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Endocrinology Vol. 147, No. 10 4638-4645
Copyright © 2006 by The Endocrine Society

The Prolactin-Deficient Mouse Has an Unaltered Metabolic Phenotype

Christopher R. LaPensee, Nelson D. Horseman, Patrick Tso, Terry D. Brandebourg, Eric R. Hugo and Nira Ben-Jonathan

Departments of Cell Biology (C.R.L., T.D.B., E.R.H., N.B.-J.), Physiology (N.D.H.), and Pathology (P.T.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Dr. Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati, 3125 Eden Avenue, Cincinnati, Ohio 45267-0521. E-mail: Nira.Ben- Jonathan{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prolactin (PRL), best recognized for its lactogenic activity, is also involved in the regulation of metabolic homeostasis in both mammalian and nonmammalian species. Although several mouse models have been used to study the metabolic functions of PRL, a clear-cut consensus has not emerged given the limited and often conflicting data. To clarify the role of PRL in metabolic homeostasis in males and nonlactating females, we used the PRL-deficient mouse. Our objectives were to compare: 1) weight gain, 2) body composition, 3) serum lipid profile, 4) circulating leptin and adiponectin levels, and 5) glucose tolerance in PRL knockout, heterozygous, and wild-type mice maintained on standard chow, high-fat, or low-fat diets. In addition, we compared the lipolytic actions of PRL using adipose tissue explants from mice, rats, and humans. We are reporting that PRL deficiency does not affect the rate of weight gain, body composition, serum lipids, or adiponectin levels in either sex on any diet. Glucose tolerance was slightly impaired in very young PRL knockout male pups but not in adults or in females at any age. Leptin was elevated in male, but not female, PRL knockout mice maintained on a low-fat diet. PRL did not affect lipolysis in adipose tissue explants from mice but significantly inhibited glycerol release from both rat and human adipose explants in a dose-dependent manner. We conclude that PRL deficiency has negligible gross metabolic effects in mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLACTIN (PRL) IS a multifunctional, 23-kDa pituitary hormone best known for its lactogenic properties. However, PRL has many physiological functions that are not limited to lactation, including osmoregulation, immune regulation, and metabolism. In lower vertebrates, PRL regulates metabolic processes such as body weight and lipid content in fish and food intake and weight gain in birds (1, 2). In addition, accumulating evidence indicates that PRL is involved in several aspects of metabolic homeostasis in nonlactating female and male mammals (3).

PRL has well-defined metabolic activities in target tissues such as the mammary gland, where it stimulates the synthesis of milk proteins, lactose, and lipids (4), the pancreas, where it promotes ß-cell growth and insulin production (5, 6), and the prostate, where it increases citrate biosynthesis (7). There is also an emerging recognition that PRL directly regulates adipose tissue function. Several isoforms of the PRL receptor (PRLR) are expressed in white and brown adipose tissue of a number of species (8, 9, 10, 11, 12). Studies with primary adipocytes, cell lines, or adipose tissue explants demonstrate that PRL suppresses lipogenesis by down-regulating lipoprotein lipase (LPL) and fatty acid synthase (10, 13, 14), whereas its lipolytic activity varies across species (15, 16, 17).

PRL also regulates the production and secretion of adipokines. Adipose tissue is a dynamic organ, acting both as an energy storage depot and as a hormone-secreting endocrine tissue. Adipokines affect metabolic homeostasis by acting on the brain, liver, pancreas, and muscle (18, 19). Recent reports indicate that PRL inhibits the production of IL-6 (8) and adiponectin (20, 21), both of which are involved in obesity-related insulin resistance. On the other hand, the relationship between PRL and leptin, a satiety factor that regulates food intake and energy expenditure, is less clear (22, 23, 24, 25).

Body weight and adiposity are coordinated in a complex manner by nutritional factors, hormonal signals, and energy expenditure. Information on the involvement of PRL with these parameters is primarily based on observations made in rats, whereby long-term elevation of circulating PRL results in increased body weight with inconsistent changes in adipose tissue mass (26, 27, 28, 29). In humans, chronic hyperprolactinemia is sometimes associated with weight gain, which can be reversed upon treatment with bromocriptine, which normalizes serum PRL levels (30, 31). However, this weight loss does not occur in all patients, is modest and delayed, and does not correlate well with the rapid suppression of serum PRL. Moreover, bromocriptine may have independent effects on weight gain and adiposity.

Mice are used extensively to study metabolic homeostasis, given the ease of generating transgenics overexpressing or lacking specific genes. Despite the use of several mouse models to examine the metabolic functions of PRL, limited and often conflicting data are available. In PRL-overexpressing females, a minor decrease in adiposity was observed with no difference in body weight (9). A 2001 study, using the PRLR-deficient mouse, reported a significant reduction in the rate of weight gain and a marked decrease in abdominal fat mass (22), whereas a more recent study found no difference in these animals (32). Paradoxically, male mice with chronic hyperprolactinemia have lower epididymal fat content (33).

