern of hormone release and obviate the need for a life-long regimen of injections. However, attempts to use WAT transplantation in animal models of leptin deficiency have given mixed results.
Transplantation of normal (Gavrilova et al. 2000), but not leptin-deficient (Colombo et al. 2002) WAT was successfully used for ameliorating metabolic abnormalities of lipodystropic mice, indicating that leptin produced by normal WAT grafts is essential for treating lipodystropic mice. Surprisingly, in contrast to lipodystropic mice, there have been no reports of successful treatment of Lepob/Lepob mice by normal WAT transplantation. Early experiments on WAT transplantation were unable to detect an improvement in metabolic and endocrine characteristics of these mice (Hausberger 1959, Ashwell et al. 1977).
The failure to demonstrate WAT graft function in Lepob/Lepob, in contrast to lipodystropic recipients, might have resulted from their highly increased fat mass and, hence, a different pattern of signals involved in WAT survival and the regulation of WAT endocrine function. Increased adiposity of Lepob/Lepob mice might also have affected their leptin sensitivity. If confirmed, such failure of WAT transplantation to treat insulin resistance and diabetes in obese subjects could significantly limit the applicability of WAT transplantation for humans, in whom these metabolic abnormalities are usually associated with obesity (Astrup & Finer 2000). We, however, hypothesized that the early studies were unsuccessful because of technical reasons, including graft rejection (Hausberger 1959) and/or insufficient graft size (Ashwell et al. 1977) and that, if modified accordingly, WAT transplantation could be successfully applied for treating metabolic abnormalities in obese Lepob/Lepob mice.
In the current study, so as to avoid immune rejection, we transplanted congenic WAT, different from the recipient’s tissue only by a narrow chromosomal region of the leptin gene, and in order to assure sufficient graft size, we transplanted gonadal fat pads from up to 8 donors into a single recipient. With these modifications, we tested whether leptin-producing WAT could prevent the development of hyperphagia, obesity, insulin resistance and infertility in Lepob/Lepob mice.
If WAT transplantation is ever to become useful for treating leptin deficiency, two important issues have to be addressed. The first is whether WAT transplants can function long enough to provide a clear alternative to regular injections and/or replaceable pumps. We addressed this issue by following the effects of WAT transplantation for up to 40 weeks. The second issue relevant to the human situation is whether subjects with a long history of leptin deficiency can still be successfully treated by WAT transplantation. We addressed this question by testing the effects of transplantation of WAT into Lepob/Lepob mice over one year of age.
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Materials and Methods |
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In the current study, we used female B6.V-Lepob/J mice purchased from The Jackson Laboratory (Bar Harbor, ME, USA). In this strain, the Lepob mutation was backcrossed to the C57BL/6J genetic background for more than 30 generations. Mice were either (1) obese, leptin-deficient Lepob/Lepob, referred to below as ob/ob mice, or (2) lean mice whose genotype was either Lepob/+ or +/+, referred to in this study as +/?.
Most of the experiments in the current study were performed on two sets of mice, introduced into the experiment at 40 days and at 13 months of age. At 40 days of age, ob/ob mice just begin to develop obesity, while at 13 months of age, mice have been severely obese for about a year. For the fertility study in mice with advanced obesity, we used 75-day-old instead of 13-month-old ob/ob females, because at that age, even normal C57BL/6J females would, for the most part, be infertile. Thus, the young mice were used to test for the preventive effects of the WAT transplantation; the older mice were used to test for the therapeutic effects, i.e. for the ability of the normal WAT transplantation to reverse the ob/ob mouse abnormalities.
For each age, we had 6 groups with 4–6 mice in each: a lean +/? control group (+/?), 4 groups of ob/ob mice transplanted with WAT from 1, 2, 4 and 8 lean +/? donor mice (FT1, FT2, FT4, FT8), and a group of ob/ob mice transplanted with leptin-deficient WAT from ob/ob donors (FT0), with a transplant volume equal to the volume of WAT from 3 lean +/? donors.
