help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Böttner, A.
Right arrow Articles by Bornstein, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Böttner, A.
Right arrow Articles by Bornstein, S. R.
Endocrinology Vol. 141, No. 11 4239-4246
Copyright © 2000 by The Endocrine Society

ARTICLES

Increased Body Fat Mass and Suppression of Circulating Leptin Levels in Response to Hypersecretion of Epinephrine in Phenylethanolamine-N-Methyltransferase (PNMT)-Overexpressing Mice1

A. Böttner, A. Haidan, G. Eisenhofer, K. Kristensen, A. L. Castle, W. A. Scherbaum, H. Schneider, G. P. Chrousos and S. R. Bornstein

Department of Internal Medicine III, University of Leipzig (A.B., A.H.), 04103 Leipzig, Germany; Intramural Research Program, National Institute of Neurological Disorders and Stroke (G.E.), National Institute of Diabetes and Digestive and Kidney Diseases (A.L.C.), and National Institute of Child Health and Human Development (G.P.C., S.R.B.), National Institutes of Health, Bethesda, Maryland 20892; Pipeline Biotech A/S (K.K.), Silkeborg, Denmark; Diabetes Research Institute (W.A.S.), Düsseldorf 40225, Germany; and Children’s Hospital, University of Erlangen-Nürnberg (H.S.), Erlangen 91054, Germany

Address all correspondence and requests for reprints to: Dr. Antje Böttner, Department of Internal Medicine III, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: antje.boettner{at}gmx.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epinephrine is a major stress hormone that plays a central role in the control of metabolic function and energy homeostasis. To evaluate the role of epinephrine and the physiological and pathophysiological consequences of sustained elevation of epinephrine on metabolic and endocrine function, we studied several metabolic parameters and circulating leptin levels in a newly developed transgenic mouse model of phenylethanolamine-N-methyltransferase (PNMT) overexpression. A 100-fold overexpression of PNMT and subsequent elevation of epinephrine levels resulted in a marked suppression of circulating leptin levels in the transgenic animals (1.14 ± 0.05 vs. 2.17 ± 0.35 ng/ml; P < 0.01), which correlated negatively with plasma epinephrine (r = -0.82; P < 0.05), thus providing evidence for an inhibitory action of epinephrine on leptin production in vivo. In parallel, we found a marked increase in the body fat content of the transgenic animals (12.54 ± 1.5 vs. 6.22 ± 0.2%; P < 0.01) that was accompanied by enlarged adipocytes, indicating an increased lipid storage in PNMT transgenic mice. Interestingly, however, transgenic animals had normal body weight and did not exhibit major alterations in carbohydrate metabolism, as evidenced by analysis of random and fasted blood glucose levels, plasma insulin and C peptide levels, and insulin tolerance test. The metabolic alterations observed were not secondary to changes in food intake or increased activity of the hypothalamic-pituitary-adrenal axis, as there were no differences in these parameters. In summary, sustained primary overproduction of epinephrine resulted in suppression of plasma leptin levels and increased lipid storage in the PNMT transgenic mice. The concerted action of the sympathoadrenal system and reduced leptin may contribute to defending energy reservoirs while maintaining a normal body weight, which may be of vital importance under conditions of stress and energy deficiency.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPINEPHRINE IS the major effector of the sympathoadrenal limb of the stress system, and as such it is involved in the regulation of metabolic, endocrine, and cardiovascular function to ensure the maintenance of body homeostasis. Epinephrine is crucially implicated in the control of energy balance through its impact on metabolic rate and direct effects on components of intermediary metabolism (1). Hence, changes in the activity of the sympathoadrenal system may influence body weight and lead to the development of obesity, which occurs as a consequence of the imbalance between energy supplies and utilization. In fact, alterations in sympathetic nervous system (SNS) function have been related to obesity in animals (2) and humans (3), and chronic stress with persisting elevation of epinephrine has been suggested to play a role in the induction of insulin resistance and obesity (4). However, data are still inconclusive, and as most studies were performed in the obese, it is not clear whether changes in sympathetic activity may be secondary to the obese state rather than being causal.

Previous studies have identified the Ob protein leptin as yet another potent regulator of body weight and energy expenditure (5). In rodents, leptin decreases body weight by inhibiting food intake and increasing energy expenditure and metabolic rate (6). The latter action of leptin is believed to be mediated to a great extent via the SNS. Recent research on the actions of leptin has changed the view from the purely adipostatic signal to a pleiotropic mediator affecting numerous biological processes distinct from those primarily related to obesity and has shown that it influences other endocrine systems, including the stress and reproductive systems as well as immune function and hemopoiesis (7, 8).

Given the great variety and importance of actions leptin exerts, the regulation of the Ob protein is now a matter of considerable interest. Indeed, abnormal regulation of the leptin gene that results in quantitative alterations of leptin secretion contributes to the development of obesity (6, 9, 10). A number of neuroendocrine, hormonal, and paracrine signals have been implicated in the regulation of leptin expression and secretion, most of which are stimulators of leptin production, such as glucocorticoids or insulin (6). Certain physiological responses, such as fasting or cold exposure, however, have been associated with a decrease in leptin production, and the SNS has been hypothesized to play a major role in this regulation (11). In fact, catecholamines and ß-agonists were reported to rapidly suppress leptin expression and secretion in vitro and in vivo, suggesting that catecholamines may represent a major controlling factor of leptin production (12). However, previous studies mainly concentrated on short-term effects of norepinephrine and pharmacological ß-agonists, whereas the effect of chronic hypercatecholaminemia and differential actions of norepinephrine and epinephrine have not been considered as yet.