To clarify the metabolic actions of PRL, we used the PRL-deficient mouse (34). A major advantage of this experimental model is that the PRLR is expressed and functional, enabling the application of replacement therapy aimed at reversing any metabolic alterations caused by PRL deficiency. Our objectives were to compare: 1) weight gain, 2) body composition, 3) serum lipid profile, 4) circulating leptin and adiponectin concentrations, and 5) glucose tolerance in PRL knockout, heterozygous, and wild-type mice fed standard chow (SC), high-fat (HF), or low-fat (LF) diets. We also examined whether PRL deficiency altered PRLR expression in adipose tissue and liver. In addition, we compared the lipolytic activity of PRL using adipose tissue explants from wild-type mice, PRL knockout mice, rats, and humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The generation of PRL knockout mice has been described previously (34). Wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice were generated by breeding +/– or –/– males with +/– females. Pups were genotyped by PCR analysis of DNA isolated from tail clippings. Primers targeted to the 5' sequence of the neomycin resistance gene (PKG-neo), inserted in the coding region of the PRL protein, amplified an expected product in +/– and –/– mice. Primers for the 5' sequence of the PRL gene, upstream of the neo insert, yielded an expected product in +/+ and +/– mice, but not in –/– mice. A common 3' primer for exon 4 of the PRL gene was used to amplify both products. Adult male rats (Sprague Dawley) were obtained from Harlan (Indianapolis, IN). Mice and rats were housed on a 12-h light, 12-h dark cycle (lights on at 0600 h), with food and water available ad libitum unless otherwise specified. Animals were fasted overnight before killed by CO2 inhalation and cervical dislocation between 0900 and 1100 h. Blood was collected by cardiac puncture and centrifuged at 5000 x g for 10 min at 4 C. Serum was kept at –80 C for later analysis. Liver and adipose tissues (epididymal or periuterine) were removed and frozen for PRLR analysis by RT-PCR. Animal protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Human patients
Fresh sc abdominal adipose tissue was obtained from patients undergoing abdominoplasty. The study was approved by the University of Cincinnati Institutional Review Board, and informed consent was obtained from each patient.

NB2 bioassay for PRL
Rat Nb2 lymphocytes were cultured as previously described (35). Briefly, cells were plated in 96-well plates (20,000 cells/well) and incubated with mouse PRL (mPRL) (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) in triplicate and mouse serum in duplicate (0.5, 1, and 2 µl). After 3 d, cell number was determined by the [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide method. The amount of PRL in the serum was calculated from the standard curve, with a lowest detectable level of 0.2 ng/ml.

Real-time PCR
Total RNA was extracted using Tri Reagent (MRC, Cincinnati, OH), and 5 µg RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) as previously described (8). Real-time PCR was performed on 200 ng cDNA using intron-spanning primers for mPRLR (forward, 5'-CAC AGT AAA TGC CAC GAA CG-3'; reverse, 5'-GGC AAC CAT TTT ACC CAC AG-3') and ß-actin as a control (forward, 5'-ACT GCT CTG GCT CCT AGC AC-3'; reverse 5'-AGT CCG CCT AGA AGC ACT TG-3'). The PRLR primers (located in the extracellular domain of the receptor) were designed so as to detect all isoforms. Products were amplified on a SmartCycler I (Cepheid, Sunnyvale, CA) using Immolase heat-activated Taq DNA polymerase (Bioline, Randolph, MA) and SYBR Green I (Invitrogen) for fluorometric product detection. Cycle parameters were 96 C for 6 min for polymerase activation, followed by 40 cycles of 95 C for 15 sec, 57 C for 15 sec, and 72 C for 25 sec, and optical read stage at 83.5 C for 6 sec. Product purity was confirmed by DNA melting curve analysis. Fold changes in gene expression were calculated from the cycle threshold measurements, using the method of Pfaffl et al. (36), and data were expressed as fold induction vs. wild-type mice.

Diets
Mice at 28 d of age were randomly divided into three experimental diet groups. SC, obtained from Harlan, was comprised of 10% fat, 20% protein, and 3% fiber. HF and LF diets were prepared by Dyets Inc. (Bethlehem, PA). The HF diet (20% fat) contained 20 g fat/100 g of food (19 g butter fat and 1 g soybean oil to provide essential fatty acids). The LF diet contained 4% fat (3 g butter fat and 1 g soybean oil). Protein, essential minerals, and vitamins were equal in the HF and LF diets. Animals were maintained on the specific diets for at least 3 months and were weighed weekly.

Body composition
Quantitative magnetic resonance (QMR; Echo Medical Systems, Houston, TX) was used for in vivo body composition analysis. Mice from each diet group were analyzed at 14 wk of age. The percent lean and fat body mass were calculated by dividing tissue mass by total body weight.

Serum lipid profile
Triglycerides were measured using a kit from Randox (Crumlin, UK). The Wako enzymatic method was used to measure phospholipids and nonesterified fatty acids (37). Cholesterol was measured by a colorimetric assay from Thermo Electron Corporation (Waltham, MA).

Leptin and adiponectin determination
Serum leptin and adiponectin were measured using ELISA kits purchased from R&D Systems (Minneapolis, MN). The lowest level of detection was 62.5 pg/ml for leptin and 160 pg/ml for adiponectin.

