Differential Regulation of Leptin Synthesis in Rats during Short-Term Hypoxia and Short-Term Carbon Monoxide Inhalation
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内分泌学杂志 2005年第1期
Department of Pediatrics, University Erlangen-Nürnberg (U.M., C.H., I.?., I.K., I.A., W.R., J.D.), 91054 Erlangen, Germany; Institute for Physiology, University Regensburg (K.-H.H.), 93040 Regensburg, Germany; Eli Lilly & Co. (W.F.B.), 61350 Bad Homburg, Germany; and Department of Pediatrics (W.F.B.), Justus Liebig University, 35390 Giessen, Germany
Address all correspondence and requests for reprints to: Dr. Udo Mei?ner, Department of Pediatrics, University of Erlangen-Nurnberg, Loschgestrasse 15, 91054 Erlangen, Germany. E-mail: udo.meissner@kinder.imed.uni-erlangen.de.
Abstract
Leptin is a circulating hormone that is secreted primarily by adipose tissue. However, recent studies have demonstrated leptin production by other tissues, including placenta, stomach, kidney, liver, and lung, a process not only activated by stimuli such as insulin or corticosteroids, but also by hypoxia, which is mediated by the hypoxia inducible factor-1. In contrast to this fact, smokers have lower plasma leptin levels. The purpose of this study was to determine whether tissue hypoxygenation [induced by lack of oxygen] or inhalation of carbon monoxide (CO) are sufficient to up-regulate leptin in fat cells as well as in peripheral organs such as lung, liver, and kidney of rats. In hypoxic rats, leptin expression was unchanged or even reduced in adipose tissue. In contrast, in liver, kidney, and lung we observed an increase in leptin expression compared with normoxic controls, whereas plasma levels were unchanged. When animals were exposed to CO, generating a functional anemia known to activate the HIF-1-dependent transcription, a significant decrease in leptin gene expression in adipose tissue and in all organs tested was observed. Plasma leptin concentrations after CO exposure were significantly diminished compared with those in control animals. These findings suggest that tissue hypoxygenation up-regulates leptin expression in nonadipose tissue. However, this is not sufficient to raise plasma leptin levels in rats. Inhalation of CO leads to a significant decrease in leptin mRNA and protein concentration in the plasma of the animals, suggesting a negative effect of CO on leptin transcription.
Introduction
THE ob GENE product leptin was identified in the mid-1990s as a predominantly adipose tissue-derived hormone that is involved in feeding and energy balance. In addition, leptin was shown to have physiological effects on tissue proliferation, the placenta, the fetus, the immune system, and a plethora of neuroendocrine functions (1). Apart from adipose tissue, leptin is synthesized by a variety of peripheral tissues, such as placenta, gastric mucosa, mammary epithelium, heart, liver, and skeletal muscle (2, 3, 4, 5, 6, 7, 8). Nutrition-dependent control of leptin expression is regulated at least in part by insulin, as shown in rats in vivo (9) and in vitro (10, 11). In addition, corticosteroids are known to stimulate leptin synthesis (12, 13).
There is evidence that hypoxic conditions enhance leptin synthesis, a process that is mediated by a hypoxia-inducible factor-1 (HIF-1)-dependent mechanism. At least one hypoxia-responsive element, located –120 to –116 bp in the leptin promoter, has been shown to play a role in this HIF-mediated effect on transcriptional regulation (14, 15, 16, 17). In humans, circulating leptin levels are increased in subjects presenting with sleep apnea (18) and hypercapnea (19). Leptin might also be an important regulator of breathing in cases of oxygen deprivation. In contrast, some reports have demonstrated that hypoxia is able to reduce plasma leptin levels in neonatal rats (kept under constant hypoxia for 7 d) in vivo (20, 21) as well as in cultured adipocytes in vitro (22).
Functional anemia, triggered by inhalation of carbon monoxide (CO), is widely used to mimic hypoxic situations. Treatment of rats with CO inhibits the degradation of HIF-1 and causes an accumulation of HIF-1 in the nuclei (23). Moreover, CO itself is able to inhibit the activity of the HIF complex (24, 25). However, no data on the effects of CO exposure on leptin gene expression have been published to date.
Based on these observations, we set out to identify the sources of leptin in rats under hypoxic conditions and during a functional, CO-inflicted anemia by asking the following questions. 1) Do short-term hypoxia and CO inhalation have any effect on the circulating levels of leptin in vivo? 2) Furthermore, does the expression of leptin mRNA change in different types of adipose tissue? 3) Finally, how is leptin gene expression regulated in the course of hypoxia in vivo in other organs, such as lung, liver, and kidneys? Resolving these issues is of importance because it is not clear whether leptin serves as a regulating agent in the course of hypoxic conditions.
Materials and Methods
In vivo protocols
All animal experiments were reviewed and performed under the approval of the national care committee following national and European law. Age- and body weight-matched Sprague Dawley rats (purchased from Charles River, Sulzfeld, Germany; 200–250 g) with free access to food and water were used for the experiments and were treated as follows. 1) In the control group, animals received no treatment (n = 4–5/group, as indicated below); 2) in the hypoxia group, the animals were placed in a gas-tight box that was continuously supplied with a gas mixture of 8% O2-92% N2 for 3, 6, or 12 h (n = 4–5/group); 3) in the CO group, the animals were placed in a gas-tight box that was continuously supplied with air plus 0.1% CO for 3, 6, or 12 h (n = 4–5/group). It has been previously shown that the hypoxic protocols applied in this study have no effect on mRNA levels of the housekeeping genes in the organs examined in our study (26).