The powerful effects of the sympathetic system and leptin on the components of energy balance make the understanding of their potential interactions in vivo a matter of considerable interest. To address this issue and to assess more precisely the role of sustained primary elevation of epinephrine levels in the regulation of body weight, metabolic function, and leptin production in vivo, we analyzed parameters of lipid and glucose metabolism and circulating leptin levels in a newly developed transgenic mouse model overexpressing phenylethanolamine-N-methyltransferase (PNMT), a cytosolic enzyme that catalyzes the formation of epinephrine from norepinephrine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
The construct used for generation of PNMT transgenic mice contained the entire coding sequence for murine PNMT (13) linked to the simian virus 40 (SV40) late promoter. The PNMT fragment was amplified by PCR from isolated murine DNA using the oligonucleotides 5'-ACT TCT AGA CAA CAG AAG CAT GAA CGG TG-3' (forward) and 5'-AGT GGA TCC GGA ACT GTC ACT TTA TTA GGT-3' (reverse). The forward primer contained the ATG codon, which has been previously demonstrated to be the major transcription initiation site by primer extension analysis (13). The identity of the PCR product was confirmed by DNA sequencing and restriction mapping. For amplification, the 1.6-kb PNMT PCR fragment was then inserted into the HincII site of the pGEM-4Z vector (Promega Corp., Madison, WI) by blunt end cloning. Subsequently, the PNMT-coding sequence was excised with BamHI and XbaI and subcloned into the XbaI and BamHI sites of the pSVL SV40 late promoter expression vector (Pharmacia Biotech, Freiburg, Germany). DNA sequencing confirmed correct introduction and direction of the construct in the vector.

Generation and maintenance of transgenic mice
The 4.1-kb transgene construct was isolated by digestion with EcoRI and SalI. Approximately 1000 copies of the purified DNA were microinjected into B6D2F1 oocytes at a concentration of 1–2 ng/µl, as described previously (14). Embryos at the 2-cell stage were transferred to postcoitum pseudopregnant NMRI females. PNMT transgenic mice were established from breeding of transgenic founders and their progeny presenting the strongest hybridization signals in Southern blot analysis to C57BL/6J mice. For Southern blot analysis, a 736-bp fragment excised from pSVL-PNMT with EcoRV and XhoI was purified and labeled with digoxigenin-deoxy (d)-UTP (Roche, Mannheim, Germany) to serve as the probe. DNA samples from tail tips were digested with EcoRV before Southern blotting. The Southern blot procedure was performed according to the manufacturer’s protocol using the DIG System for Filter Hybridization (Roche). Signals were detected by colorimetric detection with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate using the DIG Labeling and Detection Kit (Roche).

Mice were bred and maintained at the facilities of Bomholtgård Breeding & Research Center A/S (Ry, Denmark). They were housed on a 14-h light, 10-h dark schedule and were given food and water ad libitum. C57BL/6J mice of the same sex and age served as controls. Maintenance and experiments were conducted according to the principles and procedures of the NIH Guidelines for the Care and Use of Laboratory Animals.

Blood collection and tissue processing
Adult mice, aged 9–14 weeks, were anesthetized with pentobarbital sodium (50 mg/kg, ip; Sanofi Pharmaceuticals, Inc., Hanover, Germany). Blood was obtained by retroorbital phlebotomy, collected into tubes containing 2 µl heparin (5000 IU), and immediately placed on ice. After refrigerated centrifugation, plasma samples were stored in aliquots at -80 C until assayed. Tissues were quickly dissected and immediately snap-frozen in liquid nitrogen, then stored at -80 C until further processing.

RT-PCR
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and subsequently treated with deoxyribonuclease (Roche). RT was performed using the First Strand Complementary DNA (cDNA) Synthesis Kit (Pharmacia Biotech). To obtain equal concentrations of cDNA for PCR, all samples were adjusted to contain equal amounts of ß-actin. For this, ß-actin was amplified from cDNA using the following primers: forward, 5'-TGG TAA TCC TGT GGC ATC CAT G-3'; and reverse 5'-CGC AGC TCA GTA ACA GTC CG-3'; a 344-bp product was obtained (accession no. M12481). Relative concentrations of ß-actin cDNA were determined by measuring the ethidium bromide fluorescence. The cDNAs were diluted accordingly, and the ß-actin PCR was repeated until the fluorescence of the bands varied between 90–110% at maximum. Subsequently, adjusted cDNAs served as templates for PNMT PCR, producing a 1.0-kb fragment corresponding to the three exons of the murine PNMT gene. The same primers were used as those described above for the amplification of the PNMT gene.

Quantitative real-time PCR (TaqMan)
For quantification of PNMT messenger RNA (mRNA) expression, we applied the novel technique of Real-Time Quantitative PCR (TaqMan PCR) using the 7700 Sequence Detector (Perkin-Elmer Corp., PE Applied Biosystems, Foster City, CA) as described previously (15). The contents of PNMT mRNA were determined by amplification from total RNA using the following primers and the TaqMan probe designed from the mouse PNMT gene sequence (GenBank L12687) using the Primer Express program (PE Applied Biosystems): forward, 5'-GTC GGG ACG GGT TCT CAT T-3'; reverse, 5'-CCA AGA AGT CTG TCA TGG TGA TG-3'; TaqMan probe, 5' (FAM)-CTC CGG CCC CAC CAT ATA TCA GCT G-(TAMRA)-3'; for StAR mRNA quantification, 5'-CCG GAG CAG AGT GGT GTC-3' (forward) and 5'-GCC AGT GGA TGA AGC ACC AT A-3' (reverse); and probe, 5'-(FAM)-CAG AGC TGA ACA CGG CCC CAC C-(TAMRA)-3'. To control for the amount of RNA in the sample, 18S served as the housekeeping gene, which was detected with the TaqMan Ribosomal RNA control reagents (PE Applied Biosystems).

