Targeted Overexpression of Leptin to Keratinocytes in Transgenic Mice Results in Lack of Skin Phenotype but Induction of Early Leptin Resist
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《内分泌学杂志》
Epithelial Damage, Repair, and Tissue Engineering Unit (L.R., M.D.R., A.R., J.L.J., M.A.P., F.L.), Centro de Investigaciones Energeticas Medioambientales y Tecnológicas, and Fundación Marcelino Botín, 28040, Madrid, Spain
Department of Animal Pathology (A.B.), Veterinary School, University of Santiago de Compostela, 27002-Lugo, Spain
Abstract
The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopic epidermal protein overexpression need to be carefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratin K5 gene regulatory sequences. Histopathological analysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocyte-derived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3–4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance.
Introduction
LEPTIN, A MAJOR adipocyte-derived hormone, is involved in the regulation of food intake and energy expenditure (1, 2). Leptin reduces food intake by up-regulating anorexigenic neuropeptides, such as a MSH, and down-regulating orexigenic factors. Other leptin actions include those related to glucose metabolism, bone physiology, angiogenesis, and immunocompetence (3). Treatment of leptin deficiencies, occurring in both rare inherited obese and lipodystrophic patients, requires daily injections of the recombinant protein (4, 5, 6, 7, 8). Somatic gene therapy, including cutaneous gene therapy, appears a promising alternative approach for the delivery of leptin and other proteins with systemic action (9). In fact, we have provided proof of leptin deficiency correction in ob/ob mice through the use of leptin-secreting skin grafts generated from either donor transgenic mice overexpressing leptin in epidermal keratinocytes or, in a short-term manner, retrovirus-transduced human bioengineered skin (10). To achieve a safe correction of leptin deficiencies through the use of human skin as a bioreactor, the possible deleterious effects associated with overexpressing leptin in keratinocytes deserve careful assessment. In fact, the long-term constitutive effects of ectopic leptin expression by keratinocytes on epidermal physiology and the potential metabolic abnormalities associated with high levels of keratinocyte-derived circulating leptin remain to be determined. Previous studies showing leptin effects on skin involve only acute cutaneous responses to leptin including those related to wound healing improvement (11, 12). Moreover, direct proliferative effects of leptin on mouse and human keratinocytes have also been reported (13, 14).
Despite leptin’s anorexigenic action, circulating leptin levels are usually and paradoxically elevated in obese subjects (15, 16), although the basis for such leptin resistance is still poorly understood. Targeted overexpression of leptin in various transgenic mouse models has been previously reported (17, 18). Although serum leptin levels vary among the different transgenic mice, a skinny phenotype associated with low food intake and enhanced energy expenditure is the hallmark of these animals. Development of spontaneous leptin resistance in these transgenics has either not been reported [for the mice generated by Nakao and coworkers (17)] or was shown to occur at an old age in the case of those reported by Chehab and colleagues (18). However, when the latter are given a high-fat diet challenge, a leptin-resistant, obese state becomes accelerated (19).
In this study we assessed both the epidermal and metabolic effects of long-term keratinocyte-derived leptin overexpression in transgenic mice. Histopathological and marker studies revealed the lack of any distinguishable epidermal phenotype. However, the dramatic increase in serum concentration of keratinocyte-derived leptin induced early metabolic changes including leptin insensitivity and insulin resistance without severe concomitant obesity. Other leptin resistance-associated phenotypic manifestations included organomegaly, impaired wound healing, and increased bone density.
Materials and Methods
Generation of K5-leptin transgenic mice and animal care
Generation of transgenic mice on C57BL6J/DBA2J background has previously been reported (10). The presence of the transgene in founders and F1 mice was detected by EcoRI digestion of tail genomic DNA followed by Southern blot analysis, using the 5.2-kb fragment corresponding to the bovine keratin K5 promoter as a probe. Transgenic mice were used as hemizygous. Animals were maintained on autoclaved standard rodent chow (A04, Panlab, Barcelona, Spain) on a 12-h light, 12-h dark cycle. All experimental procedures were carried out according to European and Spanish laws and regulations (European convention 123, on the use and protection of vertebrate mammals used in experimentation and other scientific purposes. Spanish R.D 223/88 and O.M. 13-10-89 of the Ministry of Agricultural, Food, and Fisheries on the protection and use of animals in scientific research and internal biosafety and bioethics guidelines).
Northern blot analysis
Mouse tissues and organs were harvested immediately after dissection and were processed for total RNA extraction by the Trizol method (Gibco BRL, Life Technologies, Gaithersburg, MD) or stored at –70 C for later RNA extraction. Twenty micrograms of RNA per lane were fractionated on denaturing formaldehyde gels according to standard procedures. Northern blots were hybridized with a mouse leptin cDNA probe (10).
Skin phenotype analysis
Skin samples from newborn and adult mice were fixed either in formalin or 70% ethanol and embedded in paraffin. Routine hematoxylin and eosin (H&E) staining was performed on formalin-fixed sections. Mouse epidermal differentiation markers (keratins K5, K1, K6, and loricrin) were analyzed in ethanol fixed back-skin sections by immunoperoxidase staining using specific antibodies: mouse K5, mouse K1, mouse K6, and Loricrin (Covance, Vienna, VA). 5-Bromo-2'-deoxyuridine (BrdU) labeling and quantification were performed as previously described (20).
Body weight and food intake measurements
Body weight was measured every other day at the same time of day. For feeding studies, animals were placed in individual cages; a preweighed amount of standard rodent chow diet (A04, Panlab) was given per animal. The weight of food consumed after at least 10 d, taken at the same time of day, divided by the number of days provided a measure of food consumption per day.
Glucose and insulin tolerance tests
For the glucose tolerance test, after either a 6-h (for K5-leptin-sensitive mice and the control littermates) or overnight (for K5-leptin-resistant mice and control littermates) fast, transgenic and nontransgenic littermates were treated with an ip injection of 1.5 mg glucose per gram body weight. For the insulin tolerance test, nonfasting mice were injected ip with 1.5 mU per gram body weight of human regular insulin (Eli Lilly & Co., Indianapolis, IN). Blood glucose was sampled from the mouse tail vein immediately before and 15, 30, 60, 90, and 120 min after the injection.
In vivo nuclear magnetic resonance (NMR) determination of water/fat content.
NMR imaging experiments were performed using a BIOSPEC BMT 47/40 (Bruker, Ettlingen, Germany), operating at 4.7 Teslas, equipped with a 12-cm actively shielded gradient system. Animals were anesthetized with Avertin and placed in prone position inside a home-built birdcage coil with an inner diameter of 4 cm and a length of 8 cm. First global shimming was performed until the line width of the water peak was about 100 Hz. After the homogeneity was adjusted, a global spectrum of an unique scan was acquired and processed using the XWIN-NMR 2.6 program (Bruker). Spin-echo scout images in axial, sagittal, and coronal direction were also acquired. Water and fat peaks were plotted according to Breslow et al. (21). The NMR signal from water was well separated from that of the lipids (fat) signal. Fat and nonfat body mass were calculated by multiplying corrected body weight by the fractional volume represented by the water and lipid peak. Corrected body weight excludes gastrointestinal tract contents (estimated at 1 g) and bone and other tissue not detected by NMR (estimated at 5 and 10% for ob/ob and control animals, respectively) (22).