At the end of the experiments, the animals were killed by decapitation, and trunk blood was collected for determination of plasma leptin levels. Specimens of sc (taken from the back of the animals) and intraabdominal (mesenteric) adipose tissue, kidneys, liver, and lung were quickly removed and snap-frozen in liquid nitrogen. All organs were stored at –80 C until isolation of total RNA.
RNA isolation and RT
RNA from lung, liver, and kidney was extracted with the phenol/guanidine isothiocyanate method (27) using TRIzol reagent (Invitrogen Life Technologies, Inc., Gaithersburg, MD) as recommended by the manufacturer. In contrast to the manufacturer’s protocol, when RNA from adipose tissue was extracted, 1 ml TRIzol/100 mg adipose tissue was used. In addition, 400 μl CHCl3 were added before precipitation. DNA was digested with RQ1 deoxyribonuclease. Finally, cDNA was synthesized using a Moloney murine leukemia virus reverse transcriptase (all enzymes were purchased from Promega Corp., Karlsruhe, Germany) and was stored at –20 C until used in the PCR reaction.
Real-time PCR
To analyze the gene expression (mRNA) of leptin, the following primers were used: leptin sense, 5'-ATG ACA CCA AAA CCC TCA TCA AG-3'; leptin antisense, 5'-TGA AGT CCA AAC CGG TGA CC-3'; and leptin Taq, 5'-6-carboxy-fluorescein (FAM)-TCA ATG ACAT TTC ACA CAC GCA GTC GG-3'-6-carboxy-tetramethylrhodamine (TAMRA). The mRNAs of the hypoxanthine-guanine phosphoribosyltransferase (HPRT; sense, 5'-GCA GTA CAG CCC CAA AAT GG-3'; antisense, 5'-TCA TTA TAG TCA AGG GCA TAT CCA AC-3') and HPRT Taq (5'-FAM-TGC AAG CTT GCT GGT GAA AAG GAC CTC TC-3'-TAMRA) as well as of porphobilinogen deaminase (sense, 5'-AGA CCA TGC AGG CCA CCA-3'; antisense, 5'-CAA CCA ACT GTG GGT CAT CCT-3') and porphobilinogen deaminase Taq (5'-FAM-AGG TCC CTG TTC AGC AAG AAG ATG GTC C-3'-TAMRA) or ?-actin (sense, 5'-TGA GCT GCC TGA CGG TCA G-3'; antisense, 5'-TGC CAC AGG ATT CCA TAC CC-3'),and ?-actin Taq (5'-FAM-CAC TAT CGG CAA TGA GCG GTT CCG-3'-TAMRA) were analyzed as housekeeping genes. All assays were performed using a quantitative real-time PCR (TaqMan PCR, PerkinElmer, Palo Alto, CA), which has been previously described for the measurement of leptin gene expression in various tissues (28). All calculations were based on the Ct– method as described in detail previously (29). Briefly, using this method the amount of target mRNA is normalized to an endogenous reference sequence within the sample. In this study, calculation of leptin mRNA was performed using ?-actin, HPRT, and glyceraldehyde-3-phosphate dehydrogenase as endogenous references within the samples. The unstimulated/unincubated tissue served as the calibrator probe. The average crossing of threshold (CT) of the housekeeping gene is subtracted from the average CT of the target gene (CT); thereafter, the calibrator results are subtracted from those of the stimulus of interest (CT – CT = CT). Final mathematical operations lead to the expression of amount of target mRNA, normalized to an endogenous reference and relative to a calibrator (here unstimulated specimens): 2CT.
To exclude contamination of nonadipose tissue with adipocytes nucleic acid, all specimens used in the PCRs have been tested on the expression of adiponectin (adipose most abundant gene transcript 1, APM-1), an adipose tissue exclusive gene (30). Based on real-time PCR-based quantification, specimens from adipose tissue contained at least 250–700 (up to 2048) times more APM-1 than specimens of nonadipose tissue.
Leptin RIA
A high affinity antiserum against recombinant human leptin (provided by Dr. M. Heiman, Eli Lilly & Co., Indianapolis, IN) was produced in rabbits, using techniques previously described (31), which cross-reacted efficiently with rodent leptin. Standards and tracer were prepared by the chloramine-T method using recombinant mouse leptin (Eli Lilly & Co.). The assay buffer consisted of 0.05 mol/liter sodium phosphate (pH 7.4), 0.1 mol/liter sodium chloride, 0.1% (vol/vol) Triton X-100 (Serva, Heidelberg, Germany), and 0.05% (wt/vol) sodium azide. The assay mixture was composed of 100 μl standard or prediluted sample and 25 μl first antiserum (1:8000) in assay buffer containing 150 mg/liter rabbit -globulin (Sigma-Aldrich Corp., St. Louis, MO). After overnight incubation at room temperature, 25 μl tracer (200,000 cpm/ml assay buffer) were added, and the mixture was again incubated overnight. Separation of free and bound tracer was achieved by adding 50 μl cold 4% (wt/vol) polyethylene glycol 6000 (Serva) containing a goat antirabbit IgG serum (Diagnostic Systems Laboratories, Webster, TX). After 30 min at 4 C, the bound tracer was precipitated by centrifugation (15 min at 2000 x g), the supernatant was decanted, and the radioactivity was measured in a -counter. Maximum binding of tracer was approximately 35%. Half-maximum binding occurred at about 50 pg/ml. Sensitivity with undiluted samples was 3 pg/ml. The intra- and interassay coefficients of variation were 3.1% and 9.2%, respectively (n = 10). Excellent parallelism was obtained with serial dilutions of rat serum or plasma and also with dilutions of recombinant rat leptin (Eli Lilly & Co.). The cross-reactivity of rat leptin was 99.9%, indicating that this heterologous assay gives correct absolute values for rat leptin.