All mice were housed in a specific pathogen-free facility. Animals were fed the NIH 31 diet with 4% fat which was available ad libitum, and were maintained on a 14 h light:10 h darkness cycle, with lights on at 0700 h and off at 2100 h. Animal studies followed the guidelines of The Jackson Laboratory and The American Association for Accreditation of Laboratory Animal Care.
WAT transplantation procedure
All donor mice (+/? and ob/ob) were 2–3 months of age. Their gonadal fat pads were harvested and immediately loaded in a syringe through the plunger end, so that warm ischemia time was less than 10 min. The average volume of WAT from one donor was approximately 0.4 ml. WAT from 1 to 8 donors was transplanted to a single recipient. Recipient mice were anesthetized with isoflurane (Aerrane, Baxter Pharmaceuticals Products Inc., Deerfield, IL, USA), and multiple injections of donors’ WAT were made subcutaneously, using 16-gauge needles to minimize adipocyte damage during injections. Injection volume was kept between 100 and 150 µl per injection site. Thus, FT1 animals usually received 3–4 injections, while FT8 mice might have received up to 30 injections. The injections were made in the abdominal region first, and, if needed, additionally into the dorsal region.
For histological evaluation of transplanted tissue, grafts were removed 3–6 months after transplantation and fixed in 4% formalin in saline; 10-µm paraffin sections were stained with hematoxylin and eosin.
Determination of anti-leptin antibodies in mouse sera
Anti-leptin antibodies were measured by sandwich ELISA. Mice were bled and serum was collected and diluted 1:1000 in PBS with 1% BSA (pH 7.4). For coating with the capture antibody, a Nunc Immuno Maxisorb plate (Nalge Nunc International, Rochester, NY, USA) was incubated for 2 h at room temperature with goat anti-mouse leptin antibody (AF498, R&D Systems, Minneapolis, MN, USA) at 1 µg/ml in PBS (pH 8.0). The wells of the plate were washed and recombinant mouse leptin (498-OB, R&D Systems), 1 µg/ml in PBS (pH 8.0), was added for 18 h at 4 °C. The plate was washed, blocked by 3% BSA in PBS for 30 min, and the pre-diluted serum samples were added (50 µl per well) for 2 h at room temperature. After washing the plates, 100 µl alkaline phosphatase-conjugated goat anti-mouse Ig (A3562, Sigma, St Louis, MO, USA) diluted 1:5000 in PBS/1% BSA were added for 1 h. Finally, p-nitrophenyl phosphate (Sigma, FAST N2770) was added and the optical density was read at 405 nm.
Body weight and food consumption measurements
Food consumption for each group was measured daily in 2 separate cages, with 2–3 mice in each, by weighing the food remaining in the food hopper.
Plasma hormone and metabolite measurements
To measure plasma leptin levels, we used the Quantikine M, Mouse Leptin ELISA kit (R&D Systems). This kit detected no more than 0.15 ng/ml of a cross-reacting substance in ob/ob mice.
Insulin was measured with Sensitive Rat Insulin RIA kit (Linco Research Inc., St Charles, MO, USA). Corti-costerone was measured by an ImmuneChem double antibody corticosterone RIA 125I kit for rats and mice from ICN Biomedicals, Inc. (Costa Mesa, CA, USA). Plasma glucose was determined with a colorimetric enzymatic kit (Trinder) from Sigma-Aldrich (St Louis, MO, USA).
For the insulin tolerance test, we used 2 U human insulin/kg i.p. (Humalog, Eli Lilly, Indianapolis, IN, USA) and measured blood glucose before, and 15, 30, and 60 min after insulin injection.