Glucose tolerance test
Glucose tolerance was measured at 4, 8, and 12 wk of age. Before the test, animals were fasted overnight. Tail blood was obtained 15 min before and 15, 30, 60, and 120 min after injection of D-glucose (10% in water, 10 µl/g body weight). Blood was directly analyzed for glucose using a One Touch glucometer (Lifescan, Milpitas, CA).

Lipolysis
Rats or mice were fasted overnight before the experiment. Epididymal fat was removed, minced, and blood vessels carefully dissected out. Subcutaneous abdominal fat was obtained from patients undergoing abdominoplasty. In each case, explants (four to five pieces totaling approximately 50 mg) were incubated in Medium 199 (Cellgro, Herndon, VA) containing 1% charcoal stripped serum (Hyclone, Logan, UT). Samples were incubated with recombinant ovine PRL (for mice or rats) or human PRL (for human explants) at 0, 1, 5, and 25 ng/ml for 24 h. For basal lipolysis, explants were incubated in 200 µl Krebs-Ringers-HEPES, 1.8 mM CaCl, 1 mM sodium pyruvate, 1% charcoal stripped serum including the same PRL doses as above for 2 h. Conditioned media were collected and replaced with the same medium containing 100 nM isoproterenol for 2 h. Conditioned media were again collected and glycerol release was measured by a colorimetric assay (Sigma). Glycerol release was calculated from a standard curve and normalized by tissue weight. Data are expressed as nmol glycerol/mg tissue/2 h.

Data analysis
Statistical differences were determined by one-way ANOVA followed by Fisher LSD post hoc analysis. For glucose tolerance, the area under the curve was calculated by GraphPAD PRIZM. All experiments were repeated at least three times. P values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum PRL levels in the three mouse genotypes
The Nb2 bioassay was used to measure serum PRL levels in males and randomly cycling females. As shown in Fig. 1Go, left, Nb2 cell number showed a curvilinear relationship with the amount of mPRL from 0–75 pg/well. Addition of 0.5, 1, and 2 µl serum from +/+ males showed parallelism with the linear portion of the curve. A similar dose-response relationship was observed using serum from +/– mice (data not shown). Serum from –/– mice was below the lower limit of detection in all aliquots tested. Serum PRL concentrations were higher in females than males (Fig. 1Go, table inset), with no apparent differences between +/– and +/+ of either sex. Fig. 1Go, right, shows that PRLR expression in adipose tissue and liver was unchanged in all female genotypes. Similar results were obtained in liver and epididymal adipose tissue of males (data not shown).


Figure 1
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  		FIG. 1. Left , Determination of serum PRL levels in wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice using the Nb2 assay. Nb2 cells, rat lymphocytes that proliferate in the presence of PRL in a dose-dependent manner, were incubated with increasing concentrations of mPRL or mouse serum (0.5, 1, and 2 µl) for 3 d. Optical density was determined by the [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay. Inset, Serum PRL levels were calculated from the standard curve. Each value is mean ± SEM, n = 8 mice per group. M, Males; F, females; ND, nondetectable. Right, PRLR gene expression in liver and periuterine adipose tissue of female mice, as determined by real-time RT-PCR. Values were calculated as relative gene expression vs. that in wild-type mice. Each value is mean ± SEM, n = 8 mice per group. Similar results were obtained in males.

 
Body weight and composition
To compare the growth rate of the three genotypes, animals were placed under different diet paradigms and weighed weekly. Weaning weights of both PRL–/– males and females were comparable with +/+ and +/– litter mates. There was no difference in growth rates among the three genotypes of either females (Fig. 2AGo) or males (Fig. 2BGo) on SC. Animals on the LF diet gained weight at a slower rate than those fed SC, with no apparent differences among the three genotypes. As expected, mice on the HF diet gained considerably more (~20%) weight than those on the LF diet, but again, there were no differences in body weight among the three genotypes of either males or females. PRL–/– males on the HF diet showed a tendency for a heavier weight than +/– or +/+ mice, but the difference was not statistically significant.


Figure 2
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  		FIG. 2. Growth curves of female (A) and male (B) wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice maintained on SC, LF, or HF diets. Each value is mean ± SEM, n = 8–10 for females and 20–30 for males.

 
QMR was used to compare body composition in the HF and LF experimental groups at 14 wk of age. As expected, both males and females on the HF diet accumulated approximately twice as much body fat as those on the LF diet (Fig. 3Go, left). However, no differences in the percentage of body fat were evident among the three genotypes maintained on either diet. PRL–/– females on the HF diet showed a tendency for a higher body fat content, but the difference was significant only compared with +/– females. No significant changes in lean tissue mass were observed among all experimental groups (Fig. 3Go, right). A group of male mice on the LF diet was subjected to body composition analysis at 4, 8, and 12 wk of age. However, there were no differences in either fat or lean tissue mass among the three genotypes at any time (data not shown).


Figure 3
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  		FIG. 3. Body composition of female and male wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice maintained on the HF or LF diet for 12–14 wk. Left and right, Percent body fat and lean tissue, respectively, as determined by EchoMRI. Each value is mean ± SEM, n = 8–10 for females and 20–30 for males.