Statistical analysis
All values are presented as the mean ± SEM. Two-tailed Mann-Whitney tests with confidence intervals of 99% were performed using GraphPad PRISM (GraphPad, San Diego, CA). The threshold for significance was set at P < 0.05.
Results
Exposure of the animals to hypoxia for 3–12 h did not significantly alter leptin gene expression in sc adipose tissue (Fig. 1A). Leptin mRNA in mesenteric adipose tissue, however, was significantly reduced by hypoxia after 6 h (Fig. 1A) and by CO exposure after 3 and 6 h (Fig. 1B). The exposure of the rats to CO reduced the amount of mRNA to 23% compared with normoxic controls.
FIG. 1. Leptin gene expression in sc and mesenteric adipose tissue after short-term hypoxia. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxic conditions (8% O2; A) or CO (0.1% CO; B). After the period indicated, tissue specimens (two samples from sc and two from mesenteric adipose tissue) were collected and subjected to cDNA synthesis. The amount of leptin mRNA, relative to the levels of two housekeeping genes, was measured to calculate the relative change in induction of leptin mRNA; both housekeeping genes revealed similar results. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05; **, P < 0.01 (compared with normoxic controls).
Because leptin is found in other tissues and organs, we were interested in the ability of the kidneys to up-regulate the transcription of leptin during tissue hypoxia. When analyzing renal gene expression, we found a significant increase in leptin mRNA (3- to 6-fold induction) in animals exposed to hypoxia (Fig. 2A). Keeping the rats under room air containing CO did not alter their renal leptin expression during the first 6 h, but significantly reduced leptin mRNA to 43% of the control level after 12 h (Fig. 2B).
FIG. 2. Renal leptin gene expression after short-term hypoxia and CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (8% O2; A) or CO (0.1% CO; B). The amount of leptin mRNA, relative to the levels of two housekeeping genes, was measured to calculate the relative change in leptin mRNA. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05; **, P < 0.01 (compared with normoxic controls).
The lung is the first organ to suffer from a reduction of oxygen tension in our experimental setup. When analyzing hypoxic lung specimens, increased levels of leptin, although statistically nonsignificant, were observed after 3–6 h of hypoxia (Fig. 3A). To this end, a profound (60-fold increase) increase in leptin mRNA levels was observed after 12 h. CO-treated animals showed a significant increase in leptin message after 6 h of exposure to carbon monoxide (Fig. 3B), returning to normal after 12 h.
FIG. 3. Pulmonary leptin gene expression after short-term hypoxia and CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (8% O2; A) or CO (0.1% CO; B). The amount of leptin mRNA, relative to levels of two housekeeping genes, was measured to calculate the relative fold induction of leptin mRNA. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05 (compared with normoxic controls).
Hepatic tissue is known to synthesize leptin, and this has recently been used in clinical studies to determine liver damage under certain circumstances (32). We tested whether exposure of the animals to hypoxia or CO is able to influence hepatic mRNA synthesis of leptin. Hypoxia induced a significant approximately 7-fold increase in leptin gene expression, whereas CO had no effect (Fig. 4).
FIG. 4. Leptin expression in hepatic tissue after 12-h hypoxia or 12-h CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed to hypoxia or CO for 12 h. The amount of leptin mRNA, relative to levels of two housekeeping genes, was measured to calculate the relative change in leptin mRNA. Data normalized for ?-actin as the housekeeping gene are displayed. *, P < 0.05 (compared with normoxic controls).
Finally, after analyzing different organs for their capacity to regulate leptin transcription, we examined the effects of hypoxia as well as CO exposure on the leptin concentration in the plasma of these animals. Rats were bled immediately after exposure to hypoxia or CO and leptin in the plasma was measured by RIA. The results were normalized to 100 g live body weight (taken at the beginning of the assay) to account for differences in fat mass. When compared with that of normoxic controls, the plasma of animals kept under hypoxic conditions exhibited a trend toward higher leptin concentrations, although this was statistically not significant (0.46 vs. 0.67 μg/liter per 100 g body weight in the 6-h exposed animals; 0.44 vs. 0.79 μg/liter per 100 g body weight in the 12-h exposed animals; Fig. 5). The CO-exposed animals reacted to the stimulus with a significant reduction in circulating leptin after 12 h. Here, the plasma levels of leptin decreased to 0.17 μg/liter per 100 g body weight in contrast to 0.79 μg/liter per 100 g body weight in normoxic control animals.
FIG. 5. Differences in circulating leptin levels upon short-term hypoxia or exposure to CO. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (*) or CO (). , Results from normoxic controls were marked. Blood specimens were taken at the end of the exposure period and subjected to a specific leptin RIA. The concentration of circulating leptin was normalized to 100 g animal weight. *, P < 0.05 (compared with normoxic controls).
A summary of the effects of hypoxia or CO exposure on leptin gene expression is presented in Table 1. Neither sc adipose tissue nor mesenteric adipose tissue of animals kept under hypoxic conditions for 3–12 h responded to hypoxia with a significant up-regulation of leptin mRNA. Lungs, kidney, and liver showed significantly higher amounts of the hormone’s message compared with those in normoxic controls. Although we found an accumulation of leptin mRNA in peripheral organs, no increase in the levels of this hormone in the circulation were detected. This is consistent with a lack of hypoxia-induced transcription of leptin in adipose tissue.
ABLE 1. Effect of short-term hypoxia on CO exposure on expression of leptin in various rat tissues and plasma
Functional anemia, triggered by exposing the animals to CO, was sufficient to reduce the leptin message in mesenteric adipose tissue after a short period of exposure of the rodents to the gas. Apart from a slight, but significant, increase in pulmonary tissue after 6 h, all organs reacted to CO exposure with down-regulation of the leptin message significantly after 12 h of exposure (Table 1).