Using total RNA, a one-step RT-PCR was performed using the TaqMan Gold RT-PCR Kit (PE Applied Biosystems) according to the protocol supplied. RT and amplification of PNMT and 18S were performed in the same tube, thus minimizing random errors. Reactions contained 1 x TaqMan buffer A; 5.5 mM MgCl2; 0.3 mM each of dATP, dCTP, and dGTP; 0.6 mM dUTP; 0.4 U/µl RNase inhibitor; 0.025 U/µl AmpliTaq Gold DNA polymerase; and 0.25 U/µl MultiScribe reverse transcriptase. Primers and probes were added at the following concentrations: 900 nm for the PNMT forward and reverse primers, 200 nm for the PNMT TaqMan probe, and 50 nm for 18S primers and probe. After RT at 49 C for 30 min, AmpliTaq Gold was activated at 95 C for 10 min. Thermal cycling proceeded with 40 cycles of 95 C for 15 sec and 60 C for 1 min.

Input RNA amounts were calculated with relative standard curves for both PNMT and 18S. The amount of PNMT RNA mRNA was normalized by dividing it by the amount of 18S RNA.

Hormone measurements and analysis of metabolic parameters
For determination of catecholamine content, tissues were homogenized in 5 vol ice-cold 0.4 M perchloric acid containing 0.5 M EDTA. After refrigerated centrifugation the supernatants were stored in aliquots until assayed. Catecholamine contents of plasma and supernatants were determined by liquid chromatography with electrochemical detection after a batch alumina extraction as previously described in detail (13). Dihydroxybenzylamine was used as an internal standard. The average recovery of the internal standard was 72.32 ± 0.55%.

Other plasma hormone levels were determined using the following commercially available RIA kits according to the manufacturer’s instructions: leptin (Linco Research, Inc., St. Charles, MO), insulin and C peptide (DRG Instruments GmbH, Marburg, Germany), corticosterone (DPC, Los Angeles, CA), and ACTH (Brahms, Berlin, Germany).

Blood glucose was determined with an automated glucose oxidase method according to the manufacturer’s instructions (Care Diagnostica, Voerde, Germany). Plasma triglyceride levels were measured using a commercially available assay with colorimetric detection according to the manufacturer’s protocol (Roche). Tissue glycogen content was determined after complete enzymatic degradation to glucose with amyloglucosidase as described in detail previously (16). Glycogen levels are expressed as glucose units determined by the spectrophometric Trender reaction (Sigma, St. Louis, MO).

For evaluation of alterations in body composition, carcasses were analyzed for body fat content as described previously (17). In brief, the frozen carcasses were homogenized in the presence of liquid nitrogen. Fat content was assessed by extraction on chloroform-methanol and was subsequently determined gravimetrically.

In vivo studies on food intake and insulin tolerance
Mice were housed individually for at least 3 days before the beginning of the experiment. In this period, mice were weighed twice daily to adapt to the experimental procedure. Mice were allowed free access to a standard rodent chow and water ad libitum. Mice and food pellets were weighed at 0800 and 2000 h for 5 consecutive days. Cages were examined at each weighing for possible dispersed or hoarded food pellets, which were dried, weighed, and included in the analyses.

Insulin tolerance tests were performed on randomly fed animals. Animals were injected with 0.75 IU/kg BW human insulin into the peritoneal cavity. Blood glucose levels were measured immediately before and 15, 30 60, and 120 min after the injection.

A glucose tolerance test was performed after an overnight fast. Animals were injected with 1 mg/g BW glucose in normal saline into the peritoneal cavity. Blood glucose levels were measured immediately before and 60 and 120 min after the injection.

Histology, immunohistochemistry, electron microscopy, and morphometric analyses
Tissues were rapidly removed, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. For histological examination, 5-µm sections were stained with hematoxylin and eosin. Immunohistochemistry for PNMT was performed using a monoclonal rabbit antimouse antibody (courtesy of C. Grothe, Freiburg, Germany). Sections were deparaffinized and pretreated in the microwave in 10 mM citrate buffer, pH 6.0. Endogenous peroxidase activity was quenched by incubating sections in 0.5% hydrogen peroxide. Nonspecific binding was blocked by 3% normal goat serum (DAKO Corp., Hamburg, Germany). Sections were incubated with primary antibody diluted 1:250 at 4 C overnight. The primary antibody was detected using UniTect immunohistochemistry detection system (Dianova-Immunotech, Hamburg, Germany), and the immune complex was visualized by immersing the sections in the 3-amino-9-ethyl-carbozole chromogen system (Dianova-Immunotech). For a negative control, the primary antibody was replaced with nonimmune rabbit serum, and no staining was observed. Sections of adrenal medulla served as a positive control.

For electron microscopy, small pieces of tissue were fixed in 2.5% glutaraldehyde in 0.19 M cacodylate buffer, pH 7.4, for 3 h and postfixed in 2% OsO4 in cacodylate buffer. After washing in 0.05 M maleate buffer, pH 4.8, tissues were incubated in 2% uranyl acetate in maleate buffer, subsequently dehydrated through a graded series of ethanol, and embedded in epoxid resin. Semithin sections were stained with toluidine blue. Ultrathin sections (70 nm) stained with uranyl acetate and lead citrate were examined at 80 kV under a Philips electron microscope 301 (Philips Electronics, Mahway, NJ).