 
Serum lipid profile
Serum from males maintained on the HF, LF, or SC diets for 12–14 wk was analyzed for triglycerides, cholesterol, phospholipids, and nonesterified free fatty acids (FFAs). As shown in Fig. 4Go, there were no differences in these parameters among the three genotypes on any diet. HF food increased cholesterol levels compared with SC and LF food but without significant differences among the three genotypes. Analysis of serum from females also showed no differences, except for decreased FFA in –/– mice fed LF food (data not shown).


Figure 4
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  		FIG. 4. Serum lipids profile of male wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice maintained on the HF, SC, or LF diet for 12–14 wk. Blood was collected after an overnight fast by cardiac puncture and analyzed for phospholipids, triglycerides, cholesterol, and FFAs. Each value is mean ± SEM, n = 8 mice per group.

 
Elevated leptin in male mice on the LF diet
Serum from mice maintained on the HF and LF diets for 12–14 wk were analyzed for leptin and adiponectin. As shown in Fig. 5Go, left, leptin concentration was significantly higher (6.2 ± 0.7 ng/ml) in –/– males on the LF diet compared with +/– (3.8 ± 0.7 ng/ml) and +/+ (4.0 ± 0.6 ng/ml) mice. No differences were observed in females on the LF diet (data not shown). As expected, leptin was significantly higher in mice on the HF diet, but no differences were observed among the three genotypes in either males or females. Adiponectin levels (Fig. 5Go, right) were unchanged between –/– and +/+ males on either diet. Females had 50% higher serum adiponectin levels than males but also showed no difference among the genotypes (data not shown).


Figure 5
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  		FIG. 5. Serum leptin and adiponectin concentrations in male wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) mice maintained on the HF or LF diet for 12–14 wk. Blood was collected after an overnight fast by cardiac puncture and analyzed for leptin and adiponectin by the respective mouse-specific ELISA kits. Each value is mean ± SEM, n = 8 mice per group. *, P < 0.05 vs. +/+ or +/– mice.

 
Impaired glucose tolerance response only in 4-wk-old male mice
Males were subjected to glucose tolerance tests at 4, 8, and 12 wk of age. At all ages, fasted blood glucose levels were similar in the three genotypes. In 4-wk-old mice, peak glucose levels after ip glucose injection were comparable in all genotypes (Fig. 6Go, left). However, the total area under the curve in –/– mice was significantly (P < 0.05) higher than that in +/+ mice. By 8 and 12 wk of age, there were no differences in glucose tolerance among the three genotypes (Fig. 6Go, middle and right). At 4 and 8 wk of age, blood glucose levels after ip glucose injection were similar in all female genotypes (data not shown).


Figure 6
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  		FIG. 6. Glucose tolerance determination in wild-type (+/+), heterozygous (+/–), and PRL knockout (–/–) male mice at 4, 8, or 12 wk of age. After an overnight fast, glucose levels were measured in blood collected from the tail vein before and after an ip injection of 10% D-(+)-glucose (10 µl/g body weight). Each value is mean ± SEM, n = 8–13 mice per group. At 4 wk of age, the area under the curve was significantly (P < 0.05) higher in –/– than +/+ mice.

 
PRL inhibits lipolysis in adipose explants from rats and humans but not mice
The effect of PRL on lipolysis was compared in epididymal adipose tissue explants obtained from mice and rats and sc explants from humans. As evident in Fig. 7Go, upper panels, PRL did not affect basal or isoproterenol-stimulated lipolysis in either +/+ or –/– mice. On the other hand, PRL at physiological concentrations caused a significant, dose-dependent inhibition of both basal and isoproterenol-stimulated lipolysis in adipose explants from rats and humans (Fig. 7Go, lower panels). In all cases, isoproterenol-stimulated glycerol release was 2- to 4-fold above basal release.


Figure 7
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  		FIG. 7. Effects of PRL on basal- and isoproterenol-stimulated lipolysis in adipose tissue explants from mice, rats, and humans. Epididymal (mice and rats) and sc abdominal (nonobese women) explants were incubated with PRL (0, 1, 5, and 25, ng/ml) for 24 h. Lipolysis was determined as described in Materials and Methods. Data are expressed as nanomoles of glycerol per milligram of tissue per 2 h. Each value is mean ± SEM of five replicates. *, P < 0.05 vs. control explants. Each experiment was repeated two to three times with similar results. A similar effect of PRL on lipolysis was seen with sc abdominal explants obtained from nonobese men.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We are reporting that PRL deficiency in mice does not alter the rate of weight gain, body composition, serum lipids, or circulating adiponectin levels. There are no differences in PRLR expression in either liver or adipose tissue among the three genotypes. Glucose tolerance is impaired in very young PRL knockout males but not in adult males or females at any age. Serum leptin levels are elevated in male, but not female, PRL knockout mice maintained on the LF diet. PRL does not affect lipolysis in adipose tissue explants from mice but has a significant, dose-dependent inhibition of lipolysis in explants from rats and humans. These comprehensive studies indicate that although PRL deficiency has minor effects on leptin release and glucose tolerance, it does not result in global changes in weight gain and adiposity in mice of either sex. The suppression of lipolysis by PRL in adipose tissue from rats and humans, but not mice, gives further credence to the notion that the metabolic effects of PRL are species specific.