Discussion
Since the discovery of leptin as an adipocyte-derived, appetite-regulating hormone, additional functions of this protein and organs involved in its synthesis and secretion have been found (1). Due to the almost ubiquitous distribution of its receptors, a broad field of investigations and more dynamic views of leptin have been recently reported. It is evident that the ob gene product plays a critical role in angiogenesis, wound healing, lipolysis, and blood pressure homeostasis as well as satiety control (33, 34).
Hypoxia is a potent inducer of leptin synthesis in humans (17, 35). However, Raff and colleagues (21) have shown in newborn and juvenile rats that hypoxia can also reduce leptin concentrations in the circulation. These observations were supported by the finding that leptin production in rat adipocytes is lower when incubating the cells under hypoxic conditions (22). To shed some light on these conflicting results, we analyzed leptin gene expression and plasma levels in vivo by exposing rats to hypoxia for 3–12 h. Because sleep apnea occurring in the night hours has been associated with the induction of leptin by hypoxia, this time span seemed to be physiological for sleep phases in humans, giving us the opportunity to study the effects of short-term hypoxia in vivo (18, 19). Leptin mRNA was measured in adipose tissue and other organs. Surprisingly, leptin expression in sc tissue remained unaffected at all time points studied, whereas the mesenteric adipose tissue displayed reduced amounts of leptin mRNA after 6 h. The effect observed in rats is supported by the finding that a lowered oxygen tension in patients with cardiac or abdominal surgery has no effect on the mRNA levels of leptin within their mesenteric fat (36). In contrast, all peripheral organs tested in this study (liver, lung, and kidney) showed markedly up-regulated amounts of leptin mRNA after keeping the animals under hypoxic conditions for 12 h. In kidneys, this effect was apparent as early as 6 h of hypoxia. Nothing is known about the synthesis of leptin during hypoxia in these organs. To our knowledge, this is the first report showing changes in leptin expression in these organs in the course of hypoxia in vivo. The transcription factor HIF-1 is a key regulator of oxygen-dependent genes in these organs (37, 38). As shown by various groups, including our own, leptin is a target gene of HIF-1-mediated transcription (11, 14, 15), because these hypoxia-mediated effects seem to be regulated by at least one HIF-1-sensitive hypoxia response element on the leptin promoter.
In accordance with the publication by Hodges et al. (39), hypoxia in Sprague Dawley rats raised the pH from 7.43 to 7.50 when comparing normoxic to hypoxic animals due to the hyperventilation that causes a decrease in the partial pressure of carbon dioxide in the arterial blood (PaCO2) in these animals. We also have evidence that pH levels in vitro could not be related to differences in gene expression upon hypoxia (data not shown).
At this point one can only speculate on the physiological importance of this phenomenon. Because it is known that leptin is not only involved in body weight regulation but also plays a key role in inflammation, immunity, tissue repair, and angiogenesis, up-regulation of leptin during hypoxia in peripheral organs may support local damage repair mechanisms (40, 41). Based on our data, it seems that under general hypoxic conditions, leptin production is only enhanced locally in the peripheral organs, whereas systemic leptin, which is mainly produced by adipose tissue, remains unchanged.
By generating a functional anemia through inhalation of CO, we were able to study the effects of CO in the presence of HIF-1. It has already been shown that CO inhalation blocks HIF-1 degradation in peripheral organs such as kidney (23). Until now, no direct effect of CO inhalation on the gene expression and translation of leptin have been presented. Therefore, we investigated the effects of CO by exposing the animals to 0.1% CO throughout the experiments. All organs and adipose tissues tested (except for sc tissue, which might be the result of cell type-specific regulation of leptin transcription) reacted with a decreased transcription of the ob gene after a period of 12 h and a significant reduction of leptin concentration in the plasma of CO-exposed animals after 12 h. These results corroborate previously published observations, demonstrating diminished leptin in smokers (42), because cigarette smoke is known to contain huge amounts of CO, causing CO hemoglobinemia in smokers (43). This effect of leptin reduction in man was reversible after cessation of smoking (44). Therefore, we suggest that the inhalation of CO has a negative regulatory effect on leptin gene expression and translation not only in rodents, but also in humans, yet additional studies are necessary to collect more evidence.
These results also seem to confirm the decreased binding of HIF-1 to its DNA targets in the presence of CO which have been previously reported (24). The fact that the lung is able to up-regulate leptin gene expression after exposure to CO for 6 h indicates that pulmonary cells are still able to increase leptin transcription. This up-regulation might be induced by an accumulation of HIF-1 after CO exposure, as shown for other cells in vitro by Rosenberger and colleagues (23). In specimens taken from animals after a 12-h exposure to CO, the expression of pulmonary leptin mRNA was reduced to levels in untreated controls. Thus, additional investigations into the function of CO in the regulation of leptin transcription are required.
Three main conclusions can be drawn from these observations. First, adipose tissue does not increase leptin mRNA concentrations during the course of short-term hypoxia (from 3–12 h). Second, tissue hypoxygenation up-regulates local leptin production in the lung, kidney, and liver after 12 h, but these effects are not sufficient to raise plasma leptin levels in rats. Finally, CO is an inhibitor of leptin transcription and translation in vivo. By extrapolating these data, an additional negative effect of cigarette smoking becomes apparent: the inhibition of local leptin, a hormone that plays an important role in tissue repair.