Fat cell size was analyzed using a computer-supported imaging system (analySIS Pro 2.11, Soft Imaging GmbH, Munster, Germany) connected to a light microscope to capture gray scale microscopic images of hematoxylin-stained sections of adipose tissue with a video camera suitable for morphometric analysis. A minimum of 400 adipocytes were analyzed on different sections of each animal.

Statistical analysis
Data analysis included independent t test and Mann-Whitney test, depending on the distribution pattern of the data, and nonparametric correlation analysis (Spearman). Results are expressed as the mean ± SEM. Unless stated otherwise, statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of PNMT transgenic mice
In this study we generated transgenic mice harboring the entire gene coding for murine PNMT fused to the SV40 late promoter (Fig. 1AGo). The presence of introns in the construct has been shown to stabilize and enhance the expression of the transgene, and therefore to confer persistent and reliable gene expression (18). The transgene was stably transmitted to the offspring, and Southern blot or RT-PCR analysis of tail fragment DNA confirmed integration of the transgene into the genome in all mice used for breeding and experiments. All experiments reported in this study were performed on 9- to 14-week-old animals of the line PNMT 1815 and sex- and age-matched C57/BL6J control mice. Transgenic mice were viable and fertile and did not exhibit major phenotypic alterations, which is in line with previously established transgenic models of PNMT overexpression (19, 20).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 1. A, The 4.1-kb transgene construct containing the murine PNMT gene linked to the SV40 late promoter. RT-PCR shows expression of PNMT mRNA (upper panel) in liver (B) and adipose tissue (C) in transgenic animals and controls as indicated. Lower panel, Approximately equal amounts of ß-actin mRNA are shown (see Materials and Methods).

 
Tissue expression of PNMT and catecholamine levels
Expression of PNMT mRNA was first examined by RT-PCR as described in Materials and Methods. In the adrenal glands, products corresponding to PNMT mRNA were observed in both transgenic and control mice, as expected (data not shown). Analysis of fat and liver tissue revealed expression of PNMT mRNA in all transgenic mice analyzed, whereas no specific bands were observed in wild-type mice (Fig. 1Go, B and C). As PNMT is physiologically not present in the latter tissue, this verifies the integration of the construct into the genome and the correct transcription of the transgene.

For more definite quantification of PNMT mRNA expression, a one-step real-time RT-PCR (TaqMan) was performed, which constitutes a very accurate and reliable method for quantification of gene expression (15). High level expression of PNMT mRNA relative to ß-actin expression was confirmed in the liver (100.0 ± 38.29 vs. 0.6170 ± 0.3; P < 0.05; n = 4) and in adipose tissue of transgenic mice, whereas expression in the controls was near the detection limit (Fig. 2A).

For localization of PNMT expression in the adipose tissue, immunohistochemistry was performed using an antibody directed against murine PNMT. PNMT-immunoreactive cells were detected in the immediate vicinity of adipocytes, which themselves did not reveal positive staining. These PNMT-positive cells were clearly distinct from adipocytes, but presented as polygonal cells with a central nucleus and dense cytoplasm characteristic of neuron-like cells (Fig. 2BGo). No staining was observed in the adipose tissue of control mice.



View larger version (68K):
[in this window]
[in a new window]
  		Figure 2. PNMT expression and epinephrine production in white adipose tissue. A, Quantification of PNMT expression applying the TaqMan Real-Time PCR method, demonstrating high level expression of PNMT in adipose tissue of transgenic mice and almost undetectable PNMT mRNA levels in controls. Values are presented as relative amounts of PNMT mRNA to 18S mRNA ± SEM (n = 6). **, P < 0.01. Data are presented as mean ± SEM. B, Immunohistochemical staining for PNMT revealing PNMT-positive neuron-like cells in the immediate vicinity of adipocytes in the white adipose tissue of PNMT-overexpressing mice (magnification, x100).

 
The tissue contents of catecholamines were variable. The brain exhibited a marked increase in epinephrine content in transgenic animals (2.68 ± 0.92 vs. 036 ± 0.08 pg/mg; P < 0.05; n = 6), whereas in the adrenal gland and adipose tissue there was a mild elevation in the epinephrine content in PNMT transgenic mice [adrenal, 3.08 ± 0.21 vs. 2.15 ± 0.36 µg/adrenal (P < 0.05; n = 6); adipose tissue, 2.34 ± 0.73 vs. 1.00 ± 0.14 pg/mg (n = 5)] under basal conditions. The plasma epinephrine level was 2.30 ± 0.69 nmol/liter in the transgenic animals compared with 1.36 ± 0.12 nmol/liter in the controls, with a shift in the relative amount of plasma catecholamines in favor for epinephrine (57.0 ± 10.0% vs. 33.2 ± 4.2%; P = 0.08; n = 6).

Circulating leptin levels
Plasma leptin levels were markedly decreased in transgenic mice compared with nontransgenic mice, accounting for a reduction to 52% (Fig. 3AGo). This difference was preserved when plasma leptin levels were corrected for body weight. Leptin levels showed a positive correlation with body weight as expected (Fig. 3BGo). An even stronger, negative correlation was determined between leptin levels and plasma epinephrine levels in the transgenic mice (Fig. 3CGo), which was also preserved when leptin levels were normalized for body weight, hence indicating a suppressive effect of catecholamines on leptin production.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. A, Decreased plasma leptin levels in PNMT transgenic mice compared with normal mice (**, P < 0.01; n = 6). Data are presented as mean ±SEM. B, Positive correlation between leptin and body weight (r = 0.67; P < 0.05). C, Leptin levels showed a strong negative correlation with plasma epinephrine in the transgenic animals (r = -0.83; P < 0.05). Correlation coefficients were calculated by nonparametric correlation analysis (Spearman).