Using the very sensitive Nb2 bioassay, we confirm that PRL is undetectable in serum from –/– male or female mice. As expected, serum PRL levels are higher in wild-type or heterozygous females than males. The similarity of circulating PRL levels in +/+ and +/– mice of either sex indicates that loss of one PRL allele does not affect overall PRL production/release. However, because PRL levels may differ between wild-type and heterozygous females during the estrous cycle or under stress in either males or females, we included heterozygotes in all our studies. Notably, all of our studies were conducted during the first 5 months of life, before the formation of pituitary tumors. These develop with aging in both PRL- and PRLR-deficient mice (38, 39) and likely result in altered pituitary function.

Because the metabolic status of an organism depends on its nutritional as well as hormonal status, mice were subjected to several diet paradigms. For the longitudinal analysis of growth rates, we used a large number of mice of both sexes to ensure the significance of any observations. PRL deficiency did not affect weight gain in males or females under any diet paradigm (Fig. 2Go). As revealed by QMR, a precise tool for measuring total fat within an intact organism, PRL deficiency did not alter fat content in these mice (Fig. 3Go). Our data conflict with the report on reduced weight gain in PRLR-deficient mice (22), which was not reproduced in later studies by the same authors (32, 40). Similarly, our results do not agree with the report that both male and female PRLR-deficient mice had a marked reduction in abdominal fat mass at 8–9 months of age, as determined by gross dissection of abdominal fat (22). Indeed, a subsequent study, using dual-energy x-ray absorptiometry, revealed no difference in fat content of either 8- to 12-month-old males or 4-month-old females (32). Notably, some studies with the PRLR-deficient mouse did not take into account the confounding influence of pituitary tumors in older animals.

Studies from other laboratories also failed to find a clear relationship among PRL, weight gain, and adiposity in mice. For example, minor changes in weight gain and fat content were seen in mice made hyperprolactinemic by PRL overexpression or pituitary grafts (9, 33). Suppression of PRL with bromocriptine in ob/ob male mice caused no changes in body weight, with slight reductions in food intake and hyperglycemia (41). In contrast to mice, chronic elevation of PRL in rats has consistently been linked to increased food intake and weight gain, whereas PRL suppression resulted in the opposite outcome (26, 27, 42, 43). Sustained hyperprolactinemia in humans, caused by pituitary tumors or treatment with antipsychotic drugs, is occasionally accompanied by increased weight (30, 31, 44, 45), but it is unclear whether the elevated PRL is causative or coincidental to the weight gain.

During lactation, PRL acts as a physiological sensor that responds to the demands for milk production by partitioning lipids away from adipose tissue in favor of the mammary gland (3). In lactating animals, PRL suppresses LPL activity in adipose tissue while increasing its activity in the mammary gland (13, 46). Because LPL is the major enzyme that hydrolyzes circulating lipoprotein-triglyceride complexes, changes in its activity should have an impact on serum lipid profile. Nonetheless, it is unclear whether PRL alters lipid metabolism in nonlactating mice, with one study reporting a small reduction in serum FFA in males receiving pituitary grafts (33). Our results revealed no noticeable differences in triglycerides, cholesterol, phospholipids, or FFAs among the three genotypes in males or females on either diet (Fig. 4Go).

We observed a significant, although modest, increase in serum leptin levels in male PRL–/– mice on the LF diet but not in those on the HF diet (Fig. 5Go). Increased production of leptin driven by the HF diet-induced adiposity may have been sufficiently high to override any discernible effects of PRL deficiency. Unlike males, there was no difference in serum leptin levels between +/+ and –/– females on either diet. Our data do not support the report on lower plasma leptin levels in PRLR-deficient females (22) but agree with a later report showing no difference in leptin levels in such mice (32). Leptin secretion is differentially regulated in males and females, with estrogens stimulating and androgens inhibiting its production (47). It remains to be determined whether PRL interacts with the gonadal steroids in the regulation of leptin release. The reports of a direct effect of PRL on leptin production by adipocytes are also conflicting. In isolated mouse adipocytes, PRL alone has no effect on leptin but inhibits insulin-induced leptin production (24). In contrast, treatment with PRL and insulin increases leptin release from T37i brown adipocytes (25). These discrepancies may be due to differences in experimental design, doses of PRL or depot-specific control of leptin release.

Adiponectin production is higher in females than males and is negatively correlated with adiposity (48). An inhibitory effect of PRL on adiponectin release in female mice has been reported in two recent studies. In normal females, infusion of PRL suppressed circulating adiponectin, whereas bromocriptine injection caused an elevation (20). Serum adiponectin was significantly lower in PRL-overexpressing females, but not males; no changes were seen in male or female PRLR-deficient mice (21). PRL also inhibited adiponectin release from sc abdominal adipose explants from healthy, nonobese women (21). Our studies reveal no difference in serum adiponectin levels between PRL–/– or +/+ mice of either sex (Fig. 5Go). Interestingly, neither the nutritional status nor adiposity altered circulating adiponectin levels, although adiponectin is known to be reduced in obese individuals. Nilsson et al. (21) suggested that adiponectin levels are more reflective of insulin sensitivity rather than adiposity.