Acknowledgments
We thank Brad Taylor, Christian Plank, and Markus Schnare for critical reading of the manuscript and helpful discussions. The help of Anja Platt (University of Giessen) with the leptin RIA is gratefully acknowledged. We are indebted to Prof. Armin Kurtz (University of Regensburg) for supplying the animal care facilities and for his help with the experimental setup.
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Address all correspondence and requests for reprints to: Dr. Udo Mei?ner, Department of Pediatrics, University of Erlangen-Nurnberg, Loschgestrasse 15, 91054 Erlangen, Germany. E-mail: udo.meissner@kinder.imed.uni-erlangen.de.
Abstract
Leptin is a circulating hormone that is secreted primarily by adipose tissue. However, recent studies have demonstrated leptin production by other tissues, including placenta, stomach, kidney, liver, and lung, a process not only activated by stimuli such as insulin or corticosteroids, but also by hypoxia, which is mediated by the hypoxia inducible factor-1. In contrast to this fact, smokers have lower plasma leptin levels. The purpose of this study was to determine whether tissue hypoxygenation [induced by lack of oxygen] or inhalation of carbon monoxide (CO) are sufficient to up-regulate leptin in fat cells as well as in peripheral organs such as lung, liver, and kidney of rats. In hypoxic rats, leptin expression was unchanged or even reduced in adipose tissue. In contrast, in liver, kidney, and lung we observed an increase in leptin expression compared with normoxic controls, whereas plasma levels were unchanged. When animals were exposed to CO, generating a functional anemia known to activate the HIF-1-dependent transcription, a significant decrease in leptin gene expression in adipose tissue and in all organs tested was observed. Plasma leptin concentrations after CO exposure were significantly diminished compared with those in control animals. These findings suggest that tissue hypoxygenation up-regulates leptin expression in nonadipose tissue. However, this is not sufficient to raise plasma leptin levels in rats. Inhalation of CO leads to a significant decrease in leptin mRNA and protein concentration in the plasma of the animals, suggesting a negative effect of CO on leptin transcription.
Introduction
THE ob GENE product leptin was identified in the mid-1990s as a predominantly adipose tissue-derived hormone that is involved in feeding and energy balance. In addition, leptin was shown to have physiological effects on tissue proliferation, the placenta, the fetus, the immune system, and a plethora of neuroendocrine functions (1). Apart from adipose tissue, leptin is synthesized by a variety of peripheral tissues, such as placenta, gastric mucosa, mammary epithelium, heart, liver, and skeletal muscle (2, 3, 4, 5, 6, 7, 8). Nutrition-dependent control of leptin expression is regulated at least in part by insulin, as shown in rats in vivo (9) and in vitro (10, 11). In addition, corticosteroids are known to stimulate leptin synthesis (12, 13).
There is evidence that hypoxic conditions enhance leptin synthesis, a process that is mediated by a hypoxia-inducible factor-1 (HIF-1)-dependent mechanism. At least one hypoxia-responsive element, located –120 to –116 bp in the leptin promoter, has been shown to play a role in this HIF-mediated effect on transcriptional regulation (14, 15, 16, 17). In humans, circulating leptin levels are increased in subjects presenting with sleep apnea (18) and hypercapnea (19). Leptin might also be an important regulator of breathing in cases of oxygen deprivation. In contrast, some reports have demonstrated that hypoxia is able to reduce plasma leptin levels in neonatal rats (kept under constant hypoxia for 7 d) in vivo (20, 21) as well as in cultured adipocytes in vitro (22).
Functional anemia, triggered by inhalation of carbon monoxide (CO), is widely used to mimic hypoxic situations. Treatment of rats with CO inhibits the degradation of HIF-1 and causes an accumulation of HIF-1 in the nuclei (23). Moreover, CO itself is able to inhibit the activity of the HIF complex (24, 25). However, no data on the effects of CO exposure on leptin gene expression have been published to date.
Based on these observations, we set out to identify the sources of leptin in rats under hypoxic conditions and during a functional, CO-inflicted anemia by asking the following questions. 1) Do short-term hypoxia and CO inhalation have any effect on the circulating levels of leptin in vivo? 2) Furthermore, does the expression of leptin mRNA change in different types of adipose tissue? 3) Finally, how is leptin gene expression regulated in the course of hypoxia in vivo in other organs, such as lung, liver, and kidneys? Resolving these issues is of importance because it is not clear whether leptin serves as a regulating agent in the course of hypoxic conditions.
Materials and Methods
In vivo protocols
All animal experiments were reviewed and performed under the approval of the national care committee following national and European law. Age- and body weight-matched Sprague Dawley rats (purchased from Charles River, Sulzfeld, Germany; 200–250 g) with free access to food and water were used for the experiments and were treated as follows. 1) In the control group, animals received no treatment (n = 4–5/group, as indicated below); 2) in the hypoxia group, the animals were placed in a gas-tight box that was continuously supplied with a gas mixture of 8% O2-92% N2 for 3, 6, or 12 h (n = 4–5/group); 3) in the CO group, the animals were placed in a gas-tight box that was continuously supplied with air plus 0.1% CO for 3, 6, or 12 h (n = 4–5/group). It has been previously shown that the hypoxic protocols applied in this study have no effect on mRNA levels of the housekeeping genes in the organs examined in our study (26).
At the end of the experiments, the animals were killed by decapitation, and trunk blood was collected for determination of plasma leptin levels. Specimens of sc (taken from the back of the animals) and intraabdominal (mesenteric) adipose tissue, kidneys, liver, and lung were quickly removed and snap-frozen in liquid nitrogen. All organs were stored at –80 C until isolation of total RNA.