 
Body composition, adipose tissue, and food intake
The mean body weight did not notably differ between transgenic and nontransgenic mice (25.92 ± 1.07 vs. 23.42 ± 1.35 g; n = 6). There was, however, a striking difference in body fat content. PNMT transgenic mice had a markedly elevated fat mass, which was about double that of control mice (Fig. 4AGo). Hematoxylin-eosin staining of retroperitoneal white adipose tissue revealed normal morphology, mainly consisting of large unilocular adipocytes surrounded by a small rim of cytoplasm in both PNMT mice and controls (Fig. 4Go, C and D). On the ultrastructural level, unilocular adipocytes demonstrated large lipid vacuoles and a small edge of cytoplasm, containing few elongated mitochondria with sparse internal membranes (data not shown).



View larger version (69K):
[in this window]
[in a new window]
  		Figure 4. Parameters of body composition. A, Transgenic mice had elevated body fat content. Values are the percentage of fat mass relative to body weight (n = 6). **, P < 0.01. Data are presented as mean ±SEM. B, Morphometric analysis of white adipocytes using a computer-supported digital imaging system revealed enlarged fat cells in the transgenic animals (n = 6). *, P < 0.05. Data are presented as mean ±SEM. C and D, Hematoxylin-eosin staining of white adipose tissue depicting normal morphology of unilocular white adipocytes in control animals (C) as well as in transgenic mice (D), with enlarged adipocytes in the transgenic mice (magnification, x20).

 
Fat cell size, however, appeared to be increased in the transgenic animals. To verify this, morphometric analysis of white adipose was performed, which confirmed enlargement of adipocytes in transgenic animals compared with those in controls (Fig. 4BGo).

To determine whether the altered hormonal situation had an effect on feeding that may underlie the altered body fat contents, we determined food intake over a period of 5 days. Neither in daily food intake (4.89 ± 0.07 vs. 4.74 ± 0.16 g/day; n = 6) nor in the total amount of food consumed over 5 days (24.45 ± 0.33 vs. 23.68 ± 0.79 g/5days; n = 6) were differences between transgenic animals and controls detectable. There were also no significant differences in the plasma levels of triglycerides between transgenic and control animals (1.38 ± 0.18 vs. 1.01 ± 0.11 mmol/liter; n = 6).

Glucose metabolism
To determine whether glucose metabolism might be affected similarly in PNMT transgenic mice, several parameters of glucose metabolism were determined. There were no differences detectable in random or fasted blood glucose levels (Table 1Go). Plasma insulin and C peptide levels were increased in the transgenic animals, although statistical significance was not obtained (Table 1Go). Therefore, insulin and glucose tolerance tests were performed to detect a possible latent insulin resistance. The insulin tolerance test, however, did not reveal major disturbances in insulin action (Fig. 5Go). Even though blood glucose levels appeared to be slightly higher in the transgenic animals in the glucose tolerance test, statistical significance was not obtained (Table 1Go). Furthermore, fasting levels of blood glucose, insulin, and C peptide were determined, none of which was statistically different (Table 1Go). The content of glycogen as the primary storage form of carbohydrates was measured in the liver and skeletal muscle of the animals. Although there were no differences in muscle glycogen content between the animals, the liver glycogen content was significantly decreased in the transgenic animals (Table 1Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Parameters of glucose metabolism

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Insulin tolerance test. Blood glucose levels in response to injection of insulin (0.75 IU/kg BW) were not significantly altered in the transgenic mice, even though a small statistical significance was detected at 10 min. PNMT and controls are indicated. Values are the mean ± SEM (n = 6). *, P < 0.05, by Student’s t test).

 
Hypothalamic-pituitary-adrenal (HPA) axis
As catecholamines and leptin both interact with the HPA axis in rodents, we sought to determine whether there were any alterations in the HPA axis in the transgenic mice. Plasma corticosterone levels were 240.8 ± 40.98 ng/ml in transgenic animals and 185.0 ± 28.43 ng/ml in controls (n = 8; P = 0.61). Plasma corticosterone levels strongly correlated with plasma epinephrine levels in the transgenic animals (r = 0.93; P < 0.01; n = 8). There were no major differences in plasma ACTH levels (8.98 ± 1.56 vs. 6.72 ± 1.18 pmol/liter; n = 6) or in the expression of Steroidogenic Acute Regulatory Protein (StAR) mRNA, as determined by TaqMan PCR (100.0 ± 9.67 vs. 97.07 ± 18.84; n = 6). Hence, the HPA axis did not appear to be profoundly disturbed in PNMT transgenic mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have generated PNMT-overexpressing transgenic mice to study metabolic and endocrine consequences of chronic epinephrine hypersecretion in vivo. Primary elevation of epinephrine due to a 100-fold overexpression of the PNMT gene resulted in profound suppression of circulating leptin levels and marked lipid accumulation in the transgenic mice, whereas body weight, carbohydrate metabolism, and the HPA axis were less or not affected.

The marked reduction in circulating leptin levels with an evident and significant negative correlation between epinephrine and leptin provides strong evidence for a chronic inhibitory effect of epinephrine on leptin production in rodents in vivo. Even though we could not differentiate to what percentage this effect can be attributed to systemic vs. local paracrine consequences of PNMT overexpression and epinephrine overproduction, our findings complement reports in which ß-agonists and norepinephrine were shown to acutely reduce Ob mRNA expression and concomitant leptin secretion (12) and extend this concept by showing that epinephrine plays an important role in leptin regulation and can lead to its chronic suppression.