It is well established that lactogens promote ß-cell growth and insulin production (6, 49, 50). Indeed, PRLR deficiency results in reduced ß-cell mass and islet density and impaired glucose tolerance in adult male and female mice (40). In our case, the clearance of blood glucose after glucose injection was delayed in 4-wk-old PRL–/– males (Fig. 6Go), but this impairment did not extend to older animals. We do not know whether the transient reduction of glucose tolerance was due to a delayed maturation of pancreatic function or to a lower insulin sensitivity, and if so, why the female PRL–/– pups or adult mice did not show this impairment. Because the PRLR is expressed in the pancreas in late gestation (11), exposure to placental lactogens in utero could have supported ß-cell development in our animals, whereas the PRLR-null mice are unable to respond to either PRL or placental lactogens.

An older study reported that PRL-induced lipolysis in adipose tissue explants from virgin or pregnant mice at an extremely high dose of 5 µg/ml (15). In the present studies, when used within the physiological range (1, 5, and 25 ng/ml), PRL did not affect glycerol release from mouse epididymal adipose explants from either –/– or +/+ mice (Fig. 7Go). PRL treatment at a concentration as high as 125 ng/ml also had no effect on lipolysis in mouse adipose tissue (data not shown). In contrast, treatment with low doses of PRL inhibited basal- and isoproterenol-stimulated lipolysis in rat epididymal explants by as much as 45%, with a lesser, although significant, inhibitory effect on lipolysis in sc adipose explants obtained from women. Similar results were observed in samples obtained from men (data not shown). The ligand used to treat mouse and rat explants was ovine PRL, which is active in adipocytes from both species (14, 23). Because a 2-h incubation with PRL does not affect lipolysis in rat epididymal explants (data not shown), we speculate that PRL may act by down-regulating hormone-sensitive lipase or adipose triglyceride lipase, the critical enzymes responsible for catabolism of stored triglycerides (51, 52), rather than by affecting their phosphorylation or that of perilipin, as is the case with catecholamines (53). The mechanism by which PRL inhibits lipolysis is currently under investigation.

In conclusion, PRL is a multifunctional hormone which plays modulating and adaptive roles in many physiological systems across the animal kingdom. Among the several hundred functions ascribed to PRL, not all are equally important in any one species. For example, the osmoregulatory role of PRL is best exemplified in fish, its effects on hair follicles are more pronounced in sheep, whereas the control of corpus luteum function by PRL is unique to rodents. It is not totally surprising, therefore, that PRL appears to have gross metabolic effects in rats but not in mice. Similar to mice, there is no strong evidence to suggest that PRL is an important factor in the regulation of body weight in humans. However, PRL may play a role in other aspects of metabolic homeostasis such as lipolysis and adipokine release. Although our studies yielded mostly negative data, they are important for several reasons. First, they add to the fund of knowledge on the comparative aspects of PRL. Second, they address many published controversies with respect to the metabolic activity of PRL in the mouse. And finally, they should serve as guidance for investigators wishing to study the roles of PRL in metabolic homeostasis in their selection of the most appropriate species.


   Footnotes
 
This work was supported by National Institutes of Health Grants ES012212, CA096613, and DOD BC05725 (to N.B.-J.) and DK52134 (to N.D.H.) and Susan G. Komen Breast Cancer Foundation Grant BCRT87406 (to N.B.-J.).

The authors have nothing to disclose.

First Published Online June 29, 2006

Abbreviations: FFA, Free fatty acid; HF, high fat; LF, low fat; LPL, lipoprotein lipase; mPRL, mouse PRL; PRL, prolactin; PRLR, prolactin receptor; QMR, quantitative magnetic resonance; SC, standard chow.

Received April 13, 2006.