Glucose, leptin, insulin, adiponectin, and triglyceride measurements
Blood samples were collected by tail incision. Blood was drawn in the early to middle phase of the light cycle in the nonfasted state except when otherwise indicated in the text. Blood glucose levels were determined using an Accutrend sensor comfort digital monitor (Roche Diagnostics, S.L., Indianapolis, IN). Serum murine leptin, adiponectin and resistin levels were determined using the Quantikine murine leptin or adiponectin ELISA kits (R&D Systems, Minneapolis, MN) following manufacturers’ instructions with minor modifications. Serum samples were assayed at 1:5–1:10 dilutions. Serum insulin levels were determined using the mouse insulin ELISA kit (ELIS-7537, Peninsula Laboratories, Inc., Belmont, CA) following manufacturers’ instructions with minor modifications. Plasma triglycerides were determined using the triglyceride GPO-trinder kit (no. 337, Sigma Diagnostics, St. Louis, MO).
Necropsy and fat histochemistry
Necropsy was performed on transgenic and nontransgenic littermates. Photographs were taken to compare organ size. Samples of skeletal muscle (biceps femoris) and liver were frozen in Tissue-Tek OTC compound (Sakura, Europe B.V., Zoeterwoude, The Netherlands); sections 7 μm thick were loaded on poly-L-lysine-coated slides and stained with Sudan IV and counterstained with hematoxylin (Herxheimer’s technique) to show phospholipids in red.
Wound-healing studies
Analysis of wound healing in K5-leptin mice was performed according to Escamez et al. (20) with minor modifications. Briefly, mice were injured using 4-mm biopsy-punches (Stiefel Lab, Madrid, Spain). At 6 d post wounding, mice were killed by CO2 asphyxiation. Rectangular samples of skin containing the wounds in the center were harvested and fixed in 3.7% formaldehyde solution. After fixation the skin biopsy was embedded in paraffin. Serial 4-μm cross-sections were obtained and H&E stained. The whole sample was sectioned to determine the center of the wound and adequately monitor the healing process. The percent of reepithelialization across each wound site was measured by light microscopy using a reticule to measure the proportion of each wound that was covered by neoepidermis in relation to the entire wound length. The reepithelialization percentage was calculated by the formula: 100 x [(wound diameter-epidermal gap)/wound diameter]. The epidermal gap is the distance between opposite epithelial tongues.
Bone density studies
Contact x-rays were performed on dissected lumbar vertebrae and forelimb in wild-type and transgenic mice using a Diagnost 90S (Philips, Madrid, Spain; x-ray pictures were taken on mammography films (AD Mammo Fine; Fuji, Tokyo, Japan) at 150 kV, 1000 mA. Undecalcified 7-μm-thick sections of lumbar vertebrae were stained with the von Kossa technique to show mineralized bone in black.
Statistical analysis
Statistical analysis of the data was assessed by Student’s t test according to the recommendations of Statgraphics Plus 5.0 applications (StatPoint, Herndon, VA) used for data management. All values are expressed as the mean ± SE. Differences with a P < 0.05 were considered statistically significant. Weight and food intake studies were performed in a minimum of four animals (n 4) per group unless specified.
Results
High serum leptin levels from an epidermal source
With the aim of delivering leptin from the epidermis (10), we generated transgenic mice in which leptin expression was driven by the bovine keratin K5 gene regulatory sequences. The keratin K5-based gene construct (Fig 1A), which confers epidermal basal cell expression specificity, was similar to that used for other transgenics developed in our laboratory (23, 24, 25). Two fertile male transgenic founders, L1 and L2, transmitted the transgene to one of eight and one of 10 of their progeny, respectively, suggesting mosaic transgene integration. Southern blot analysis of genomic DNA from both founders and F1 transgenic progenies confirmed mosaicism (Fig. 1B). In fact, both founders contained an average of 10 transgene copies/cell, whereas L1 and L2 F1 transgenic mice contained 20 and 30 copies of the transgene, respectively (Fig. 1B). Transgene expression specificity was assessed by Northern blot analysis of RNA from selected organs. The presence of a 1.6-kb corresponding to transgenic leptin mRNA was detected, as anticipated, only in transgenic mouse skin (Fig. 1C) but not in muscle, liver, or kidney, in which the keratin K5 gene promoter is not active. A weak 4.5-kb band corresponding to the endogenous leptin mRNA, probably due to traces of adipose tissue, was present only in samples from control animals. At the protein level, leptin was measured by ELISA in both primary keratinocyte culture supernatants (10) and serum (10 and Fig. 1D). Levels of 80 ± 8 and 110 ± 21 ng/ml were found in animals from lines L1 and L2, respectively. These leptin serum concentrations are higher than those previously reported for other leptin transgenic mice (17, 18). Comparison between serum leptin levels from L1 and L2 founders, and their transgenic offspring, also confirmed the mosaicism of founder mice at the protein level, although the increase in serum leptin levels in transgenic F1 mice was not proportional to the increase in transgene copy number (data not shown).
Leptin overexpression does not affect epidermal homeostasis
To assess whether leptin overexpression by keratinocytes may be affecting skin homeostasis, we performed a routine histological examination of skin from K5-leptin transgenics and wild-type littermates at different ages. H&E staining of back skin sections did not show apparent differences in interfollicular epidermis or epidermal appendages (hair follicle and sweat glands) (Fig. 2, A and B, H&E panels). Because keratin K5 expression begins in the mouse embryo at d 11.5 (23), it would be expected that any direct effect due to continuous leptin overexpression driven by the K5 promoter was visible at birth. However, the only clear differential feature, namely the absence of paniculum adiposum in the dermis, was detected only in adult mice (Fig. 2B, K5-leptin panels), reflecting at the cutaneous level, the generalized fat tissue depletion secondary to the systemic leptin action (see below). To assess for putative subtle changes in epidermal tissue, undetectable at the H&E level, we examined the expression of epidermal cell differentiation markers in both newborn and adult K5-leptin mice and control littermates. Thus, immunoperoxidase staining for keratins K5 and K1 revealed the normal basal and suprabasal expression pattern, respectively (Fig. 2, A and B, K1 and K5 panels). Also, keratin K6 expression, a sensitive marker of hyperproliferative situations in interfollicular epidermis, was restricted to its normal localization in the inner root sheath of hair follicles (Fig. 2, A and B, K6 panels). The expression pattern of loricrin, a late differentiation marker was also similar for both K5-leptin and control mice, although more widely expressed in newborn skin, consistent with the normal acanthotic phenotype of neonatal mouse epidermis (Fig. 2, A and B, loricrin panels). The proliferative status of K5-leptin skin was studied through BrdU incorporation. Quantification of BrdU-labeled cells in interfollicular epidermis detected by immunoperoxidase (Fig. 2C, newborn and adult mice panels) showed no statistically significant differences between wild-type and K5-leptin mice at either newborn or adult age (Fig. 2D).