Estrous cyclicity measurements
Estrous cycle stages were determined from vaginal smears as described by Nelson and colleagues (1982). Briefly, smears were obtained daily between 1000 and 1300 h. The fire-polished tip of a Pasteur pipette was placed at the vaginal orifice, and a drop of saline was expelled into the vagina, aspirated back, and transferred to a microscope glass. Dry smears were fixed and stained in 2% Giemsa blood stain. Smears were examined microscopically at x 40 magnification and were classified into seven stages: diestrus/proestrus, proestrus, proestrus/estrus, estrus, metestrus 1, metestrus 2, and diestrus. An estrous cycle was defined as the period between two successive proestrous smears.
Data analysis
Data are expressed as means ± standard error. To assess the effectiveness of WAT transplantation, we used the t-test to compare all transplanted groups with the FT0 group. To assess whether the transplantation resulted in a complete reversal of the ob/ob phenotype, we used the t-test to compare transplanted groups with the lean +/? group.
For body weight, food consumption, and plasma glucose, the analyses were carried out on untransformed data. Because the variance strongly correlated with the mean for insulin and corticosterone data, we applied the t-test to log-transformed values for these analyses. Average daily corticosterone levels were calculated by the trapezoid rule (http://archives.math.utk.edu/visual.calculus/4/approx.1/index.html) on the log-transformed values. Fertility data were analyzed by the Chi-square test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histological evaluation of adipose tissue grafts 3 months after transplantation reveals that, while some fibroblasts are present, adipocytes are the predominant cell type with characteristic unilocular lipid droplet and eccentric nucleus (Fig. 1A
). A significant degree of vascularization is present (Fig. 1B is a higher magnification of the area in the frame on Fig. 1A showing erythrocytes in the lumen of a venule).
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WAT transplantation at 40 days of age
We transplanted WAT from 1, 2, 4, or 8 lean +/? donors into 40-day-old female ob/ob recipients (the FT1, FT2, FT4, and FT8 groups respectively). One group of ob/ob recipients was also transplanted with WAT from ob/ob mice (FT0 group). Figure 2 shows plasma leptin levels in ob/ob recipients 9–14 weeks after transplantation. All groups transplanted with +/? WAT had leptin levels significantly higher than the one transplanted with ob/ob WAT (P<0.001; t-test). Recipients of WAT from 4 +/? donors had higher leptin levels than recipients of WAT from 1 donor (P<0.001; t-test), and recipients of WAT from 8 donors had higher leptin levels than recipients of WAT from 1 (P<0.001), 2 (P<0.001) and 4 (P<0.01) donors. While plasma leptin levels increased with the amount of transplanted WAT, even mice transplanted with WAT from 8 donors had leptin levels of 1.32 ± 0.04 ng/ml, i.e. only 20% of the 6.72 ± 1.30 ng/ml plasma levels found in lean, age-matched +/? control mice (n=6).
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shows FT0 (center) and FT8 (right) mice 9 months after transplantation; both are compared with an age-matched +/? mouse (left). Even transplantation of WAT from a single donor(FT1), resulting in leptin levels of less than 8% of those in the lean age-matched +/? mice, significantly decelerated body weight gain to about halfway between obese and normal (Fig. 3B). Moreover, WAT from 4 and 8 donors, creating plasma leptin levels of only 14% and 20% respectively of the normal, completely prevented body weight gain 16 weeks following transplantation – the period when even lean +/? mice, with leptin levels five- to sevenfold higher, were gaining weight. Sixteen weeks after transplantation, the FT4 mice weighed 35.2 ± 1.3 g and FT8 mice weighed 33.0 ± 0.6 g, only 5.5 and 3.3 g respectively more than lean +/? mice. At the same time, the differences in body weights between the FT4 and FT8 groups and the FT0 group were more than 30 g. Thirty-nine weeks after the transplantation, body weight of the FT4 group seemed to plateau at 39.8 ± 0.8 g; the FT8 group plateaued at 37.4 ± 0.9 g, i.e. much closer to 29.5 ± 0.6 g – the weight of lean +/? mice, than to 85.3 ± 1.5 g – the weight of the FT0 mice (Fig. 3B). Transplantation of ob/ob WAT did not have a significant effect on body weight and 8 weeks after transplantation, for example, FT0 and aged-matched intact ob/ob mice weighed 58.1 ± 0.7 g and 58.7 ± 1.0 g respectively.