Accepted for publication June 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Meier AH 1969 Diurnal variations of metabolic responses to prolactin in lower vertebrates. Gen Comp Endocrinol 12:55–62
  2. Buntin JD, Hnasko RM, Zuzick PH 1999 Role of the ventromedial hypothalamus in prolactin-induced hyperphagia in ring doves. Physiol Behav 66:255–261[CrossRef][Medline]
  3. Ben Jonathan N, Hugo ER, Brandebourg TD, Lapensee CR 2006 Focus on prolactin as a metabolic hormone. Trends Endocrinol Metab 117:110–116
  4. Neville MC, McFadden TB, Forsyth I 2002 Hormonal regulation of mammary differentiation and milk secretion. J Mammary Gland Biol Neoplasia 7:49–66[CrossRef][Medline]
  5. Sorenson RL, Brelje TC 1997 Adaptation of islets of Langerhans to pregnancy: ß-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res 29:301–307[Medline]
  6. Sorenson RL, Brelje TC, Hegre OD, Marshall S, Anaya P, Sheridan JD 1987 Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells. Endocrinology 121:1447–1453[Abstract]
  7. Costello LC, Franklin RB 2002 Testosterone and prolactin regulation of metabolic genes and citrate metabolism of prostate epithelial cells. Horm Metab Res 34:417–424[CrossRef][Medline]
  8. Hugo ER, Brandebourg TD, Comstock CE, Gersin KS, Sussman JJ, Ben Jonathan N 2006 LS14: a novel human adipocyte cell line that produces prolactin. Endocrinology 147:306–313[Abstract/Free Full Text]
  9. Ling C, Hellgren G, Gebre-Medhin M, Dillner K, Wennbo H, Carlsson B, Billig H 2000 Prolactin (PRL) receptor gene expression in mouse adipose tissue: increases during lactation and in PRL-transgenic mice. Endocrinology 141:3564–3572[Abstract/Free Full Text]
  10. Ling C, Svensson L, Oden B, Weijdegard B, Eden B, Eden S, Billig H 2003 Identification of functional prolactin (PRL) receptor gene expression: PRL inhibits lipoprotein lipase activity in human white adipose tissue. J Clin Endocrinol Metab 88:1804–1808[Abstract/Free Full Text]
  11. Royster M, Driscoll P, Kelly PA, Freemark M 1995 The prolactin receptor in the fetal rat: cellular localization of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation. Endocrinology 136:3892–3900[Abstract]
  12. Symonds ME, Phillips ID, Anthony RV, Owens JA, McMillen IC 1998 Prolactin receptor gene expression and fetal adipose tissue. J Neuroendocrinol 10:885–890[CrossRef][Medline]
  13. Flint DJ, Binart N, Kopchick J, Kelly P 2003 Effects of growth hormone and prolactin on adipose tissue development and function. Pituitary 6:97–102[CrossRef][Medline]
  14. Hogan JC, Stephens JM 2005 The regulation of fatty acid synthase by STAT5A. Diabetes 54:1968–1975[Abstract/Free Full Text]
  15. Fielder PJ, Talamantes F 1987 The lipolytic effects of mouse placental lactogen II, mouse prolactin, and mouse growth hormone on adipose tissue from virgin and pregnant mice. Endocrinology 121:493–497[Abstract]
  16. Houseknecht KL, Bauman DE, Vernon RG, Byatt JC, Collier RJ 1996 Insulin-like growth factors-I and -II, somatotropin, prolactin, and placental lactogen are not acute effectors of lipolysis in ruminants. Domest Anim Endocrinol 13:239–249[CrossRef][Medline]
  17. Fortun-Lamothe L, Langin D, Lafontan M 1996 Influence of prolactin on in vivo and in vitro lipolysis in rabbits. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 115:141–147[CrossRef][Medline]
  18. Ahima RS, Saper CB, Flier JS, Elmquist JK 2000 Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 21:263–307[CrossRef][Medline]
  19. Ahima RS, Flier JS 2000 Adipose tissue as an endocrine organ. Trends Endocrinol Metab 11:327–332[CrossRef][Medline]
  20. Combs TP, Berg AH, Rajala MW, Klebanov S, Iyengar P, Jimenez-Chillaron JC, Patti ME, Klein SL, Weinstein RS, Scherer PE 2003 Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin. Diabetes 52:268–276[Abstract/Free Full Text]
  21. Nilsson L, Binart N, Bohlooly Y, Bramnert M, Egecioglu E, Kindblom J, Kelly PA, Kopchick JJ, Ormandy CJ, Ling C, Billig H 2005 Prolactin and growth hormone regulate adiponectin secretion and receptor expression in adipose tissue. Biochem Biophys Res Commun 331:1120–1126[CrossRef][Medline]
  22. Freemark M, Fleenor D, Driscoll P, Binart N, Kelly P 2001 Body weight and fat deposition in prolactin receptor-deficient mice. Endocrinology 142:532–537[Abstract/Free Full Text]
  23. Gualillo O, Lago F, Garcia M, Menendez C, Senaris R, Casanueva FF, Dieguez C 1999 Prolactin stimulates leptin secretion by rat white adipose tissue. Endocrinology 140:5149–5153[Abstract/Free Full Text]
  24. Ling C, Billig H 2001 PRL receptor-mediated effects in female mouse adipocytes: PRL induces suppressors of cytokine signaling expression and suppresses insulin-induced leptin production in adipocytes in vitro. Endocrinology 142:4880–4890[Abstract/Free Full Text]
  25. Viengchareun S, Bouzinba-Segard H, Laigneau JP, Zennaro MC, Kelly PA, Bado A, Lombes M, Binart N 2004 Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes. J Mol Endocrinol 33:679–691[Abstract/Free Full Text]