Thinness and increased glucose metabolism in young adult leptin-sensitive K5-leptin mice
Body weight curves for mice of L1 transgenic line are shown in Fig. 3A. At birth both L1 and L2 transgenic mice were indistinguishable from nontransgenic littermates; however, weight curves for control and transgenic groups clearly deviated from each other before weaning (Fig. 3A). At 3 wk of age, transgenic animals weighed about one fourth less than control littermates (males: 24 ± 1.8%; females: 21.4 ± 5.22%) and could be clearly distinguished from controls, not only by their thinness but also by the presence of a marked depression in the cervical area due in turn to an almost complete lack of brown adipose tissue in the interscapular region (Fig. 3B, arrow). As predicted, white adipose tissue was also markedly reduced in the sc pad and other sites (Figs. 2B and 3B). Quantitative fat and lean mass determination using NMR analysis (Fig. 3, D and E) in 6-wk-old animals revealed a 4-fold decrease in adipose tissue mass with respect to control littermates (0.4 ± 0.1 vs. 1.7 ± 0.2 g; P = 0.019). Analysis at 8 wk of age demonstrated a slight, although significant, reduction in food intake in transgenic animals, compared with control littermates (Fig. 3C). Transgenic mouse mortality was particularly severe in line L2 and occurred early after weaning affecting both male and female animals. Conversely, in line L1, mortality occurred mostly in female transgenic mice. Given the high mortality rate of line L2, detailed studies were performed only in line L1, although both transgenic lines shared identical behavior, thus ruling out phenotypic effects due to transgene integration. The finding that some animals entered into convulsions at the end of the light cycle suggested a severe hypoglycemic state. In fact, animals were rescued from convulsions by ip glucose injections, and mortality was highly reduced (particularly in line L1) thereafter by adding glucose [1.5% (wt/vol)] to the drinking water. The striking increase in glucose metabolism was also evident because 8-wk-old transgenic animals did not survive an overnight fasting. Thus, the glucose tolerance tests had to be performed only after a 6-h fasting, which resulted in basal blood glucose values of 28 ± 8.1 mg/dl, compared with 79 ± 4.5 mg/dl in control littermates (Fig. 3, F and G).
Development of an early leptin-insensitivity state in K5-leptin mice
Although several features of young adult K5-leptin transgenic mice resembled those of the skinny mouse phenotype previously described by Nakao’s group (17), it was noticeable early on that, in contrast to that reported for these transgenic mice, a gradual body weight gain occurred in the standard diet-fed K5-leptin transgenic mice (Fig. 4A). By 3 months of age, most male transgenic mice had reached control body weight values. By 6 months, mean transgenic mice body weight exceeded that of the controls by only 14% (Fig. 4A and data not shown). To determine whether such a weight gain was due to changes in feeding behavior, food intake was studied. At 6 months of age, transgenic mice reached food consumption values similar to those of control littermates (Fig. 4B), indicating moderate leptin insensitivity in relation to the weight-reducing effect observed at a younger age. Food intake values remained constant at older ages, even for 9-month-old mice (data not shown). NMR analysis of whole-body fat and lean mass content showed a 19% increase (7.9 ± 2.1 vs. 6.4 ± 0, P = 0.423, n = 4) in adiposity in aged (9–11 months old) K5-leptin mice and a similar fat distribution (Fig. 4C).
Whereas weight-gain increase occurred early in males, i.e. at 12–14 wk of age, the same phenomenon was observed only in female K5-leptin mice at 22–25 wk of age (a 10- to 11-wk delay in relation to males). Thus, by 23–24 wk of age, the average weight of K5-leptin female mice was similar to that of control littermates (Fig. 4D). Remarkably, however, pregnancy (including offspring delivery and a 2-wk recovery time) during the leptin-sensitive stage completely blunted the delay needed to develop leptin insensitivity in female K5-leptin mice Thus, whereas similar body weight was observed in 14-wk-old female K5-leptin transgenics (when compared with control littermates after pregnancy), a significantly lower body weight was still found in virgin K5-leptin mice at the same age (Fig. 4F).
Because insulin resistance usually coexists with leptin insensitivity in obesity, we thus speculated that the moderate leptin insensitivity observed in the K5-lep-resistant mice at the level of weight-reducing action might also affect glucose metabolism. In fact, when glucose tolerance tests were performed, impairment of glucose use was found in transgenic mice (males and females) older than 6 months (Fig. 5A). Moreover, insulin resistance occurred in all transgenic mice as revealed by the insulin tolerance tests (Fig. 5B). However, serum insulin levels ranged from moderately high to very high levels (Fig. 5C). The latter was closely similar to those found in the ob/ob mice (data not shown).
To assess whether leptin insensitivity in aged K5-leptin mice was not a consequence of transgene expression loss, we determined serum leptin concentrations in both 6- and 9-month-old animals. Consistent with results in other K5-driven transgenic models (23, 24, 25), we found that keratinocyte-derived leptin was contributing to the sustained hyperleptinemia at all ages (Fig. 5D).
To gain further insight on the possible interplay between different adipokines regarding leptin sensitive and resistant stages, we also measured both adiponectin and resistin serum levels in K5-leptin animals at different ages. As previously described (26, 27), serum adiponectin levels in K5-leptin mice and controls showed sexually dimorphism. Thus, young (6 wk old) K5-leptin mice showed a reduction in serum adiponectin that was more pronounced in male animals (females: 12.2 ± 0.7 vs. 9.9 ± 1.2 μg/ml, P < 0.05; males: 6.3 ± 1.2 vs. 4.0 ± 0.7 μg/ml, P < 0.05) (Fig. 5E). However, these changes were blunted in aged K5-leptin (10 months old) mice. These results are consistent with those reported recently by Ueno et al. (28) in a leptin (overexpression) gene therapy setting.
In contrast, serum resistin levels exhibited a highly variable behavior and did not show any consistent change in either young or aged K5-leptin mice in comparison with the values of their respective controls (data not shown).
Organ, fat changes, and wound healing impairment associated with leptin/insulin resistance in resistant K5-leptin mice
Necropsy study of animals with the most severe hyperinsulinemia showed marked organomegaly affecting, particularly, the heart, male sexual glands, and liver (Fig. 6A). Also, pancreatic islets were remarkably enlarged to such an extent that they could be grossly seen by the naked eye (Fig 6A, white arrows, and data not shown). To test whether the leptin resistance state could affect also fat metabolism and fat deposition, both plasma triglyceride analysis and Sudan IV staining of muscle sections were performed. Remarkably, resistant K5-leptin mice showed reduced (although not statistically significant) levels of plasma triglycerides, compared with control littermates (Fig. 6B). Consistently, Sudan IV staining revealed a slight decrease in the number and size of phospholipid droplets in K5-leptin mice, compared with the wild-type littermates (Fig. 6C). Both wild-type and K5-leptin mice showed most of the fat droplets located at the periphery of the muscular fiber, in striking contrast to the intracellular location of fat observed in the ob/ob mice (Fig. 6C).
Impaired wound healing response is a hallmark of leptin-deficient ob/ob mice (11). Considering that severe leptin resistance could be regarded, in a relaxed way, as a deficiency, we wanted to assess the skin wound healing response of aged (5 months old) K5-leptin mice.
Animals with a moderate insulin resistance were chosen for this study to minimize the effects of impaired glucose use on wound closure. Analysis at 6 d after an excisional wound, showed a degree of healing impairment similar to that of ob/ob mice (Fig. 7A). Histological examination and quantitative measure of reepithelialization, at the center of the wounds, showed a 69% respect to control littermates (Fig. 7, B and C).