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). All mice, except the FT1 group, had a significantly lower food consumption than the FT0 group (* – significantly different from FT0 (P<0.05; t-test)). The effect was so robust that it could be detected with food consumption measured only in 2 cages per group. Food consumption was not, however, fully normalized and the FT2 and the FT8 groups still had a significantly higher food consumption than lean +/? mice (# – significantly different from +/? (P<0.05; t-test)).
The ob mutation on the C57BL/6J genetic background is characterized by hyperinsulinemia and normoglycemia (with only a transient hyperglycemia early in life (Coleman & Hummel 1973)). Thus, we tested whether WAT transplantation would prevent the development of hyper-insulinemia. Figure 4A illustrates that nonfasting insulin levels were brought to normal in the FT2, FT4, and FT8 groups by 9–14 weeks after the transplantation. Transplantation of WAT from even a single donor (FT1), resulting in less than 8% of normal plasma leptin, was sufficient to lower insulin levels by approximately fivefold in ob/ob mice, indicating that insulin sensitivity was improved. In contrast, insulin levels in ob/ob mice transplanted with ob/ob WAT were not different from those in age-matched intact ob/ob mice, 26.3 ± 8.0 ng/ml and 19.9 ± 2.3 ng/ml respectively, suggesting that the transplantation procedure itself did not have any long-lasting effect on insulin levels.
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, * – FT0 significantly different from FT2 and +/? (P<0.05; t-test)).
Hypercorticism is an important feature of the ob/ob mouse phenotype (Naeser 1974, Dubuc et al. 1975). We measured the levels of the mouse major glucocorticoid, corticosterone, 7–9 months after transplantation. Because of the pronounced diurnal variation, corticosterone measurements were taken around the time of minimum, 0800 h, and around the time of peak, 1800 and 2100 h, concentrations. At all time points, corticosterone levels were significantly higher in the FT0 group than in lean, +/? mice and in any WAT-transplanted mice (data not shown). Average diurnal corticosterone levels showed a very similar response to WAT transplantation. All transplanted groups had significantly lower corticosterone levels than the FT0 group, and none of the groups, except the FT2 group, differed in corticosterone levels from the +/? mice (Fig. 5; * – significantly different from FT0 (P<0.05; t-test), # – significantly different from +/? (P<0.05; t-test)).
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To confirm that estrous cyclicity was indicative of full reproductive competence, we mated a separate group of ob/ob mice, 6 weeks after they had been transplanted with WAT from 4 donors. Nine out of twelve transplanted mice became pregnant and produced pups when mated with proven breeder C57BL/6J males – results similar to those in lean +/? mice where 11 out of 12 mice became pregnant (
2, P>0.05). Litter size was slightly lower in FT mice than in +/? mice, 5.9 ± 0.4 and 7.9 ± 0.7 pups respectively (P<0.05). It is noteworthy, however, that while pups from all newly found litters from +/? dams had visible gastric milk content, pups in a half of the litters (4 out of 9) from the transplanted females had empty stomachs, seemingly due to the females’ failure to lactate.
We also tested whether WAT transplantation could restore fertility in ob/ob mice that had already developed substantial obesity. For that study, we used 75-day-old female ob/ob mice with a body weight of 56.1 ± 0.9 g. The mice were transplanted with WAT from 4 lean +/? donors; 3 months later, when their body weight was 43.4 ± 1.0 g, they were tested for fertility. All 7 transplanted mice became pregnant and continued to produce litters for the next 5–7 months. Average first litter size was 6.6 ± 1.0 pups, not statistically different from 7.2 ± 1.1 pups in the age-matched +/? mice. In this case, all first litters of transplanted mothers died because, seemingly, females did not lactate. It is noteworthy, however, that some of the consequent pregnancies resulted in a small number of pups (1–2) being weaned.