  26. Baptista T, de Baptista EA, Lalonde J, Plamondon J, Kin NM, Beaulieu S, Joober R, Richard D 2004 Comparative effects of the antipsychotics sulpiride and risperidone in female rats on energy balance, body composition, fat morphology and macronutrient selection. Prog Neuropsychopharmacol Biol Psychiatry 28:1305–1311[CrossRef][Medline]
  27. Byatt JC, Staten NR, Salsgiver WJ, Kostelc JG, Collier RJ 1993 Stimulation of food intake and weight gain in mature female rats by bovine prolactin and bovine growth hormone. Am J Physiol 264:E986–E992
  28. Gerardo-Gettens T, Moore BJ, Stern JS, Horwitz BA 1989 Prolactin stimulates food intake in a dose-dependent manner. Am J Physiol 256:R276–R280
  29. Sauve D, Woodside B 2000 Neuroanatomical specificity of prolactin-induced hyperphagia in virgin female rats. Brain Res 868:306–314[CrossRef][Medline]
  30. Doknic M, Pekic S, Zarkovic M, Medic-Stojanoska M, Dieguez C, Casanueva F, Popovic V 2002 Dopaminergic tone and obesity: an insight from prolactinomas treated with bromocriptine. Eur J Endocrinol 147:77–84[Abstract]
  31. Greenman Y, Tordjman K, Stern N 1998 Increased body weight associated with prolactin secreting pituitary adenomas: weight loss with normalization of prolactin levels. Clin Endocrinol (Oxf) 48:547–553[CrossRef][Medline]
  32. Fleenor D, Oden J, Kelly PA, Mohan S, Alliouachene S, Pende M, Wentz S, Kerr J, Freemark M 2005 Roles of the lactogens and somatogens in perinatal and postnatal metabolism and growth: studies of a novel mouse model combining lactogen resistance and growth hormone deficiency. Endocrinology 146:103–112[Abstract/Free Full Text]
  33. Matsuda M, Mori T, Sassa S, Sakamoto S, Park MK, Kawashima S 1996 Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice. Life Sci 58:1171–1177[CrossRef][Medline]
  34. Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M, Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16:6926–6935[CrossRef][Medline]
  35. Zinger M, McFarland M, Ben-Jonathan N 2003 Prolactin expression and secretion by human breast glandular and adipose tissue. J Clin Endocrinol Metab 88:689–696[Abstract/Free Full Text]
  36. Pfaffl MW, Horgan GW, Dempfle L 2002 Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36
  37. Takayama M, Itoh S, Nagasaki T, Tanimizu I 1977 A new enzymatic method for determination of serum choline-containing phospholipids. Clin Chim Acta 79:93–98[CrossRef][Medline]
  38. Cruz-Soto ME, Scheiber MD, Gregerson KA, Boivin GP, Horseman ND 2002 Pituitary tumorigenesis in prolactin gene-disrupted mice. Endocrinology 143:4429–4436[Abstract/Free Full Text]
  39. Schuff KG, Hentges ST, Kelly MA, Binart N, Kelly PA, Iuvone PM, Asa SL, Low MJ 2002 Lack of prolactin receptor signaling in mice results in lactotroph proliferation and prolactinomas by dopamine-dependent and -independent mechanisms. J Clin Invest 110:973–981[CrossRef][Medline]
  40. Freemark M, Avril I, Fleenor D, Driscoll P, Petro A, Opara E, Kendall W, Oden J, Bridges S, Binart N, Breant B, Kelly PA 2002 Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology 143:1378–1385[Abstract/Free Full Text]
  41. Cincotta AH, Tozzo E, Scislowski PW 1997 Bromocriptine/SKF38393 treatment ameliorates obesity and associated metabolic dysfunctions in obese (ob/ob) mice. Life Sci 61:951–956[CrossRef][Medline]
  42. Moore BJ, Gerardo-Gettens T, Horwitz BA, Stern JS 1986 Hyperprolactinemia stimulates food intake in the female rat. Brain Res Bull 17:563–569[CrossRef][Medline]
  43. Noel MB, Woodside B 1993 Effects of systemic and central prolactin injections on food intake, weight gain, and estrous cyclicity in female rats. Physiol Behav 54:151–154[CrossRef][Medline]
  44. Kopelman PG 2000 Physiopathology of prolactin secretion in obesity. Int J Obes Relat Metab Disord 24(Suppl 2):S104–S108
  45. Baptista T, Lacruz A, Meza T, Contreras Q, Delgado C, Mejias MA, Hernandez L 2001 Antipsychotic drugs and obesity: is prolactin involved? Can J Psychiatry 46:829–834[Medline]
  46. Ros M, Lobato MF, Garcia-Ruiz JP, Moreno FJ 1990 Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation. Mol Cell Biochem 93:185–194[Medline]
  47. Mystkowski P, Schwartz MW 2000 Gonadal steroids and energy homeostasis in the leptin era. Nutrition 16:937–946[CrossRef][Medline]
  48. Berg AH, Combs TP, Scherer PE 2002 ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab 13:84–89[CrossRef][Medline]
  49. Brelje TC, Scharp DW, Lacy PE, Ogren L, Talamantes F, Robertson M, Friesen HG, Sorenson RL 1993 Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy. Endocrinology 132:879–887[Abstract]
  50. Brelje TC, Parsons JA, Sorenson RL 1994 Regulation of islet ß-cell proliferation by prolactin in rat islets. Diabetes 43:263–273[Abstract]
  51. Zechner R, Strauss JG, Haemmerle G, Lass A, Zimmermann R 2005 Lipolysis: pathway under construction. Curr Opin Lipidol 16:333–340[Medline]
  52. Yeaman SJ 2004 Hormone-sensitive lipase: new roles for an old enzyme. Biochem J 379:11–22[CrossRef][Medline]
  53. Holm C 2003 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Trans 31:1120–1124[Medline]



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