Changes in bone density associated with leptin resistance in K5-leptin transgenic mice
Considering the role of leptin in bone physiology (29, 30, 31), we finally tested whether manifestations of leptin resistance in 9-month-old K5-leptin mice would include also changes at the level of bone architecture. In fact, x-ray analysis and von Kossa staining of undecalcified bones revealed, respectively, a highly increased bone density and augmented mineralization in all leptin-resistant K5-leptin male and female mice analyzed (Fig. 8). These findings parallel the observed effects in the bones of ob/ob mice as a consequence of leptin absence (Fig. 8) (25, 26, 27).
Discussion
With the purpose of delivering leptin from an epidermal source for cutaneous gene therapy of leptin deficiencies, we generated transgenic mice-producing leptin ectopically from keratinocytes via the expression of a mouse leptin cDNA under the control of keratin K5 gene regulatory sequences (10). Thus, small skin pieces taken from these animals and grafted to immunodeficient ob/ob mouse recipients were sufficient to correct leptin deficiency (10). Because we seek long-term correction of leptin deficiencies using human keratinocyte grafts, we decided to study how long-term ectopic leptin expression from keratinocytes could affect epidermal homeostasis and characterize the metabolic effects of keratinocyte-derived circulating leptin. Histological and molecular assessment of skin differentiation and proliferation in K5-leptin mice at both newborn and adult age support the assertion of leptin as an innocuous factor for epidermal cells. Previous reports showed acute beneficial effects of leptin in skin undergoing wound healing (11, 12). Although not carefully addressed, these studies suggested a direct response of keratinocytes to leptin that implies signal transduction from a leptin receptor in the keratinocytes rather than a systemic effect. In fact, Frank and colleagues (13, 14) have shown ObR protein expression in keratinocytes. Assuming a putative leptin signaling in keratinocytes, the fact that we do not detect effects of leptin overexpression on epidermis may be associated with the lack of skin challenge. In fact, transgenic mice overexpressing an activated form of signal transducer and activator of transcription-3, a signal transducer of leptin action, in the epidermis did not exhibit any overt phenotype. However, either a wound healing or tumor promotion challenge in these animal results in the development of a psoriasiform phenotype (32). Work by Sierra-Honigmann et al. (12) showed evidence of dramatic leptin mRNA and protein induction from skin cells, although these authors did not clearly specify whether leptin comes from epidermal or mesenchymal skin cells. In repeated experiments we were unable to detect leptin expression from normal nontransgenic mouse keratinocytes in culture, a condition that resembles a wound healing-like situation (10) (Rico, L., unpublished observations).
Despite its anorexigenic action, plasma leptin is usually and paradoxically elevated in obese subjects (15, 16). The basis for such leptin resistance is still poorly understood. Leptin-resistant obesity frequently coexists with insulin resistance, although the molecular mechanisms underlying that link have also remained elusive. Animal models of diet-induced obesity recapitulate both leptin and insulin resistance (33). However, the presence of large fat deposits in adipose tissue and other organs and the concomitant alterations in other adipose hormones such as adiponectin and resistin may interfere with the pure effects of leptin. Previous studies with transgenic mice, in which leptin gene overexpression has been targeted to the liver (17) or adipose tissue (18), have either not reported leptin insensitivity/resistance or showed it to occur at older ages and on high-fat diet challenge, respectively. An explanation for the early appearance of leptin insensitivity/resistance in our system would probably involve the higher serum leptin levels, compared with those achieved in the other leptin transgenic mouse models. Support for this hypothesis has been provided recently from a study showing increased susceptibility for diet-induced obesity in high-fat diet-fed transgenic mice overexpressing leptin from adipose tissue (19).
Analysis of serum adiponectin, an adipokine whose decrease has been implicated in obesity-associated insulin resistance (34), revealed age-dependent changes that appeared to better correlate with changes in fat depots rather than with the state of leptin/insulin sensitivity. A decrease in serum adiponectin, as observed in young K5-leptin mice, has recently been described also as a consequence of leptin overexpression through intracerebrocranial injection of an adeno-associated viral vector encoding leptin (28). Whether adiponectin acts independently of leptin, which appears to be the driving force in the progression toward insulin resistance in our model, deserves further investigation.
Recent evidence points to a major hypothalamic role in the control of leptin and insulin interactions (35, 36, 37, 38). We are tempted to speculate that the development of insulin resistance in K5-leptin transgenic mice may occur primarily at the hypothalamic level. On the one hand, transgenic mice are not severely obese and present normal fat distribution. In this regard, it has been demonstrated that transplantation of mouse adipose tissue, from leptin-deficient ob/ob mice to lipodystrophic mice, does not correct for insulin resistance despite ameliorating hypertriglyceridemia (39). On the other hand, female K5-leptin mice develop either late leptin and insulin resistance or an early one, only in the case that pregnancy has occurred, which points to hypothalamus-pituitary axis modulation of the response. Recently, Bates et al. (40) reported that signal transducer and activator of transcription-3 signaling disruption through a gene knock-in approach abrogated leptin regulation of energy balance but had a less severe effect on fertility and glycemic control. Our results also suggest that independent regulation of such phenomena may occur. Thus, whereas moderate insensitivity to the anorexigenic effect of leptin appears to affect all K5-leptin transgenic mice in a similar way, variability occurs particularly in the severity of insulin resistance/hyperinsulinemia. In fact, our model (a F1, hybrid genetic background C57BL6J/DBA2) recapitulates the human situation in which obesity-related hyperleptinemia is accompanied by a whole spectrum of insulin sensitivity. In this regard, animals with the highest hyperinsulinemia showed marked organomegaly. Whether this is a consequence of either combined hyperleptinemia and hyperinsulinemia or hyperinsulinemia alone would possibly require the use of transgenic mice with inducible leptin overexpression, a model currently not available.
Wound healing impairment is one of the major problems associated with diabetes, and diabetic animals such as the ob/ob and db/db mice serve as models of delayed wound healing (11). In the ob/ob mice, leptin treatment restores wound healing to control values, an effect seemingly independent of improved glycemic control when a topical, acute leptin treatment is used (11). The fact that moderately hyperinsulinemic K5-leptin mice showed a remarkable wound healing impairment similar to that of ob/ob mice suggest that this leptin resistance-associated feature could be also dissociated from adiposity and/or glucose metabolism control (see below).
Recently it has also been demonstrated that leptin levels control bone mass, and the central leptin action associated with bone formation is independent of food intake control and is linked to sympathetic autonomous nervous system activation (29, 30, 31). We have found increased bone formation in leptin-resistant K5-leptin animals similar to that found in the leptin null ob/ob mice, indicating that severe leptin insensitivity occurs for this hypothalamic-sympathetic driven effect. Due to the large number of animals needed, experiments aimed at matching different leptin resistance traits have not been performed. However, it is conceivable that, based on our results and the recent evidence gathered on this issue, the different manifestations of leptin resistance (regarding weight reducing action, insulin action, fertility, wound healing, and bone metabolism) depend on discrete pathways differentially controlled by leptin. Accordingly, the K5-leptin transgenic mice may constitute an excellent model to study the mechanisms of pure leptin resistance and test pharmacological approaches to counteract global or individual leptin resistance phenotypes. Overall, we show here that, with clinical purposes in mind, high levels of keratinocyte-derived leptin appear not to affect skin functionality, although caution, regarding gene therapy vector performances and graft size, is needed to avoid excessive leptin dosage leading to resistance.
Acknowledgments
We are indebted to Blanca Duarte and Almudena Holguin for expert technical assistance. We also thank Isabel de los Santos and Pilar Hernandez for histology and Jesus Martinez Palacio for animal care.