Despite the marked reduction in leptin levels in our transgenic mice, their body weights remained surprisingly similar to those of control mice, which is at variance with what was expected from numerous observations that leptin deficiency or dysregulation leads to adiposity (9, 10). However, PNMT mice showed a considerably greater amount of body fat mass. This increase in adipose tissue mass was primarily due to an enlarged size of white adipocytes, which implies a greater degree of lipid storage and/or decreased lipolysis in the transgenic animals. This increased lipid storage may be explained by the differential down-regulation of adrenergic receptors in adipose tissue. In contrast to ß1- and ß2-receptors, which promote the lipolytic actions of catecholamines and are readily down-regulated in states of chronic stimulation, the ß3- and {alpha}2-receptors that mediate leptin suppression (21) and antilipolytic effects (22), respectively, are fairly resistant to agonist-induced desensitization (23), which consequently leads to lipid storage and leptin suppression (24). Other studies showed that obesity is indeed associated with reduced ß2-receptor sensitivity of adipocytes (25) and an impaired lipolytic response to adrenergic stimulation (26). Furthermore, the epinephrine-induced attenuation of leptin action with subsequent inhibition of the latter’s autocrine antilipolytic effects (27) may contribute to the observed shift from lipid depletion to increased storage in the face of chronically elevated epinephrine levels.

Body weight and adiposity are markedly influenced by the rate of food consumption, which is influenced by catecholamines and leptin (21). We, therefore, analyzed food intake to assess whether the elevated fat storage observed was secondary to an increased supply of nutrients. Our results show that sustained hypersecretion of epinephrine has no apparent effect on feeding and that the observed metabolic alterations are thus not likely to be primarily attributed to changes in nutrient input.

The impact of chronic hypercatecholaminemia on glucose metabolism was less apparent. Analysis of basal as well as fasted blood glucose levels yielded substantially the same results in transgenic and control mice. Although there were no differences in muscle glycogen content, the glycogen content of the liver was significantly decreased in the transgenic animals, which may be attributed to acute effects of catecholamines on carbohydrate metabolism on the liver. There is also further support for differential effects of leptin, which primarily affects glycogen degradation in the liver, but not in the muscle in the fed to fasted transition (28).

Catecholamines inhibit insulin secretion, whereas visceral obesity and chronic stress, on the other hand, have been linked to insulin resistance (4). In our model of PNMT overexpression, however, basal and fasted plasma levels of insulin and C peptide showed only a subtle tendency to be elevated. To finally rule out impaired insulin tolerance in these mice, we performed an insulin tolerance test, which did not reveal major impairment in the efferent actions of insulin. Thus, glucose metabolism did not appear to be severely affected by the combined alterations of the sympathoadrenal and leptin systems, although this may not be finally ruled out by our data.

The SNS and the HPA axis exert mutual regulatory influences on each other’s function (29, 30). We, therefore, tested the possibility that a secondarily increased activity of the HPA axis in PNMT transgenic mice may have been an underlying mechanism of the metabolic findings in our model. However, we did not find profound alterations in parameters of the HPA axis, suggesting that the HPA axis in these mice either is not severely affected or may have adapted to the persistent elevation of epinephrine.

The roles of the SNS and the adrenal medulla in the development of adiposity at this point remain conjectural, with both a decrease and an increase in sympathetic activity having been associated with obesity (2, 3). In our model of primary elevation of epinephrine, no significant changes in body weight or carbohydrate metabolism were observed, thus indicating that at least an increased sympathetic activity does not constitute a major primary pathogenic factor for excessive body weight gain or diabetes. However, parameters related to lipid metabolism were abnormal, reflecting increased lipid accumulation. The sympathoadrenal system is particularly involved in the initiation of the adaptive response to fasting (31). A decline in leptin secretion that promotes partitioning of energy toward fat is also supposed to play a crucial role in this response, and in fact, previous studies showed that leptin levels are decreased in fasting (32), most likely as a result of suppression of leptin levels by increased sympathetic activity (11). Hence, our observations are well in line with this concept. Considering that the inability to meet energy requirements is a more severe threat to survival for rodents than the metabolic consequences of overfeeding, these mechanisms are of substantial physiological importance (33).

Both catecholamines and leptin independently adapt metabolic functions to the prevalent state of energy demand or storage (1, 34, 35). Furthermore, catecholamines and leptin regulate each other, with catecholamines exerting an inhibitory influence on leptin production, and leptin, in turn, stimulating sympathetic activity (36, 37). Interestingly, we found recently that leptin is expressed in the adrenal glands of mice (38), and given the direct stimulatory action of leptin on catecholamine secretion from the adrenal medulla (39), one may surmise that an additional intraadrenal regulatory loop exists. Hence, catecholamines and leptin are not only closely linked to the regulation of metabolic function, but may comprise parts of a regulatory circuit, with their concerted actions responding to exogenous stimuli affecting either system, with the aim of maintaining body weight homeostasis (40). This may be of particular clinical importance under circumstances in which the saving of energy is vital; these are often conditions associated with hyperactivation of the sympathoadrenal and systemic sympathetic system and altered leptin homeostasis, such as stress, fasting, or cardiac dysfunction (32, 41, 42). Insufficiency or dysfunction of such a regulatory loop may indeed be involved in the development of obesity, as was similarly hypothesized recently (10, 40), and, moreover, may be involved in several disease states unrelated to obesity (43, 44, 45).