WAT transplantation at 13 months of age
To test whether late WAT transplantation may reverse the ob/ob phenotype, we transplanted WAT from 1, 2, 4, or 8 young lean, +/? donors into 13-month-old ob/ob recipients (FT1, FT2, FT4, and FT8 groups respectively). Figure 6 shows plasma leptin levels in ob/ob recipients 10 weeks after transplantation. Groups transplanted with WAT from 2, 4, or 8 donors had leptin levels significantly higher than the group transplanted with WAT from 1 donor (P<0.05; t-test). While leptin levels increased with the amount of transplanted WAT (from 2 to 8 donors), the differences among FT2, FT4 and FT 8 groups were not statistically significant (P>0.05). Plasma leptin levels were similar to those achieved with the transplantation into young recipients (see Fig. 2). However, even mice transplanted with WAT from 8 donors had leptin levels of 1.32 ± 0.35 ng/ml, i.e. only 15% of 8.67 ± 2.57 ng/ml, the plasma levels found in lean, age-matched +/? control mice.
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). Eighteen weeks after transplantation, the FT4 and FT8 mice lost 40% and 50% respectively of their initial body weight. In contrast to young animals, however, WAT from 1 donor (the FT1 group) was not sufficient to affect the body weight of middle-aged recipients.
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, * –significantly different from FT0 (P<0.05; t-test), # –significantly different from +/? (P<0.05; t-test)). As in young animals, the transplantation procedure had little effect on insulin levels, which were 17.9 ± 8.0 ng/ml and 23.9 ± 5.9 ng/ml in FT0 and in intact ob/ob mice respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current study is the first to show that in Lepob/Lepob mice, WAT transplantation is effective as both a preventive and a curative measure. In young, 40-day-old animals, WAT transplantation prevented further development of obesity and mostly normalized food consumption. It also normalized insulin and corticosterone levels. Finally, in both 40-day-old mice with mild obesity and 75-day-old mice with substantial obesity, WAT transplantation led to fertility.
In 13-month-old mice weighing over 80 g, transplantation of as little as 2–3 ml normal WAT led to an up to two-fold reduction in body weight, and to the normalization of insulin levels.
Thus, WAT transplantation markedly improved, or even completely reversed, various aspects of the Lepob/Lepob phenotype. The ameliorating effect of WAT transplantation persisted for an extended period of at least 20 to 40 weeks. The procedure was effective in both young mice and mice with a long history of leptin deficiency. Finally, WAT transplantation had a pronounced effect on the Lepob/Lepob phenotype, despite relatively low leptin levels of only 10% to 20% of those in the +/? mice.
The molecular nature of the Lepob/Lepob phenotype remained unknown until the leptin gene was cloned (Zhang et al. 1994). The parabiosis study, in which a Lepob/Lepob partner of a lean animal lost weight, suggested that a missing circulating anorectic factor was the primary defect in the Lepob/Lepob mouse (Coleman 1973). However, the lack of the effect of grafted WAT on the Lepob/Lepob phenotype (Hausberger 1959) and a WAT cross-transplantation study (Ashwell et al. 1977), seemingly excluded WAT as a site of the primary defect. Most likely, previous attempts to detect the effect of WAT transplantation on the Lepob/Lepob recipient failed due either to immune rejection, when transplantation was attempted in genetically heterogeneous animals (Hausberger 1959), or to the small size of the graft (Ashwell et al. 1977).
In this study, for transplantation we used WAT from congenic mice, differing genetically from the ob/ob mouse only in having at least one normal copy of the leptin gene. Since transplantation of syngeneic, leptin-deficient WAT into ob/ob mice (the FT0 group) did not affect body weight, insulin levels and fertility as the transplantation of a similar volume of normal WAT did, the effects of transplantation should be attributed to leptin produced by normal adipocytes.