Footnotes
This work was supported in part by Grant SAF 2004-07717 from the Spanish Ministry for Education and Science.
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; H&E, hematoxylin and eosin; NMR, nuclear magnetic resonance.
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Department of Animal Pathology (A.B.), Veterinary School, University of Santiago de Compostela, 27002-Lugo, Spain
Abstract
The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopic epidermal protein overexpression need to be carefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratin K5 gene regulatory sequences. Histopathological analysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocyte-derived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3–4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance.
Introduction
LEPTIN, A MAJOR adipocyte-derived hormone, is involved in the regulation of food intake and energy expenditure (1, 2). Leptin reduces food intake by up-regulating anorexigenic neuropeptides, such as a MSH, and down-regulating orexigenic factors. Other leptin actions include those related to glucose metabolism, bone physiology, angiogenesis, and immunocompetence (3). Treatment of leptin deficiencies, occurring in both rare inherited obese and lipodystrophic patients, requires daily injections of the recombinant protein (4, 5, 6, 7, 8). Somatic gene therapy, including cutaneous gene therapy, appears a promising alternative approach for the delivery of leptin and other proteins with systemic action (9). In fact, we have provided proof of leptin deficiency correction in ob/ob mice through the use of leptin-secreting skin grafts generated from either donor transgenic mice overexpressing leptin in epidermal keratinocytes or, in a short-term manner, retrovirus-transduced human bioengineered skin (10). To achieve a safe correction of leptin deficiencies through the use of human skin as a bioreactor, the possible deleterious effects associated with overexpressing leptin in keratinocytes deserve careful assessment. In fact, the long-term constitutive effects of ectopic leptin expression by keratinocytes on epidermal physiology and the potential metabolic abnormalities associated with high levels of keratinocyte-derived circulating leptin remain to be determined. Previous studies showing leptin effects on skin involve only acute cutaneous responses to leptin including those related to wound healing improvement (11, 12). Moreover, direct proliferative effects of leptin on mouse and human keratinocytes have also been reported (13, 14).
Despite leptin’s anorexigenic action, circulating leptin levels are usually and paradoxically elevated in obese subjects (15, 16), although the basis for such leptin resistance is still poorly understood. Targeted overexpression of leptin in various transgenic mouse models has been previously reported (17, 18). Although serum leptin levels vary among the different transgenic mice, a skinny phenotype associated with low food intake and enhanced energy expenditure is the hallmark of these animals. Development of spontaneous leptin resistance in these transgenics has either not been reported [for the mice generated by Nakao and coworkers (17)] or was shown to occur at an old age in the case of those reported by Chehab and colleagues (18). However, when the latter are given a high-fat diet challenge, a leptin-resistant, obese state becomes accelerated (19).
In this study we assessed both the epidermal and metabolic effects of long-term keratinocyte-derived leptin overexpression in transgenic mice. Histopathological and marker studies revealed the lack of any distinguishable epidermal phenotype. However, the dramatic increase in serum concentration of keratinocyte-derived leptin induced early metabolic changes including leptin insensitivity and insulin resistance without severe concomitant obesity. Other leptin resistance-associated phenotypic manifestations included organomegaly, impaired wound healing, and increased bone density.
Materials and Methods
Generation of K5-leptin transgenic mice and animal care
Generation of transgenic mice on C57BL6J/DBA2J background has previously been reported (10). The presence of the transgene in founders and F1 mice was detected by EcoRI digestion of tail genomic DNA followed by Southern blot analysis, using the 5.2-kb fragment corresponding to the bovine keratin K5 promoter as a probe. Transgenic mice were used as hemizygous. Animals were maintained on autoclaved standard rodent chow (A04, Panlab, Barcelona, Spain) on a 12-h light, 12-h dark cycle. All experimental procedures were carried out according to European and Spanish laws and regulations (European convention 123, on the use and protection of vertebrate mammals used in experimentation and other scientific purposes. Spanish R.D 223/88 and O.M. 13-10-89 of the Ministry of Agricultural, Food, and Fisheries on the protection and use of animals in scientific research and internal biosafety and bioethics guidelines).
Northern blot analysis
Mouse tissues and organs were harvested immediately after dissection and were processed for total RNA extraction by the Trizol method (Gibco BRL, Life Technologies, Gaithersburg, MD) or stored at –70 C for later RNA extraction. Twenty micrograms of RNA per lane were fractionated on denaturing formaldehyde gels according to standard procedures. Northern blots were hybridized with a mouse leptin cDNA probe (10).
Skin phenotype analysis
Skin samples from newborn and adult mice were fixed either in formalin or 70% ethanol and embedded in paraffin. Routine hematoxylin and eosin (H&E) staining was performed on formalin-fixed sections. Mouse epidermal differentiation markers (keratins K5, K1, K6, and loricrin) were analyzed in ethanol fixed back-skin sections by immunoperoxidase staining using specific antibodies: mouse K5, mouse K1, mouse K6, and Loricrin (Covance, Vienna, VA). 5-Bromo-2'-deoxyuridine (BrdU) labeling and quantification were performed as previously described (20).
Body weight and food intake measurements
Body weight was measured every other day at the same time of day. For feeding studies, animals were placed in individual cages; a preweighed amount of standard rodent chow diet (A04, Panlab) was given per animal. The weight of food consumed after at least 10 d, taken at the same time of day, divided by the number of days provided a measure of food consumption per day.
Glucose and insulin tolerance tests
For the glucose tolerance test, after either a 6-h (for K5-leptin-sensitive mice and the control littermates) or overnight (for K5-leptin-resistant mice and control littermates) fast, transgenic and nontransgenic littermates were treated with an ip injection of 1.5 mg glucose per gram body weight. For the insulin tolerance test, nonfasting mice were injected ip with 1.5 mU per gram body weight of human regular insulin (Eli Lilly & Co., Indianapolis, IN). Blood glucose was sampled from the mouse tail vein immediately before and 15, 30, 60, 90, and 120 min after the injection.
In vivo nuclear magnetic resonance (NMR) determination of water/fat content.
NMR imaging experiments were performed using a BIOSPEC BMT 47/40 (Bruker, Ettlingen, Germany), operating at 4.7 Teslas, equipped with a 12-cm actively shielded gradient system. Animals were anesthetized with Avertin and placed in prone position inside a home-built birdcage coil with an inner diameter of 4 cm and a length of 8 cm. First global shimming was performed until the line width of the water peak was about 100 Hz. After the homogeneity was adjusted, a global spectrum of an unique scan was acquired and processed using the XWIN-NMR 2.6 program (Bruker). Spin-echo scout images in axial, sagittal, and coronal direction were also acquired. Water and fat peaks were plotted according to Breslow et al. (21). The NMR signal from water was well separated from that of the lipids (fat) signal. Fat and nonfat body mass were calculated by multiplying corrected body weight by the fractional volume represented by the water and lipid peak. Corrected body weight excludes gastrointestinal tract contents (estimated at 1 g) and bone and other tissue not detected by NMR (estimated at 5 and 10% for ob/ob and control animals, respectively) (22).