RNA isolation and RT
RNA from lung, liver, and kidney was extracted with the phenol/guanidine isothiocyanate method (27) using TRIzol reagent (Invitrogen Life Technologies, Inc., Gaithersburg, MD) as recommended by the manufacturer. In contrast to the manufacturer’s protocol, when RNA from adipose tissue was extracted, 1 ml TRIzol/100 mg adipose tissue was used. In addition, 400 μl CHCl3 were added before precipitation. DNA was digested with RQ1 deoxyribonuclease. Finally, cDNA was synthesized using a Moloney murine leukemia virus reverse transcriptase (all enzymes were purchased from Promega Corp., Karlsruhe, Germany) and was stored at –20 C until used in the PCR reaction.
Real-time PCR
To analyze the gene expression (mRNA) of leptin, the following primers were used: leptin sense, 5'-ATG ACA CCA AAA CCC TCA TCA AG-3'; leptin antisense, 5'-TGA AGT CCA AAC CGG TGA CC-3'; and leptin Taq, 5'-6-carboxy-fluorescein (FAM)-TCA ATG ACAT TTC ACA CAC GCA GTC GG-3'-6-carboxy-tetramethylrhodamine (TAMRA). The mRNAs of the hypoxanthine-guanine phosphoribosyltransferase (HPRT; sense, 5'-GCA GTA CAG CCC CAA AAT GG-3'; antisense, 5'-TCA TTA TAG TCA AGG GCA TAT CCA AC-3') and HPRT Taq (5'-FAM-TGC AAG CTT GCT GGT GAA AAG GAC CTC TC-3'-TAMRA) as well as of porphobilinogen deaminase (sense, 5'-AGA CCA TGC AGG CCA CCA-3'; antisense, 5'-CAA CCA ACT GTG GGT CAT CCT-3') and porphobilinogen deaminase Taq (5'-FAM-AGG TCC CTG TTC AGC AAG AAG ATG GTC C-3'-TAMRA) or ?-actin (sense, 5'-TGA GCT GCC TGA CGG TCA G-3'; antisense, 5'-TGC CAC AGG ATT CCA TAC CC-3'),and ?-actin Taq (5'-FAM-CAC TAT CGG CAA TGA GCG GTT CCG-3'-TAMRA) were analyzed as housekeeping genes. All assays were performed using a quantitative real-time PCR (TaqMan PCR, PerkinElmer, Palo Alto, CA), which has been previously described for the measurement of leptin gene expression in various tissues (28). All calculations were based on the Ct– method as described in detail previously (29). Briefly, using this method the amount of target mRNA is normalized to an endogenous reference sequence within the sample. In this study, calculation of leptin mRNA was performed using ?-actin, HPRT, and glyceraldehyde-3-phosphate dehydrogenase as endogenous references within the samples. The unstimulated/unincubated tissue served as the calibrator probe. The average crossing of threshold (CT) of the housekeeping gene is subtracted from the average CT of the target gene (CT); thereafter, the calibrator results are subtracted from those of the stimulus of interest (CT – CT = CT). Final mathematical operations lead to the expression of amount of target mRNA, normalized to an endogenous reference and relative to a calibrator (here unstimulated specimens): 2CT.
To exclude contamination of nonadipose tissue with adipocytes nucleic acid, all specimens used in the PCRs have been tested on the expression of adiponectin (adipose most abundant gene transcript 1, APM-1), an adipose tissue exclusive gene (30). Based on real-time PCR-based quantification, specimens from adipose tissue contained at least 250–700 (up to 2048) times more APM-1 than specimens of nonadipose tissue.
Leptin RIA
A high affinity antiserum against recombinant human leptin (provided by Dr. M. Heiman, Eli Lilly & Co., Indianapolis, IN) was produced in rabbits, using techniques previously described (31), which cross-reacted efficiently with rodent leptin. Standards and tracer were prepared by the chloramine-T method using recombinant mouse leptin (Eli Lilly & Co.). The assay buffer consisted of 0.05 mol/liter sodium phosphate (pH 7.4), 0.1 mol/liter sodium chloride, 0.1% (vol/vol) Triton X-100 (Serva, Heidelberg, Germany), and 0.05% (wt/vol) sodium azide. The assay mixture was composed of 100 μl standard or prediluted sample and 25 μl first antiserum (1:8000) in assay buffer containing 150 mg/liter rabbit -globulin (Sigma-Aldrich Corp., St. Louis, MO). After overnight incubation at room temperature, 25 μl tracer (200,000 cpm/ml assay buffer) were added, and the mixture was again incubated overnight. Separation of free and bound tracer was achieved by adding 50 μl cold 4% (wt/vol) polyethylene glycol 6000 (Serva) containing a goat antirabbit IgG serum (Diagnostic Systems Laboratories, Webster, TX). After 30 min at 4 C, the bound tracer was precipitated by centrifugation (15 min at 2000 x g), the supernatant was decanted, and the radioactivity was measured in a -counter. Maximum binding of tracer was approximately 35%. Half-maximum binding occurred at about 50 pg/ml. Sensitivity with undiluted samples was 3 pg/ml. The intra- and interassay coefficients of variation were 3.1% and 9.2%, respectively (n = 10). Excellent parallelism was obtained with serial dilutions of rat serum or plasma and also with dilutions of recombinant rat leptin (Eli Lilly & Co.). The cross-reactivity of rat leptin was 99.9%, indicating that this heterologous assay gives correct absolute values for rat leptin.
Statistical analysis
All values are presented as the mean ± SEM. Two-tailed Mann-Whitney tests with confidence intervals of 99% were performed using GraphPad PRISM (GraphPad, San Diego, CA). The threshold for significance was set at P < 0.05.