As leptin is a major adipostatic hormone, we expected the normal transplants to be stimulated through feedback mechanisms eventually to support normal leptin levels. This, however, was not the case as even ob/ob mice transplanted with WAT from 8 lean +/? donors failed to achieve normal leptin levels. Several factors might have contributed to this apparent discrepancy. First, we might not have used an optimal WAT for transplantation. Even though plasma leptin levels were reported not to be different in +/+ and Lepob/+ mice (Chung et al. 1998), under the conditions of high leptin demand per unit of tissue, Lepob/+ WAT might possibly not perform as well as +/+ WAT. Also, despite seemingly normal morphology, transplant viability and transplantation site might not be optimal. Another contributing factor might be the transplants’ limited capacity for growth. Finally, leptin may not be the only adipostatic hormone, and endogenous WAT, although leptin deficient, might have overproduced some other adipostatic hormones. To distinguish between these possibilities, different transplantation modalities should be attempted, including transplantation into leptin-deficient lipodystropic mice.
Interestingly, the Lepob/Lepob phenotype was significantly ameliorated, or even reversed, at leptin levels several-fold lower than those found in lean +/? mice. These results may indicate either that even low leptin levels are suficient for normal physiology or that mice never exposed to leptin have a very high sensitivity to the hormone. If the latter explanation were correct, elevated sensitivity to leptin would be expected to dissipate after an extended period of exposure to leptin. The effect of transplants on body weight, however, remained stable for as long as 9 months (Fig. 3B), thus arguing against this explanation. Low levels of transgenically expressed human leptin (supposedly from birth) have also been shown to normalize physiology in mice having no endogenous mouse leptin. Such mice were somewhat heavier than nontransgenic lean controls, but they were fertile and had normal insulin and corticosterone levels (Ioffe et al. 1998). While the above-mentioned observations support the notion that even low leptin levels are practically sufficient for normal physiology, we still cannot exclude a possibility that some developmental imprinting makes Lepob/Lepob mice permanently hypersensitive to leptin (Bouret et al. 2004).
The majority of abnormalities characteristic of ob/ob mice, such as extreme obesity and insulin resistance, were readily preventable in 40-day-old and reversible in 13-month-old mice. Similar, very low leptin levels (see Figs 2 and 6) were effective in restoring insulin sensitivity in young and in old ob/ob mice. This observation suggests that the lack of life-long exposure to leptin prevented the development of leptin resistance with age in ob/ob mice in contrast to some other rodents (Ma et al. 2002). The lack of leptin at early ages, up to 40 days, may, however, result in some permanent developmental defects, including compromised lactation.
A WAT transplantation technique similar to the one described in this study may be used for assessing the relative contribution of adipocyte-derived endocrine factors to metabolic regulation. For example, it has been shown that in lipodystropic mice, insulin resistance and hyperglycemia may be substantially alleviated by transplanting normal, but not leptin-deficient WAT (Gavrilova et al. 2000, Colombo et al. 2002). The technique may also be used for detecting novel adipocyte-derived endocrine factors, as has recently been reported for acyl CoA:diacylglycerol acyltransferase 1 knock-out mice (Chen et al. 2003).
Our results extend the applicability of WAT transplantation from treating lipodystropy to preventing and treating the Lepob/Lepob syndrome. Thus, the excessive adiposity per se does not seem to have an adverse effect on the effectiveness of WAT transplantation to cure adipose-related endocrine abnormalities. WAT transplantation may, in the future, become a better option than a hormone replacement therapy because it provides the advantage of continuous hormone release without the disadvantages of daily injections and the repeated monitoring and adjusting of an exogenous hormone.
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Acknowledgements |
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References |
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Received in final form 26 March 2005
Accepted 28 April 2005
Made available online as an Accepted Preprint 9 May 2005
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