Glucose, leptin, insulin, adiponectin, and triglyceride measurements
Blood samples were collected by tail incision. Blood was drawn in the early to middle phase of the light cycle in the nonfasted state except when otherwise indicated in the text. Blood glucose levels were determined using an Accutrend sensor comfort digital monitor (Roche Diagnostics, S.L., Indianapolis, IN). Serum murine leptin, adiponectin and resistin levels were determined using the Quantikine murine leptin or adiponectin ELISA kits (R&D Systems, Minneapolis, MN) following manufacturers’ instructions with minor modifications. Serum samples were assayed at 1:5–1:10 dilutions. Serum insulin levels were determined using the mouse insulin ELISA kit (ELIS-7537, Peninsula Laboratories, Inc., Belmont, CA) following manufacturers’ instructions with minor modifications. Plasma triglycerides were determined using the triglyceride GPO-trinder kit (no. 337, Sigma Diagnostics, St. Louis, MO).
Necropsy and fat histochemistry
Necropsy was performed on transgenic and nontransgenic littermates. Photographs were taken to compare organ size. Samples of skeletal muscle (biceps femoris) and liver were frozen in Tissue-Tek OTC compound (Sakura, Europe B.V., Zoeterwoude, The Netherlands); sections 7 μm thick were loaded on poly-L-lysine-coated slides and stained with Sudan IV and counterstained with hematoxylin (Herxheimer’s technique) to show phospholipids in red.
Wound-healing studies
Analysis of wound healing in K5-leptin mice was performed according to Escamez et al. (20) with minor modifications. Briefly, mice were injured using 4-mm biopsy-punches (Stiefel Lab, Madrid, Spain). At 6 d post wounding, mice were killed by CO2 asphyxiation. Rectangular samples of skin containing the wounds in the center were harvested and fixed in 3.7% formaldehyde solution. After fixation the skin biopsy was embedded in paraffin. Serial 4-μm cross-sections were obtained and H&E stained. The whole sample was sectioned to determine the center of the wound and adequately monitor the healing process. The percent of reepithelialization across each wound site was measured by light microscopy using a reticule to measure the proportion of each wound that was covered by neoepidermis in relation to the entire wound length. The reepithelialization percentage was calculated by the formula: 100 x [(wound diameter-epidermal gap)/wound diameter]. The epidermal gap is the distance between opposite epithelial tongues.
Bone density studies
Contact x-rays were performed on dissected lumbar vertebrae and forelimb in wild-type and transgenic mice using a Diagnost 90S (Philips, Madrid, Spain; x-ray pictures were taken on mammography films (AD Mammo Fine; Fuji, Tokyo, Japan) at 150 kV, 1000 mA. Undecalcified 7-μm-thick sections of lumbar vertebrae were stained with the von Kossa technique to show mineralized bone in black.
Statistical analysis
Statistical analysis of the data was assessed by Student’s t test according to the recommendations of Statgraphics Plus 5.0 applications (StatPoint, Herndon, VA) used for data management. All values are expressed as the mean ± SE. Differences with a P < 0.05 were considered statistically significant. Weight and food intake studies were performed in a minimum of four animals (n 4) per group unless specified.
Results
High serum leptin levels from an epidermal source
With the aim of delivering leptin from the epidermis (10), we generated transgenic mice in which leptin expression was driven by the bovine keratin K5 gene regulatory sequences. The keratin K5-based gene construct (Fig 1A), which confers epidermal basal cell expression specificity, was similar to that used for other transgenics developed in our laboratory (23, 24, 25). Two fertile male transgenic founders, L1 and L2, transmitted the transgene to one of eight and one of 10 of their progeny, respectively, suggesting mosaic transgene integration. Southern blot analysis of genomic DNA from both founders and F1 transgenic progenies confirmed mosaicism (Fig. 1B). In fact, both founders contained an average of 10 transgene copies/cell, whereas L1 and L2 F1 transgenic mice contained 20 and 30 copies of the transgene, respectively (Fig. 1B). Transgene expression specificity was assessed by Northern blot analysis of RNA from selected organs. The presence of a 1.6-kb corresponding to transgenic leptin mRNA was detected, as anticipated, only in transgenic mouse skin (Fig. 1C) but not in muscle, liver, or kidney, in which the keratin K5 gene promoter is not active. A weak 4.5-kb band corresponding to the endogenous leptin mRNA, probably due to traces of adipose tissue, was present only in samples from control animals. At the protein level, leptin was measured by ELISA in both primary keratinocyte culture supernatants (10) and serum (10 and Fig. 1D). Levels of 80 ± 8 and 110 ± 21 ng/ml were found in animals from lines L1 and L2, respectively. These leptin serum concentrations are higher than those previously reported for other leptin transgenic mice (17, 18). Comparison between serum leptin levels from L1 and L2 founders, and their transgenic offspring, also confirmed the mosaicism of founder mice at the protein level, although the increase in serum leptin levels in transgenic F1 mice was not proportional to the increase in transgene copy number (data not shown).
Leptin overexpression does not affect epidermal homeostasis
To assess whether leptin overexpression by keratinocytes may be affecting skin homeostasis, we performed a routine histological examination of skin from K5-leptin transgenics and wild-type littermates at different ages. H&E staining of back skin sections did not show apparent differences in interfollicular epidermis or epidermal appendages (hair follicle and sweat glands) (Fig. 2, A and B, H&E panels). Because keratin K5 expression begins in the mouse embryo at d 11.5 (23), it would be expected that any direct effect due to continuous leptin overexpression driven by the K5 promoter was visible at birth. However, the only clear differential feature, namely the absence of paniculum adiposum in the dermis, was detected only in adult mice (Fig. 2B, K5-leptin panels), reflecting at the cutaneous level, the generalized fat tissue depletion secondary to the systemic leptin action (see below). To assess for putative subtle changes in epidermal tissue, undetectable at the H&E level, we examined the expression of epidermal cell differentiation markers in both newborn and adult K5-leptin mice and control littermates. Thus, immunoperoxidase staining for keratins K5 and K1 revealed the normal basal and suprabasal expression pattern, respectively (Fig. 2, A and B, K1 and K5 panels). Also, keratin K6 expression, a sensitive marker of hyperproliferative situations in interfollicular epidermis, was restricted to its normal localization in the inner root sheath of hair follicles (Fig. 2, A and B, K6 panels). The expression pattern of loricrin, a late differentiation marker was also similar for both K5-leptin and control mice, although more widely expressed in newborn skin, consistent with the normal acanthotic phenotype of neonatal mouse epidermis (Fig. 2, A and B, loricrin panels). The proliferative status of K5-leptin skin was studied through BrdU incorporation. Quantification of BrdU-labeled cells in interfollicular epidermis detected by immunoperoxidase (Fig. 2C, newborn and adult mice panels) showed no statistically significant differences between wild-type and K5-leptin mice at either newborn or adult age (Fig. 2D).