Results
Exposure of the animals to hypoxia for 3–12 h did not significantly alter leptin gene expression in sc adipose tissue (Fig. 1A). Leptin mRNA in mesenteric adipose tissue, however, was significantly reduced by hypoxia after 6 h (Fig. 1A) and by CO exposure after 3 and 6 h (Fig. 1B). The exposure of the rats to CO reduced the amount of mRNA to 23% compared with normoxic controls.
FIG. 1. Leptin gene expression in sc and mesenteric adipose tissue after short-term hypoxia. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxic conditions (8% O2; A) or CO (0.1% CO; B). After the period indicated, tissue specimens (two samples from sc and two from mesenteric adipose tissue) were collected and subjected to cDNA synthesis. The amount of leptin mRNA, relative to the levels of two housekeeping genes, was measured to calculate the relative change in induction of leptin mRNA; both housekeeping genes revealed similar results. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05; **, P < 0.01 (compared with normoxic controls).
Because leptin is found in other tissues and organs, we were interested in the ability of the kidneys to up-regulate the transcription of leptin during tissue hypoxia. When analyzing renal gene expression, we found a significant increase in leptin mRNA (3- to 6-fold induction) in animals exposed to hypoxia (Fig. 2A). Keeping the rats under room air containing CO did not alter their renal leptin expression during the first 6 h, but significantly reduced leptin mRNA to 43% of the control level after 12 h (Fig. 2B).
FIG. 2. Renal leptin gene expression after short-term hypoxia and CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (8% O2; A) or CO (0.1% CO; B). The amount of leptin mRNA, relative to the levels of two housekeeping genes, was measured to calculate the relative change in leptin mRNA. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05; **, P < 0.01 (compared with normoxic controls).
The lung is the first organ to suffer from a reduction of oxygen tension in our experimental setup. When analyzing hypoxic lung specimens, increased levels of leptin, although statistically nonsignificant, were observed after 3–6 h of hypoxia (Fig. 3A). To this end, a profound (60-fold increase) increase in leptin mRNA levels was observed after 12 h. CO-treated animals showed a significant increase in leptin message after 6 h of exposure to carbon monoxide (Fig. 3B), returning to normal after 12 h.
FIG. 3. Pulmonary leptin gene expression after short-term hypoxia and CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (8% O2; A) or CO (0.1% CO; B). The amount of leptin mRNA, relative to levels of two housekeeping genes, was measured to calculate the relative fold induction of leptin mRNA. Data normalized to ?-actin as the housekeeping gene are displayed. *, P < 0.05 (compared with normoxic controls).
Hepatic tissue is known to synthesize leptin, and this has recently been used in clinical studies to determine liver damage under certain circumstances (32). We tested whether exposure of the animals to hypoxia or CO is able to influence hepatic mRNA synthesis of leptin. Hypoxia induced a significant approximately 7-fold increase in leptin gene expression, whereas CO had no effect (Fig. 4).
FIG. 4. Leptin expression in hepatic tissue after 12-h hypoxia or 12-h CO exposure. Age-, sex-, and weight-matched groups of four or five animals were exposed to hypoxia or CO for 12 h. The amount of leptin mRNA, relative to levels of two housekeeping genes, was measured to calculate the relative change in leptin mRNA. Data normalized for ?-actin as the housekeeping gene are displayed. *, P < 0.05 (compared with normoxic controls).
Finally, after analyzing different organs for their capacity to regulate leptin transcription, we examined the effects of hypoxia as well as CO exposure on the leptin concentration in the plasma of these animals. Rats were bled immediately after exposure to hypoxia or CO and leptin in the plasma was measured by RIA. The results were normalized to 100 g live body weight (taken at the beginning of the assay) to account for differences in fat mass. When compared with that of normoxic controls, the plasma of animals kept under hypoxic conditions exhibited a trend toward higher leptin concentrations, although this was statistically not significant (0.46 vs. 0.67 μg/liter per 100 g body weight in the 6-h exposed animals; 0.44 vs. 0.79 μg/liter per 100 g body weight in the 12-h exposed animals; Fig. 5). The CO-exposed animals reacted to the stimulus with a significant reduction in circulating leptin after 12 h. Here, the plasma levels of leptin decreased to 0.17 μg/liter per 100 g body weight in contrast to 0.79 μg/liter per 100 g body weight in normoxic control animals.
FIG. 5. Differences in circulating leptin levels upon short-term hypoxia or exposure to CO. Age-, sex-, and weight-matched groups of four or five animals were exposed for 3, 6, or 12 h to hypoxia (*) or CO (). , Results from normoxic controls were marked. Blood specimens were taken at the end of the exposure period and subjected to a specific leptin RIA. The concentration of circulating leptin was normalized to 100 g animal weight. *, P < 0.05 (compared with normoxic controls).
A summary of the effects of hypoxia or CO exposure on leptin gene expression is presented in Table 1. Neither sc adipose tissue nor mesenteric adipose tissue of animals kept under hypoxic conditions for 3–12 h responded to hypoxia with a significant up-regulation of leptin mRNA. Lungs, kidney, and liver showed significantly higher amounts of the hormone’s message compared with those in normoxic controls. Although we found an accumulation of leptin mRNA in peripheral organs, no increase in the levels of this hormone in the circulation were detected. This is consistent with a lack of hypoxia-induced transcription of leptin in adipose tissue.
ABLE 1. Effect of short-term hypoxia on CO exposure on expression of leptin in various rat tissues and plasma
Functional anemia, triggered by exposing the animals to CO, was sufficient to reduce the leptin message in mesenteric adipose tissue after a short period of exposure of the rodents to the gas. Apart from a slight, but significant, increase in pulmonary tissue after 6 h, all organs reacted to CO exposure with down-regulation of the leptin message significantly after 12 h of exposure (Table 1).