In summary, primary overproduction of epinephrine in PNMT transgenic mice resulted in a marked suppression of circulating leptin levels, providing strong evidence for a long-term role of epinephrine in the regulation of leptin production in rodents in vivo. The differentially regulated tone of the sympathetic system in accordance with the attenuated action of leptin, in turn, may contribute to increased lipid accumulation while preserving normal body weight and glucometabolic parameters in states of chronic hypercatecholaminemia. The sympathoadrenal system and leptin may thus constitute a dynamic and closely interconnected system of vital importance in an energy-deficient or otherwise stressful environment. Such genetically engineered animals may be a valuable tool to deduce the complexity of these interactions and to better understand the impact of the adrenergic system on metabolic function (46).


   Acknowledgments
 
We thank Drs. E. Junger and L. Herberg for their most helpful advice, and Drs. P. Rother and W. Wolff for their support with the morphometric analyses. The expert technical assistance of S. Laue, R. Tauchnitz, and D. Hooper is gratefully acknowledged.


   Footnotes
 
1 This work was supported by a grant from the Boehringer Ingelheim GmbH Fonds (to A.B.), grants from the Deutsche Forschungsgemeinschaft (to S.R.B. and W.A.S.), and a Heisenberg grant (to S.R.B.; Bo 1141/6–1). Back