Thinness and increased glucose metabolism in young adult leptin-sensitive K5-leptin mice
Body weight curves for mice of L1 transgenic line are shown in Fig. 3A. At birth both L1 and L2 transgenic mice were indistinguishable from nontransgenic littermates; however, weight curves for control and transgenic groups clearly deviated from each other before weaning (Fig. 3A). At 3 wk of age, transgenic animals weighed about one fourth less than control littermates (males: 24 ± 1.8%; females: 21.4 ± 5.22%) and could be clearly distinguished from controls, not only by their thinness but also by the presence of a marked depression in the cervical area due in turn to an almost complete lack of brown adipose tissue in the interscapular region (Fig. 3B, arrow). As predicted, white adipose tissue was also markedly reduced in the sc pad and other sites (Figs. 2B and 3B). Quantitative fat and lean mass determination using NMR analysis (Fig. 3, D and E) in 6-wk-old animals revealed a 4-fold decrease in adipose tissue mass with respect to control littermates (0.4 ± 0.1 vs. 1.7 ± 0.2 g; P = 0.019). Analysis at 8 wk of age demonstrated a slight, although significant, reduction in food intake in transgenic animals, compared with control littermates (Fig. 3C). Transgenic mouse mortality was particularly severe in line L2 and occurred early after weaning affecting both male and female animals. Conversely, in line L1, mortality occurred mostly in female transgenic mice. Given the high mortality rate of line L2, detailed studies were performed only in line L1, although both transgenic lines shared identical behavior, thus ruling out phenotypic effects due to transgene integration. The finding that some animals entered into convulsions at the end of the light cycle suggested a severe hypoglycemic state. In fact, animals were rescued from convulsions by ip glucose injections, and mortality was highly reduced (particularly in line L1) thereafter by adding glucose [1.5% (wt/vol)] to the drinking water. The striking increase in glucose metabolism was also evident because 8-wk-old transgenic animals did not survive an overnight fasting. Thus, the glucose tolerance tests had to be performed only after a 6-h fasting, which resulted in basal blood glucose values of 28 ± 8.1 mg/dl, compared with 79 ± 4.5 mg/dl in control littermates (Fig. 3, F and G).
Development of an early leptin-insensitivity state in K5-leptin mice
Although several features of young adult K5-leptin transgenic mice resembled those of the skinny mouse phenotype previously described by Nakao’s group (17), it was noticeable early on that, in contrast to that reported for these transgenic mice, a gradual body weight gain occurred in the standard diet-fed K5-leptin transgenic mice (Fig. 4A). By 3 months of age, most male transgenic mice had reached control body weight values. By 6 months, mean transgenic mice body weight exceeded that of the controls by only 14% (Fig. 4A and data not shown). To determine whether such a weight gain was due to changes in feeding behavior, food intake was studied. At 6 months of age, transgenic mice reached food consumption values similar to those of control littermates (Fig. 4B), indicating moderate leptin insensitivity in relation to the weight-reducing effect observed at a younger age. Food intake values remained constant at older ages, even for 9-month-old mice (data not shown). NMR analysis of whole-body fat and lean mass content showed a 19% increase (7.9 ± 2.1 vs. 6.4 ± 0, P = 0.423, n = 4) in adiposity in aged (9–11 months old) K5-leptin mice and a similar fat distribution (Fig. 4C).
Whereas weight-gain increase occurred early in males, i.e. at 12–14 wk of age, the same phenomenon was observed only in female K5-leptin mice at 22–25 wk of age (a 10- to 11-wk delay in relation to males). Thus, by 23–24 wk of age, the average weight of K5-leptin female mice was similar to that of control littermates (Fig. 4D). Remarkably, however, pregnancy (including offspring delivery and a 2-wk recovery time) during the leptin-sensitive stage completely blunted the delay needed to develop leptin insensitivity in female K5-leptin mice Thus, whereas similar body weight was observed in 14-wk-old female K5-leptin transgenics (when compared with control littermates after pregnancy), a significantly lower body weight was still found in virgin K5-leptin mice at the same age (Fig. 4F).
Because insulin resistance usually coexists with leptin insensitivity in obesity, we thus speculated that the moderate leptin insensitivity observed in the K5-lep-resistant mice at the level of weight-reducing action might also affect glucose metabolism. In fact, when glucose tolerance tests were performed, impairment of glucose use was found in transgenic mice (males and females) older than 6 months (Fig. 5A). Moreover, insulin resistance occurred in all transgenic mice as revealed by the insulin tolerance tests (Fig. 5B). However, serum insulin levels ranged from moderately high to very high levels (Fig. 5C). The latter was closely similar to those found in the ob/ob mice (data not shown).
To assess whether leptin insensitivity in aged K5-leptin mice was not a consequence of transgene expression loss, we determined serum leptin concentrations in both 6- and 9-month-old animals. Consistent with results in other K5-driven transgenic models (23, 24, 25), we found that keratinocyte-derived leptin was contributing to the sustained hyperleptinemia at all ages (Fig. 5D).
To gain further insight on the possible interplay between different adipokines regarding leptin sensitive and resistant stages, we also measured both adiponectin and resistin serum levels in K5-leptin animals at different ages. As previously described (26, 27), serum adiponectin levels in K5-leptin mice and controls showed sexually dimorphism. Thus, young (6 wk old) K5-leptin mice showed a reduction in serum adiponectin that was more pronounced in male animals (females: 12.2 ± 0.7 vs. 9.9 ± 1.2 μg/ml, P < 0.05; males: 6.3 ± 1.2 vs. 4.0 ± 0.7 μg/ml, P < 0.05) (Fig. 5E). However, these changes were blunted in aged K5-leptin (10 months old) mice. These results are consistent with those reported recently by Ueno et al. (28) in a leptin (overexpression) gene therapy setting.
In contrast, serum resistin levels exhibited a highly variable behavior and did not show any consistent change in either young or aged K5-leptin mice in comparison with the values of their respective controls (data not shown).
Organ, fat changes, and wound healing impairment associated with leptin/insulin resistance in resistant K5-leptin mice
Necropsy study of animals with the most severe hyperinsulinemia showed marked organomegaly affecting, particularly, the heart, male sexual glands, and liver (Fig. 6A). Also, pancreatic islets were remarkably enlarged to such an extent that they could be grossly seen by the naked eye (Fig 6A, white arrows, and data not shown). To test whether the leptin resistance state could affect also fat metabolism and fat deposition, both plasma triglyceride analysis and Sudan IV staining of muscle sections were performed. Remarkably, resistant K5-leptin mice showed reduced (although not statistically significant) levels of plasma triglycerides, compared with control littermates (Fig. 6B). Consistently, Sudan IV staining revealed a slight decrease in the number and size of phospholipid droplets in K5-leptin mice, compared with the wild-type littermates (Fig. 6C). Both wild-type and K5-leptin mice showed most of the fat droplets located at the periphery of the muscular fiber, in striking contrast to the intracellular location of fat observed in the ob/ob mice (Fig. 6C).
Impaired wound healing response is a hallmark of leptin-deficient ob/ob mice (11). Considering that severe leptin resistance could be regarded, in a relaxed way, as a deficiency, we wanted to assess the skin wound healing response of aged (5 months old) K5-leptin mice.
Animals with a moderate insulin resistance were chosen for this study to minimize the effects of impaired glucose use on wound closure. Analysis at 6 d after an excisional wound, showed a degree of healing impairment similar to that of ob/ob mice (Fig. 7A). Histological examination and quantitative measure of reepithelialization, at the center of the wounds, showed a 69% respect to control littermates (Fig. 7, B and C).
Changes in bone density associated with leptin resistance in K5-leptin transgenic mice
Considering the role of leptin in bone physiology (29, 30, 31), we finally tested whether manifestations of leptin resistance in 9-month-old K5-leptin mice would include also changes at the level of bone architecture. In fact, x-ray analysis and von Kossa staining of undecalcified bones revealed, respectively, a highly increased bone density and augmented mineralization in all leptin-resistant K5-leptin male and female mice analyzed (Fig. 8). These findings parallel the observed effects in the bones of ob/ob mice as a consequence of leptin absence (Fig. 8) (25, 26, 27).
Discussion
With the purpose of delivering leptin from an epidermal source for cutaneous gene therapy of leptin deficiencies, we generated transgenic mice-producing leptin ectopically from keratinocytes via the expression of a mouse leptin cDNA under the control of keratin K5 gene regulatory sequences (10). Thus, small skin pieces taken from these animals and grafted to immunodeficient ob/ob mouse recipients were sufficient to correct leptin deficiency (10). Because we seek long-term correction of leptin deficiencies using human keratinocyte grafts, we decided to study how long-term ectopic leptin expression from keratinocytes could affect epidermal homeostasis and characterize the metabolic effects of keratinocyte-derived circulating leptin. Histological and molecular assessment of skin differentiation and proliferation in K5-leptin mice at both newborn and adult age support the assertion of leptin as an innocuous factor for epidermal cells. Previous reports showed acute beneficial effects of leptin in skin undergoing wound healing (11, 12). Although not carefully addressed, these studies suggested a direct response of keratinocytes to leptin that implies signal transduction from a leptin receptor in the keratinocytes rather than a systemic effect. In fact, Frank and colleagues (13, 14) have shown ObR protein expression in keratinocytes. Assuming a putative leptin signaling in keratinocytes, the fact that we do not detect effects of leptin overexpression on epidermis may be associated with the lack of skin challenge. In fact, transgenic mice overexpressing an activated form of signal transducer and activator of transcription-3, a signal transducer of leptin action, in the epidermis did not exhibit any overt phenotype. However, either a wound healing or tumor promotion challenge in these animal results in the development of a psoriasiform phenotype (32). Work by Sierra-Honigmann et al. (12) showed evidence of dramatic leptin mRNA and protein induction from skin cells, although these authors did not clearly specify whether leptin comes from epidermal or mesenchymal skin cells. In repeated experiments we were unable to detect leptin expression from normal nontransgenic mouse keratinocytes in culture, a condition that resembles a wound healing-like situation (10) (Rico, L., unpublished observations).
Despite its anorexigenic action, plasma leptin is usually and paradoxically elevated in obese subjects (15, 16). The basis for such leptin resistance is still poorly understood. Leptin-resistant obesity frequently coexists with insulin resistance, although the molecular mechanisms underlying that link have also remained elusive. Animal models of diet-induced obesity recapitulate both leptin and insulin resistance (33). However, the presence of large fat deposits in adipose tissue and other organs and the concomitant alterations in other adipose hormones such as adiponectin and resistin may interfere with the pure effects of leptin. Previous studies with transgenic mice, in which leptin gene overexpression has been targeted to the liver (17) or adipose tissue (18), have either not reported leptin insensitivity/resistance or showed it to occur at older ages and on high-fat diet challenge, respectively. An explanation for the early appearance of leptin insensitivity/resistance in our system would probably involve the higher serum leptin levels, compared with those achieved in the other leptin transgenic mouse models. Support for this hypothesis has been provided recently from a study showing increased susceptibility for diet-induced obesity in high-fat diet-fed transgenic mice overexpressing leptin from adipose tissue (19).
Analysis of serum adiponectin, an adipokine whose decrease has been implicated in obesity-associated insulin resistance (34), revealed age-dependent changes that appeared to better correlate with changes in fat depots rather than with the state of leptin/insulin sensitivity. A decrease in serum adiponectin, as observed in young K5-leptin mice, has recently been described also as a consequence of leptin overexpression through intracerebrocranial injection of an adeno-associated viral vector encoding leptin (28). Whether adiponectin acts independently of leptin, which appears to be the driving force in the progression toward insulin resistance in our model, deserves further investigation.
Recent evidence points to a major hypothalamic role in the control of leptin and insulin interactions (35, 36, 37, 38). We are tempted to speculate that the development of insulin resistance in K5-leptin transgenic mice may occur primarily at the hypothalamic level. On the one hand, transgenic mice are not severely obese and present normal fat distribution. In this regard, it has been demonstrated that transplantation of mouse adipose tissue, from leptin-deficient ob/ob mice to lipodystrophic mice, does not correct for insulin resistance despite ameliorating hypertriglyceridemia (39). On the other hand, female K5-leptin mice develop either late leptin and insulin resistance or an early one, only in the case that pregnancy has occurred, which points to hypothalamus-pituitary axis modulation of the response. Recently, Bates et al. (40) reported that signal transducer and activator of transcription-3 signaling disruption through a gene knock-in approach abrogated leptin regulation of energy balance but had a less severe effect on fertility and glycemic control. Our results also suggest that independent regulation of such phenomena may occur. Thus, whereas moderate insensitivity to the anorexigenic effect of leptin appears to affect all K5-leptin transgenic mice in a similar way, variability occurs particularly in the severity of insulin resistance/hyperinsulinemia. In fact, our model (a F1, hybrid genetic background C57BL6J/DBA2) recapitulates the human situation in which obesity-related hyperleptinemia is accompanied by a whole spectrum of insulin sensitivity. In this regard, animals with the highest hyperinsulinemia showed marked organomegaly. Whether this is a consequence of either combined hyperleptinemia and hyperinsulinemia or hyperinsulinemia alone would possibly require the use of transgenic mice with inducible leptin overexpression, a model currently not available.
Wound healing impairment is one of the major problems associated with diabetes, and diabetic animals such as the ob/ob and db/db mice serve as models of delayed wound healing (11). In the ob/ob mice, leptin treatment restores wound healing to control values, an effect seemingly independent of improved glycemic control when a topical, acute leptin treatment is used (11). The fact that moderately hyperinsulinemic K5-leptin mice showed a remarkable wound healing impairment similar to that of ob/ob mice suggest that this leptin resistance-associated feature could be also dissociated from adiposity and/or glucose metabolism control (see below).
Recently it has also been demonstrated that leptin levels control bone mass, and the central leptin action associated with bone formation is independent of food intake control and is linked to sympathetic autonomous nervous system activation (29, 30, 31). We have found increased bone formation in leptin-resistant K5-leptin animals similar to that found in the leptin null ob/ob mice, indicating that severe leptin insensitivity occurs for this hypothalamic-sympathetic driven effect. Due to the large number of animals needed, experiments aimed at matching different leptin resistance traits have not been performed. However, it is conceivable that, based on our results and the recent evidence gathered on this issue, the different manifestations of leptin resistance (regarding weight reducing action, insulin action, fertility, wound healing, and bone metabolism) depend on discrete pathways differentially controlled by leptin. Accordingly, the K5-leptin transgenic mice may constitute an excellent model to study the mechanisms of pure leptin resistance and test pharmacological approaches to counteract global or individual leptin resistance phenotypes. Overall, we show here that, with clinical purposes in mind, high levels of keratinocyte-derived leptin appear not to affect skin functionality, although caution, regarding gene therapy vector performances and graft size, is needed to avoid excessive leptin dosage leading to resistance.
Acknowledgments
We are indebted to Blanca Duarte and Almudena Holguin for expert technical assistance. We also thank Isabel de los Santos and Pilar Hernandez for histology and Jesus Martinez Palacio for animal care.
Footnotes
This work was supported in part by Grant SAF 2004-07717 from the Spanish Ministry for Education and Science.
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; H&E, hematoxylin and eosin; NMR, nuclear magnetic resonance.
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