Discussion
Since the discovery of leptin as an adipocyte-derived, appetite-regulating hormone, additional functions of this protein and organs involved in its synthesis and secretion have been found (1). Due to the almost ubiquitous distribution of its receptors, a broad field of investigations and more dynamic views of leptin have been recently reported. It is evident that the ob gene product plays a critical role in angiogenesis, wound healing, lipolysis, and blood pressure homeostasis as well as satiety control (33, 34).
Hypoxia is a potent inducer of leptin synthesis in humans (17, 35). However, Raff and colleagues (21) have shown in newborn and juvenile rats that hypoxia can also reduce leptin concentrations in the circulation. These observations were supported by the finding that leptin production in rat adipocytes is lower when incubating the cells under hypoxic conditions (22). To shed some light on these conflicting results, we analyzed leptin gene expression and plasma levels in vivo by exposing rats to hypoxia for 3–12 h. Because sleep apnea occurring in the night hours has been associated with the induction of leptin by hypoxia, this time span seemed to be physiological for sleep phases in humans, giving us the opportunity to study the effects of short-term hypoxia in vivo (18, 19). Leptin mRNA was measured in adipose tissue and other organs. Surprisingly, leptin expression in sc tissue remained unaffected at all time points studied, whereas the mesenteric adipose tissue displayed reduced amounts of leptin mRNA after 6 h. The effect observed in rats is supported by the finding that a lowered oxygen tension in patients with cardiac or abdominal surgery has no effect on the mRNA levels of leptin within their mesenteric fat (36). In contrast, all peripheral organs tested in this study (liver, lung, and kidney) showed markedly up-regulated amounts of leptin mRNA after keeping the animals under hypoxic conditions for 12 h. In kidneys, this effect was apparent as early as 6 h of hypoxia. Nothing is known about the synthesis of leptin during hypoxia in these organs. To our knowledge, this is the first report showing changes in leptin expression in these organs in the course of hypoxia in vivo. The transcription factor HIF-1 is a key regulator of oxygen-dependent genes in these organs (37, 38). As shown by various groups, including our own, leptin is a target gene of HIF-1-mediated transcription (11, 14, 15), because these hypoxia-mediated effects seem to be regulated by at least one HIF-1-sensitive hypoxia response element on the leptin promoter.
In accordance with the publication by Hodges et al. (39), hypoxia in Sprague Dawley rats raised the pH from 7.43 to 7.50 when comparing normoxic to hypoxic animals due to the hyperventilation that causes a decrease in the partial pressure of carbon dioxide in the arterial blood (PaCO2) in these animals. We also have evidence that pH levels in vitro could not be related to differences in gene expression upon hypoxia (data not shown).
At this point one can only speculate on the physiological importance of this phenomenon. Because it is known that leptin is not only involved in body weight regulation but also plays a key role in inflammation, immunity, tissue repair, and angiogenesis, up-regulation of leptin during hypoxia in peripheral organs may support local damage repair mechanisms (40, 41). Based on our data, it seems that under general hypoxic conditions, leptin production is only enhanced locally in the peripheral organs, whereas systemic leptin, which is mainly produced by adipose tissue, remains unchanged.
By generating a functional anemia through inhalation of CO, we were able to study the effects of CO in the presence of HIF-1. It has already been shown that CO inhalation blocks HIF-1 degradation in peripheral organs such as kidney (23). Until now, no direct effect of CO inhalation on the gene expression and translation of leptin have been presented. Therefore, we investigated the effects of CO by exposing the animals to 0.1% CO throughout the experiments. All organs and adipose tissues tested (except for sc tissue, which might be the result of cell type-specific regulation of leptin transcription) reacted with a decreased transcription of the ob gene after a period of 12 h and a significant reduction of leptin concentration in the plasma of CO-exposed animals after 12 h. These results corroborate previously published observations, demonstrating diminished leptin in smokers (42), because cigarette smoke is known to contain huge amounts of CO, causing CO hemoglobinemia in smokers (43). This effect of leptin reduction in man was reversible after cessation of smoking (44). Therefore, we suggest that the inhalation of CO has a negative regulatory effect on leptin gene expression and translation not only in rodents, but also in humans, yet additional studies are necessary to collect more evidence.
These results also seem to confirm the decreased binding of HIF-1 to its DNA targets in the presence of CO which have been previously reported (24). The fact that the lung is able to up-regulate leptin gene expression after exposure to CO for 6 h indicates that pulmonary cells are still able to increase leptin transcription. This up-regulation might be induced by an accumulation of HIF-1 after CO exposure, as shown for other cells in vitro by Rosenberger and colleagues (23). In specimens taken from animals after a 12-h exposure to CO, the expression of pulmonary leptin mRNA was reduced to levels in untreated controls. Thus, additional investigations into the function of CO in the regulation of leptin transcription are required.
Three main conclusions can be drawn from these observations. First, adipose tissue does not increase leptin mRNA concentrations during the course of short-term hypoxia (from 3–12 h). Second, tissue hypoxygenation up-regulates local leptin production in the lung, kidney, and liver after 12 h, but these effects are not sufficient to raise plasma leptin levels in rats. Finally, CO is an inhibitor of leptin transcription and translation in vivo. By extrapolating these data, an additional negative effect of cigarette smoking becomes apparent: the inhibition of local leptin, a hormone that plays an important role in tissue repair.
Acknowledgments
We thank Brad Taylor, Christian Plank, and Markus Schnare for critical reading of the manuscript and helpful discussions. The help of Anja Platt (University of Giessen) with the leptin RIA is gratefully acknowledged. We are indebted to Prof. Armin Kurtz (University of Regensburg) for supplying the animal care facilities and for his help with the experimental setup.
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