Received May 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Goldstein DS 1995 Stress, Catecholamines, and Cardiovascular Disease. Oxford University Press, New York
  2. Bray GA 1990 Obesity-a state of reduced sympathetic activity and normal or high adrenal activity (the autonomic and adrenal hypothesis revisited). Int J Obes 14:77–92
  3. Macdonald IA 1995 Advances in our understanding of the role of the sympathetic nervous system in obesity. Int J Obes Relat Metab Disord 19:S2–S7
  4. McEwen BS 1998 Protective and damaging effects of stress mediators. N Engl J Med 338:171–179[Free Full Text]
  5. Campfield LA, Smith FJ, Burn P 1996 The ob protein (leptin) pathway: a link between adipose tissue mass and central neural networks. Horm Metab Res 28:619–632[Medline]
  6. Housknecht KL, Baile CA, Matteri RL, Spurlock ME 1998 The biology of leptin: a review. J Anim Sci 76:1405–1420[Abstract/Free Full Text]
  7. Himms-Hagen J 1999 Physiological roles of the leptin endocrine system: differences between mice and humans. Crit Rev Clin Lab Sci 36:575–655[CrossRef][Medline]
  8. Glasow A, Haidan A, Hilbers U, Breidert M, Gillespie J, Scherbaum WA, Chrousos GP, Bornstein SR 1998 Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab 83:4459–4466[Abstract/Free Full Text]
  9. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543[Abstract/Free Full Text]
  10. Ioffe E, Moon B, Connolly E, Friedman JM 1998 Abnormal regulation of the leptin gene in the pathogenesis of obesity. Proc Natl Acad Sci USA 95:11852–11857[Abstract/Free Full Text]
  11. Hardie LJ, Rayner DV, Holmes S, Trayhurn P 1996 Circulating leptin levels are modulated by fasting, cold exposure and insulin administration in lean but not Zucker (fa/fa) rats as measured by ELISA. Biochem Biophys Res Commun 223:660–665[CrossRef][Medline]
  12. Trayhurn P, Duncan JS, Hoggard N, Rayner DV 1998 Regulation of leptin production: a dominant role for the sympathetic nervous system? Proc Nutr Soc 57:413–419[CrossRef][Medline]
  13. Morita S, Kobayashi K, Hidaka H, Nagatsu T 1992 Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase. Mol Brain Res 13:313–319[Medline]
  14. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH 1980 Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77:7380–7384[Abstract/Free Full Text]
  15. Heid CA, Stevens J, Livak KJ, Williams PM 1996 Real time quantitative PCR. Genome Res 6:986–994[Abstract/Free Full Text]
  16. Passonneau JV, Lauderdale VR 1974 A comparison of three methods of glycogen measurement in tissues. Anal Biochem 60:405–412[CrossRef][Medline]
  17. Trayhurn P, James WP, Gurr MI 1979 Studies on the body composition, fat distribution and fat cell size and number of ‘Ad’, a new obese mutant mouse. Br J Nutr 41:211–221[CrossRef][Medline]
  18. Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD 1988 Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 85:836–840[Abstract/Free Full Text]
  19. Cadd GG, Hoyle GW, Quaife CJ, Marck B, Matsumoto AM, Brinster RL, Palmiter RD 1992 Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice. Mol Endocrinol 6:1951–1960[Abstract]
  20. Kobayashi K, Sasaoka T, Morita S, Nagatsu I, Iguchi A, Kurosawa Y, Fujita K, Nomura T, Kimura M, Katsuki M, Nagatsu T 1992 Genetic alteration of catecholamine specificity in transgenic mice. Proc Natl Acad Sci USA 89:1631–1635[Abstract/Free Full Text]
  21. Mantzoros CS, Qu D, Frederich RC, Susulic VS, Lowell BB, Maratos-Flier E, Flier JS 1996 Activation of ß(3) adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 45:909–914[Abstract]
  22. Lafontan M, Bousquet-Melou A, Galitzky J, Barbe P, Carpene C, Langin D, Berlan M, Valet P, Castan I, Bouloumie A 1995 Adrenergic receptors and fat cells: differential recruitment by physiological amines and homologous regulation. Obes Res 3:507S–514S
  23. Vicario PP, Candelore MR, Schaeffer MT, Kelly L, Thompson GM, Brady EJ, Saperstein R, MacIntyre DE, Tota LM, Cascieri MA 1998 Desensitization of ß3-adrenergic receptor-stimulated adenylyl cyclase activity and lipolysis in rats. Life Sci 62:627–638[CrossRef][Medline]
  24. Liggett SB, Freedman NJ, Schwinn DA, Lefkowitz RJ 1993 Structural basis for receptor subtype-specific regulation revealed by a chimeric ß3/ß2-adrenergic receptor. Proc Natl Acad Sci USA 90:3665–3669[Abstract/Free Full Text]
  25. Reynisdottir S, Wahrenberg H, Carlstrom K, Rossner S, Arner P 1994 Catecholamine resistance in fat cells of women with upper-body obesity due to decreased expression of ß2-adrenoceptors. Diabetologia 37:428–435[Medline]
  26. Bougneres P, Stunff CL, Pecqueur C, Pinglier E, Adnot P, Ricquier D 1997 In vivo resistance of lipolysis to epinephrine. A new feature of childhood onset obesity. J Clin Invest 99:2568–2573[Medline]
  27. Siegrist-Kaiser CA, Pauli V, Juge-Aubry CE, Boss O, Pernin A, Chin WW, Cusin I, Rohner-Jeanrenaud F, Burger AG, Zapf J, Meier CA 1997 Direct effects of leptin on brown and white adipose tissue. J Clin Invest 100:2858–2864[Medline]
  28. O’Doherty RM, Anderson PR, Zhao AZ, Bornfeldt KE, Newgard CB 1999 Sparing effect of leptin on liver glycogen stores in rats during the fed-to-fasted transition. Am J Physiol 277:E544–E550
  29. Chrousos GP, Gold PW 1992 The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 267:1244–1252[Abstract]
  30. Ehrhart-Bornstein M, Hinson JP, Bornstein SR, Scherbaum WA, Vinson GP 1998 Intraadrenal interactions in the regulation of adrenocortical steroidogenesis. Endocr Rev 19:101–143[Abstract/Free Full Text]
  31. Young JB, Rosa RM, Landsberg L 1984 Dissociation of sympathetic nervous system and adrenal medullary responses. Am J Physiol 247:E35–E40
  32. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS 1996 Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252[CrossRef][Medline]
  33. Schwartz MW, Seeley RJ 1997 Neuroendocrine responses to starvation and weight loss. N Engl J Med 336:1802–1811[Free Full Text]
  34. Flier JS 1998 Whats in a name? In search of leptins physiological role. J Clin Endocrinol Metab 83:1407–1413[Free Full Text]
  35. Wang MY, Lee Y, Unger RH 1999 Novel form of lipolysis induced by leptin. J Biol Chem 274:17541–17544[Abstract/Free Full Text]
  36. Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS 1996 Role of leptin in fat regulation. Nature 380:677–677[CrossRef][Medline]
  37. Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI 1997 Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 100:270–278[Medline]
  38. Böttner, A, Scherbaum WA, Chrousos GP, and Bornstein SR Leptin expression in the mouse adrenal gland. 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p P2–404 (Abstract)
  39. Takekoshi K, Motooka M, Isobe K, Nomura F, Manmoku T, Ishii K, Nakai T 1999 Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. Biochem Biophys Res Commun 261:426–431[CrossRef][Medline]
  40. Tataranni PA 1998 From physiology to neuroendocrinology: a reappraisal of risk factors of body weight gain in humans. Diabetes Metab 24:108–115[Medline]
  41. Bornstein SR 1997 Is leptin a stress related peptide? Nat Med 3:937–937[CrossRef][Medline]
  42. Eisenhofer G, Friberg P, Rundqvist B, Quyyumi AA, Lambert G, Kaye DM, Kopin IJ, Goldstein DS, Esler MD 1996 Cardiac sympathetic nerve function in congestive heart failure. Circulation 93:1667–1676[Abstract/Free Full Text]
  43. Bornstein SR, Licinio J, Tauchnitz R, Engelmann L, Negrao AB, Gold P, Chrousos GP 1998 Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm, in cortisol and leptin secretion. J Clin Endocrinol Metab 83:280–283[Abstract/Free Full Text]
  44. Böttner A, Eisenhofer G, Friberg P, Rundqvist B, Bornstein SR 1999 Hyperleptinaemia does not correlate with plasma catecholamine levels in chronic heart failure. Eur Heart J 20:1051–1052[Free Full Text]
  45. Böttner A, Eisenhofer G, Torpy DJ, Ehrhart-Bornstein M, Keiser HR, Chrousos GP, Bornstein SR 1999 Lack of leptin suppression in response to hypersecretion of catecholamines in pheochromocytoma patients. Metabolism 48:543–555[CrossRef][Medline]
  46. Bornstein SR, Böttner A, Chrousos GP 1999 Knocking out the stress response. Mol Psychiatry 4:403–407[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
P. Keller, C. Keller, A. Steensberg, L. E. Robinson, and B. K. Pedersen
Leptin gene expression and systemic levels in healthy men: effect of exercise, carbohydrate, interleukin-6, and epinephrine
J Appl Physiol, May 1, 2005; 98(5): 1805 - 1812.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Deng, K. Muthu, R. Gamelli, R. Shankar, and S. B. Jones
Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis
Am J Physiol Cell Physiol, September 1, 2004; 287(3): C730 - C736.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
E. Charmandari, M. Weise, S. R. Bornstein, G. Eisenhofer, M. F. Keil, G. P. Chrousos, and D. P. Merke
Children with Classic Congenital Adrenal Hyperplasia Have Elevated Serum Leptin Concentrations and Insulin Resistance: Potential Clinical Implications
J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2114 - 2120.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Böttner, A.
Right arrow Articles by Bornstein, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Böttner, A.
Right arrow Articles by Bornstein, S. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals