J. Anim Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ramsay, T. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ramsay, T. G.
J. Anim. Sci. 2005. 83:2066-2074
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Porcine preadipocyte proliferation and differentiation: A role for leptin?1

T. G. Ramsay2,3

ARS, USDA, Growth Biology Laboratory, Beltsville MD 20705


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig s.c. adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on six-well tissue culture plates and proliferated to confluency in 10% (vol/vol) fetal bovine serum (FBS) in Dulbecco’s modified Eagle medium/F12 (DMEM/F12; 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol) and recombinant porcine leptin at concentrations of 0 to 1,000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 µM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL). The GPDH and LPL activities are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 h after seeding cells with 10% FBS in DMEM/F12 in 25-cm2 tissue culture flasks, cells were switched to 5% FBS and supplemented with 0 to 1,000 ng of porcine leptin or 1,000 ng of murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 h on d 4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1 µM dexamethasone) for 7 d. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone, or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL. Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone, or IGF-I; however, leptin at 1,000 ng/mL stimulated a 30% increase in preadipocyte proliferation (P = 0.007; n = 6) and a 27% increase in stromal cell proliferation (P < 0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.

Key Words: Adipocyte • Cell Culture • Differentiation • Leptin • Proliferation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Adipose tissue produces a variety of hormones and growth factors that can then act on the tissue to alter both metabolism and growth of adipocytes (Kim and Moustaid-Moussa, 2000Go; Guerre-Millo, 2004Go). For example, tumor necrosis factor (Hube and Hauner, 1999Go) and IGF-I (Frick et al., 2000Go; Hausman et al., 2001Go) have been demonstrated to alter metabolic activity of adipose tissue as well as the proliferation and/or differentiation of preadipocytes, thereby affecting the total quantity of adipose tissue.

Leptin also is a paracrine functioning hormone produced by porcine adipose tissue (Ramsay et al., 1998Go) that can subsequently act on the tissue to alter lipid accretion by altering lipolysis and lipogenesis (Ramsay, 2001Go, 2004Go). Two recent reports have suggested that leptin can induce the differentiation of human (Aprath-Husmann et al., 2001Go) and rat (Machinal-Quelin et al., 2002Go) preadipocytes. Machinal-Quelin et al. (2002)Go also reported that leptin is mitogenic for preadipocytes derived from rat s.c. adipose tissue; however, it is unknown whether leptin is adipogenic for porcine adipose tissue-derived cells. A recent report indicated that peripheral leptin may stimulate apoptosis in adipocytes (Della-Fera et al., 2003Go), which would imply that leptin may decrease the proliferative capacity of the preadipocyte. The present study was designed to test the hypothesis that porcine leptin inhibits the proliferation and promotes the differentiation of the porcine preadipocyte.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Cell Culture
Dorsal s.c. adipose tissue was obtained from between the shoulder blades of 1- to 4-d-old, female, crossbred pigs (Yorkshire x Landrace) following i.v. pentobarbital sodium administration (100 mg/kg BW). Primary cultures containing pig preadipocytes were prepared by methods previously published (Ramsay et al., 1989aGo). Animal procedures were approved by the Beltsville Animal Care and Use Committee. Briefly, tissue was minced into sections of approximately 1 mm2 with scissors and then incubated with 5 mL/g of tissue in a digestion buffer comprising Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Gibco, Grand Island, NY); 100 mM HEPES; and 1.5% BSA (wt/vol, fatty acid free; Sigma A-6003, Sigma-Aldrich, St. Louis, MO), pH 7.4, containing 2 mg/mL collagenase (Type 1; Worthington Biomedical Corp., Lakewood, NJ). A fivefold excess of digestion buffer (room temperature, excluding collagenase) was added to the digestion flask after 45 min of incubation at 37°C in a shaking water bath (90 oscillations per min). Flask contents were mixed and filtered through nylon screens with 250- and 20-µm mesh openings to remove undigested tissue and large cell aggregates. The filtered cells were centrifuged at 500 x g for 10 min to separate the floating adipocytes from the pellet of stromal-vascular cells. The stromal-vascular cells were then incubated with erythrocyte lysis buffer (0.154 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) at room temperature (Hauner et al., 1989Go) for 10 min, followed by centrifugation at 500 x g for 10 min. The stromal vascular cell pellet was washed with DMEM/F12, centrifuged, and resuspended in DMEM/F12 containing 10% fetal bovine serum (FBS; vol/vol; Sigma-Aldrich).

Aliquots of the stromal-vascular fraction were removed, stained with Rappaport’s stain and counted on a hemocytometer. Cells were then diluted in DMEM/F12 containing 10% FBS (vol/vol), 10,000 U/L of penicillin sodium, 100 mg of streptomycin sulfate/L, and 250 µg of amphotericin B/L (plating medium). Cells were seeded on six-well tissue culture plates for differentiation analysis or 25-cm2 tissue culture flasks (for proliferation analysis) at a density of 1.0 x 104 cells/cm2 in plating medium. Cells were maintained at 37°C in a humidified, 5% CO2 atmosphere.

Differentiation Analysis
Six-well plates were maintained in plating medium until confluency (d 5 to 6 of culture), with medium changed every 2 d. At confluency, plating medium was replaced with test medium comprising DMEM/F12 containing 2.5% porcine serum (vol/vol; Sigma-Aldrich), 10,000 U/L of penicillin sodium, 100 mg of streptomycin sulfate/L, and 250 µg of amphotericin B/L. This served as both a control medium and a basal medium for hormone supplementation. Porcine leptin was added to the basal medium at doses of 1, 10, 100, or 1,000 ng/mL. Recombinant porcine leptin was prepared and acquired from A. Gertler (Rehovot, Israel; Raver et al., 2000). Test media were changed on alternate days until d 7 postconfluency, when enzyme analysis was performed to measure the extent of differentiation. This experiment was repeated five times with cultures derived from five different animals.

Further experiments used hormones in combination with the leptin dose response trial to assess interactive effects upon adipocyte differentiation. Insulin (100 nM), dexamethasone (1 µM), and IGF-1 (250 ng/mL) were all tested in combination with doses of 1, 10 , 100, and 1,000 ng of porcine leptin/mL. These hormones have all been demonstrated to be adipogenic for pig preadipocytes (Ramsay et al., 1989aGo,bGo). Test media were changed on alternate days until d 7 postconfluency, when enzyme analysis was performed. These experiments were repeated four times with cultures derived from four different animals.

Enzyme Analysis
Cell cultures for analysis of GPDH were homogenized by sonic dismembranation in 1.0 mL of ice-cold homogenizing buffer. Homogenizing buffer was identical to that described by Ramsay et al. (1987)Go and comprised 0.25 M sucrose, 1 mM EDTA, 5 mM Tris base, and 1 mM dithiothreitol at pH 7.4.

Sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) activity was determined by a modification of the procedure of Wise and Green (1978)Go as described previously by Ramsay et al. (1987)Go. The activity of lipoprotein lipase (LPL; EC 3.1.1.34) was assessed according to the procedures of Nilsson-Ehle and Schotz (1976)Go. These enzymes have been demonstrated to be associated with the differentiation of porcine preadipocytes (Ramsay et al., 1989aGo,bGo). Assays were linear for sample concentration and time. Variations between duplicate determinations never exceeded 4% for the culture analyses. Enzymatic data were calculated as follows: GPDH in nmol NADH oxidized·min–1·µg–1 culture protein; and LPL in nmol 3H-oleic acid released·30 min–1·µg–1 culture protein. Protein in cell homogenates was determined by a modified Lowry procedure following NaOH solubilization of trichloroacetic acid-precipitated material (Nerakur et al., 1991). Bovine serum albumin was used as the protein standard.

Proliferation Analysis
Twenty-four hours following the seeding of cells in 25-cm2 flasks, plating medium was replaced with test medium comprising DMEM/F12 with 5% fetal bovine serum (vol/vol; Sigma-Aldrich), 10,000 U/L of penicillin sodium, 100 mg of streptomycin sulfate/L, and 250 µg of amphotericin B/L. This served as both a control medium and a basal medium for hormone supplementation.

Test media comprised basal medium supplemented with 10, 100, or 1,000 ng/mL of porcine leptin or 1,000 ng/mL of mouse leptin (R&D Systems, Minneapolis MN). Porcine insulin (1 µM; Sigma-Aldrich) served as a positive control. Quadruplicate flasks were incubated with each of these test media for 5 d, with medium changes every day. On d 4 of culture, proliferation of preadipocytes and stromal cells was measured by 3H-thymidine incorporation according to the procedures of Ramsay et al. (1987Go, 1989c)Go. Quadruplicate flasks were labeled for 24 h with 0.2 µCi of [H]-thymidine in 4 mL of test media per flask. Cultures were rinsed three times with 5 mL of Earle’s salt solution after labeling and refed with 4 mL of fresh, unlabeled test media. At confluency (d 6), cultures were induced to differentiate and accumulate lipid using DMEM/F12 medium containing 2.5% pig serum (Sigma-Aldrich), 100 nM insulin, and 1 µM dexamethasone. Differentiation medium was changed on alternate days.

After 7 d of lipid filling, cultures were washed free of this medium, incubated overnight in DMEM/F12 containing 2% pig serum and then adipocytes were separated from stromal cells by Percoll density gradient centrifugation (Ramsay et al., 1987Go; 1989cGo). Cells were harvested from the culture flasks on d 13 with Hank’s salt solution containing 0.22% crystalline trypsin, 0.02% collagenase, and 0.5% BSA. Adipocytes were separated from undifferentiated preadipocytes and stromal-vascular cells by centrifugation on a solution of Percoll and Hank’s salt solution with a density of 1.02. The isolated cell fractions were placed in scintillation vials for determination of 3H-thymidine incorporation. Differentiated preadipocytes are adipocytes that formed in culture from replicating 3H-thymidine-labeled preadipocytes. Any preexisting adipocytes do not incorporate 3H-thymidine. The 7-d period following exposure to differentiation medium allows labeled preadipocytes to differentiate and accumulate sufficient lipid for separation from undifferentiated cells on a Percoll density gradient. Passage of cells isolated from the adipocyte fraction yields secondary cultures containing <5% stromal cells that can be induced to form adipocytes. The proliferation analysis was performed with six different cell cultures derived from six different pigs.

Leptin Receptor mRNA Detection
Three separate cell isolates from three different pigs were cultured under conditions identical to those described above. At d 4 of culture (1 d before confluency), total RNA was harvested from three flasks from each culture. Following differentiation with 2.5% PS, 100 nM insulin, and 1 µM dexamethasone, an additional three flasks from each culture were harvested at 48 h after introduction of the differentiation medium (early differentiation). Finally, three more flasks from each culture were harvested 5 d after introduction of differentiation medium (middle-late differentiation).

Total RNA was isolated using TRI reagent according to the manufacturer’s protocol (Sigma-Aldrich). Integrity of RNA was assessed via agarose gel electrophoresis and RNA concentration and purity were determined spectrophotometrically using A260 and A280 measurements. Reverse-transcription reactions (20 µL) consisted of 1 µg of total RNA, 50 U of SuperScript II reverse transcriptase (Invitrogen/Life Technologies, Carlsbad, CA), 40 U of an RNAse inhibitor (Invitrogen/Life Technologies), 0.5 mmol/L dNTP, and 100 ng of random hexamer primers. Polymerase chain reaction was performed in 25 µL containing 20 mmol/L Tris-HCl, pH 8.4, 50 mmol/L KCl, 1.0 µL of the reverse transcription reaction, 1.0 U of Platinum Taq DNA polymerase (Hot Start, Invitrogen/Life Technologies), 0.2 mmol/L deoxyribonucleotide triphosphate, 2.0 mmol/L Mg++ (Invitrogen/Life Technologies), and 10 pmol of the leptin receptor specific primers. Thermal cycling parameters were as follows: 1 cycle at 94°C for 2 min, followed by 30 cycles, 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 8 min. The primers for the long-form leptin receptor (Ob-Rb) were used to generate a 396-bp product: 5'-CAGTGAC ATTTGGCCCTCTT-3' (forward), 5'-AGGCCTGGGTT TCTATCTCC-3' (reverse). The PCR products were run in a 1% agarose gel stained with ethidium bromide. The Ob-Rb amplicon was excised from an agarose gel, reamplified, and run through a GenElute PCR cleanup kit (Sigma-Aldrich). The Ob-Rb amplicon was sequenced to confirm identity using automated fluorescent DNA sequencing (ABI 310, Perkin Elmer Applied Biosystems, Foster City, CA).

Statistical Analyses
The experimental model for these experiments was a completely randomized design. Data were normalized by converting data to percents, relative to basal medium (0 ng/mL leptin), to account for culture-to-culture variation. Data were analyzed by one-way ANOVA using SigmaStat software (SPSS Science, Chicago, IL). Mean separation was analyzed using Student-Newman-Keuls test. Means were defined as significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Porcine leptin in the concentration range of 1 to 1,000 ng/mL had no effect on the differentiation of the porcine preadipocyte as monitored by the activities of the differentiation associated enzymes GPDH and LPL (Figure 1Go). The activity of neither enzyme was elevated in cultures incubated with leptin relative to cultures exposed to basal medium (GPDH, P = 0.229; LPL, P = 0.538). The combination of 100 nM insulin and 1 µM dexamethasone (I+D) was included as a positive control for differentiation induction. This combination produced a 20-fold increase in GPDH activity (P = 0.003) and a fivefold increase in LPL activity (P = 0.010).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Relative enzymatic differentiation in primary cultures of porcine adipose tissue following incubation with 0 to 1,000 ng/mL porcine leptin or the combination of 100 nM insulin and 1 µM dexamethasone (I+D). Solid bars represent sn-glycerol-3 phosphate dehydrogenase (GPDH) activity, whereas hatched bars represent lipoprotein lipase (LPL) activity. Cultures were maintained on treatment media from confluency (d 5 to 6) until harvest (d 14). Mean ± SE for five trials; values are expressed as percentage of activity of cultures exposed to 2.5% pig serum without supplemental leptin (100% GPDH = 17.0 ± 1.8 nmol NADH oxidized·min–1·µg–1 culture protein; 100% LPL = 876 ± 274 nmol 3H-oleic acid released·30 min–1·µg–1 culture protein). Means for GPDH activity that do not have a common lower case letter differ, P < 0.05 (n = 5). Means for LPL activity that do not have a common capital letter differ, P < 0.05 (n = 5).

 
Porcine leptin at concentrations from 1 to 1,000 ng/mL in combination with insulin (100 nM) had no effect on preadipocyte differentiation (Figure 2Go). Insulin produced an approximately 120% increase in GPDH activity (P = 0.004), whereas leptin had no additive or negative effect on this insulin response (P = 0.504). Insulin promoted an increase in LPL activity of 82% (P = 0.046), whereas leptin had no effect on this response to insulin (P = 0.724).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 2. Relative enzymatic differentiation in primary cultures of porcine adipose tissue following incubation with 0 to 1,000 ng/mL porcine leptin and 100 nM porcine insulin. Solid bars represent sn-glycerol-3 phosphate dehydrogenase (GPDH) activity, whereas hatched bars represent lipoprotein lipase (LPL) activity. Cultures were maintained on treatment media from confluency (d 5 to 6) until harvest (d 14). Mean ± SE for four trials; values are expressed as percentage of activity of cultures exposed to 2.5% pig serum without supplemental hormones (100% GPDH = 32.7 ± 7.8 nmol NADH oxidized·min–1·µg–1 culture protein; 100% LPL = 505 ± 76 nmol 3H-oleic acid released·30 min–1·µg–1 culture protein). Means for GPDH activity that do not have a common lower case letter differ, P < 0.05 (n = 4). Means for LPL activity that do not have a common capital letter differ, P < 0.05 (n = 4).

 
Porcine leptin at concentrations from 10 to 1,000 ng/mL in combination with dexamethasone (1 µM), had no effect on preadipocyte differentiation (Figure 3Go). Dexamethasone alone promoted an approximately 250% increase in GPDH activity (P = 0.048) and an approximately 190% increase in LPL activity (P = 0.022), whereas leptin had no effect relative to this dexamethasone response (GPDH, P = 0.790; LPL, P = 0.730).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. Relative enzymatic differentiation in primary cultures of porcine adipose tissue following incubation with 0 to 1,000 ng/mL porcine leptin and 1 µM dexamethasone (Dex). Solid bars represent sn-glycerol-3 phosphate dehydrogenase (GPDH) activity, whereas hatched bars represent lipoprotein lipase (LPL) activity. Cultures were maintained on treatment media from confluency (d 5 to 6) until harvest (d 14). Mean ± SE for five trials; values are expressed as percentage of activity of cultures exposed to 2.5% pig serum without supplemental hormones (100% GPDH = 23.2 ± 8.9 nmol NADH oxidized·min–1·µg–1 culture protein; 100% LPL = 564 ± 277 nmol 3H-oleic acid released·30 min–1·µg–1 culture protein). Means for GPDH activity that do not have a common lower case letter differ, P < 0.05 (n = 4). Means for LPL activity that do not have a common capital letter differ, P < 0.05 (n = 4).

 
Insulin-like growth factor-1 (250 ng/mL) stimulated the differentiation of the porcine preadipocyte (Figure 4Go). Insulin-like growth factor I induced an 84% increase in GPDH activity (P = 0.041) and a 76% increase in LPL activity (P = 0.029). Leptin had no effect on IGF-I-induced adipocyte differentiation within the concentration range tested (GPDH, P = 0.562; LPL, P = 0.202).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Relative enzymatic differentiation in primary cultures of porcine adipose tissue following incubation with 0 to 1,000 ng/mL porcine leptin and 250 ng/mL IGF-1. Solid bars represent sn-glycerol-3 phosphate dehydrogenase (GPDH) activity, whereas hatched bars represent lipoprotein lipase (LPL) activity. Cultures were maintained on treatment media from confluency (d 5 to 6) until harvest (d 14). Mean ± SE for four trials; values are expressed as percentage of activity of cultures exposed to 2.5% pig serum without supplemental hormones (100% GPDH = 16.4 ± 2.0 nmol NADH oxidized·min–1·µg–1 culture protein; 100% LPL = 871 ± 229 nmol 3H-oleic acid released·30 min–1·µg–1 culture protein). Means for GPDH activity that do not have a common lower case letter differ, P < 0.05 (n = 45). Means for LPL activity that do not have a common capital letter differ, P < 0.05 (n = 4).

 
Porcine leptin at 1,000 ng/mL stimulated preadipocyte proliferation by 30% within the primary cultures of porcine adipose tissue (P = 0.007), as measured by 3H-thymidine incorporation (Figure 5Go). Lower concentrations had no detectable effect on proliferation (P = 0.269). Mouse leptin produced a similar response (22% greater than controls; P = 0.002). Insulin served as a positive control for the assay and produced a 67% increase in preadipocyte incorporation of 3H-thymidine (P < 0.001). Leptin had a similar mitogenic action on the stromal vascular cells (P < 0.001). Porcine leptin at 1,000 ng/mL induced a 27% increase in stromal vascular cell proliferation. Mouse leptin produced a similar response (34% greater than controls, P < 0.001). Insulin caused a doubling in 3H-thymidine incorporation (P < 0.001).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. Relative 3H-thymidine incorporation in primary cultures of porcine adipose tissue following incubation with 0 to 1,000 ng/mL porcine leptin, 1,000 ng/mL mouse leptin or 1 µM porcine insulin. Solid bars represent preadipocyte proliferation while hatched bars represent stromal-vascular cell proliferation. Quadruplicate flasks were labeled for 24 h with 0.2 µCi [H]-thymidine on d 4 of culture. At confluency (d 6), cultures were induced to differentiate and accumulate lipid. After 7 d of lipid filling, adipocytes were separated from undifferentiated preadipocytes and stromal-vascular cells by centrifugation on a solution of Percoll and Hank’s salt solution with a density of 1.02. The isolated cell fractions were placed in scintillation vials for determination of 3H-thymidine incorporation. Mean ± SE for six trials; values are expressed as percentage of 3H-thymidine incorporation into cultures exposed to 5% fetal bovine serum without supplemental hormones. Means for preadipocyte proliferation that do not have a common lowercase letter differ, P < 0.05 (n = 6). Means for stromal-vascular cell proliferation that do not have a common capitol letter differ, P < 0.05 (n = 6).

 
The absence of a significant effect of leptin on differentiation in these primary cultures led to the possibility that the leptin receptor may not be expressed by these cells in culture. The presence of Ob-Rb was examined in three separate cultures from three different pigs (Figure 6Go). The Ob-Rb was detected in all cultures at each stage of development: preconfluency, 48 h after induction of differentiation, and 5 d after induction of differentiation.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 6. Porcine long-form leptin receptor (Ob-Rb) mRNA abundance in primary cultures derived from neonatal subcutaneous adipose tissue. One microgram of total RNA from cell cultures was used in reverse transcription-PCR with cDNA primers derived from the complete coding sequence for the porcine Ob-Rb (GenBank AF092422) to generate a 396 bp product. Lanes 1 to 3 = proliferating cells; Lanes 4 to 6 = cells following 48 h of differentiation; Lanes 7 to 9 = cells following 5 d of differentiation. mw = 100- to 1,000-bp molecular weight standards.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Leptin is a protein secreted from adipose tissue with effects on feeding behavior, reproduction, and immune status (Zhang et al., 1994Go; Considine and Caro; 1997Go; Hwang et al., 1997Go). Leptin protein has been demonstrated to be expressed by porcine adipose tissue, and porcine leptin has been shown to affect feeding behavior in swine (Barb et al., 1998Go; Ramsay et al., 1998Go). Leptin receptor mRNA (long form) has been detected in a variety of porcine tissues, including adipose tissue (Lin et al., 2000Go), suggesting that leptin can interact with the adipocyte to alter metabolic activity. Recent studies have demonstrated that leptin can alter the metabolic activity of the porcine adipocyte (Ramsay, 2001Go, 2004Go). Shifts in the metabolic activity of adipose tissue can have a dramatic effect on the overall size of the adipose tissue.

The present study was conducted to determine whether the other component that regulates size of the adipose tissue is affected by leptin: the proliferation and differentiation of the preadipocyte. Regulation of the proliferation and differentiation of the preadipocyte has been well characterized (Hausman et al., 2001Go). Nonetheless, despite more than 8,000 published articles related to the topic of leptin, very few have examined a possible role for leptin to affect the processes of preadipocyte proliferation or differentiation.

Machinal-Quelin et al. (2002)Go used GPDH activity and LPL mRNA expression to demonstrate that murine leptin can promote the differentiation of the rat preadipocyte. They demonstrated that 48-h exposure to 10 nM leptin could produce a 50% increase in GPDH activity and a threefold increase in LPL mRNA levels. This adipogenic activity may function through peroxisome proliferator-activated receptor-{gamma}, as the expression of this transcription factor was increased by 50% (Machinal-Quelin et al., 2002Go). The present study could not replicate these observations.

Aprath-Husmann et al. (2001)Go reported that 500 ng/mL of human recombinant leptin could accelerate the differentiation of the human preadipocyte as indicated by a 50 to 80% increase in GPDH activity; however, the authors noted that the final number of formed adipocytes was unchanged at the end of their 18-d culture period with leptin. This finding would imply that there was only a transient effect on differentiation, accelerating the process but not promoting the formation of new adipocytes. Data from the present study agree with the observation of Aprath-Husmann et al. (2001)Go; porcine leptin did not alter the differentiation of the porcine preadipocyte. Leptin in a concentration range of 1 to 1,000 ng/mL of medium could not affect the differentiation program as assessed by changes in both GPDH and LPL activity. Leptin was used in combination with other hormones to determine whether there might be a synergistic or inhibitory effect of leptin on differentiation. Other hormones have been demonstrated to function synergistically to promote differentiation; insulin and dexamethasone promote higher differentiation rates than each hormone alone (Ramsay et al., 1989aGo), and as seen in the present study, insulin and dexamethasone in combination in the first experiment produced much higher levels of GPDH and LPL activity than insulin in the second experiment or dexamethasone in the third experiment. Nonetheless, leptin did not have synergistic interactions with insulin, dexamethasone, or IGF-I and their actions on the differentiation program, nor did leptin have any detectable inhibitory activity on insulin-, dexamethasone-, or IGF-I induced differentiation. Leptin has been reported to inhibit the differentiation of human marrow preadipocytes (Thomas et al., 1999Go) and has been shown to inhibit insulin action at the porcine adipocyte (Ramsay, 2001Go, 2004Go). Nevertheless, these inhibitory actions do not seem to extend to the porcine subcutaneous preadipocyte.

The inability of leptin to affect differentiation was surprising based on the previous studies with human and rat adipose tissue. The most evident cause would be that the leptin receptor is not expressed in the adipose tissue that contains the preadipocyte. However, Chen et al. (2000)Go and Lin et al. (2000)Go reported the expression of leptin receptor mRNA in porcine adipose tissue. In addition, previous studies using immunocytochemistry with primary cultures of human adipose tissue have demonstrated that leptin receptors are expressed on both adipocytes and stromal vascular cells during culture (Bornstein et al., 2000Go). Machinal-Quelin et al. (2002)Go reported that Ob-Rb was expressed in primary cultures derived from rat s.c. adipose tissue at confluence and after differentiation. The results of the present study support the finding of Bornstein et al. (2000)Go and Machinal-Quelin et al. (2002)Go, as leptin receptor mRNA expression was detected during all stages of development of the primary cultures of pig adipose cultures. Therefore, the inability to differentiate in response to leptin may be due to factors other than the receptor, although detection of receptor message does not demonstrate that functional receptors are present on the cells. Other factors may include a potential immaturity in the leptin-signaling pathways of the porcine preadipocyte relative to the adipocyte in the pig. Previous studies have demonstrated that pig adipocytes differentiated in primary culture respond to leptin with changes in lipogenic (Ramsay, 2003Go) and lipolytic activity (Ramsay, 2001Go). Therefore, the inability to respond to leptin must be specific to the stromal vascular cells and preadipocytes; however, this would require further studies to identify the specific mechanism and signaling pathway as multiple pathways are involved (Machinal-Quelin et al., 2002Go).

Alternatively, the potential interference of factors present in either fetal bovine serum or pig serum used in the culture medium cannot be excluded. However, previous studies using serum-containing medium have detected lipolytic (Ramsay, 2001Go) and antilipogenic (Ramsay, 2003Go, 2004Go) responses to leptin by porcine adipocyte cultures. This finding suggests that the presence of serum may not have a significant effect on the leptin response by these cultures, although it cannot be excluded. Alternatively, suppressors of cytokine signaling (SOCS) has been implicated in the interference of leptin signaling in adipose tissue of obese animals (Wang et al., 2000Go) and in the hypothalamus (Bjoerbaek et al., 1998Go). These SOCS function by binding to phosphorylated tyrosine residues on Janus-activated kinase proteins (Endo et al., 1997Go), and thereby inhibit cytokine signaling (including leptin). Although SOCS have been demonstrated to alter the intracellular signaling of leptin under specific conditions, previous in vitro studies have demonstrated that chronic incubation with leptin can still induce proliferation and differentiation of human (Aprath-Husmann et al., 2001Go) and rodent preadipocytes (Machinal-Quelin et al., 2002Go) at the concentration range used in the present study. Therefore, the inability of porcine leptin to affect porcine preadipocyte proliferation cannot be the result of SOCS activity, unless the inhibitory effects of SOCS are specific to leptin signaling in the pig preadipocyte; however, differences due to species variability or specific culture conditions cannot be excluded.

Preadipocyte and stromal vascular proliferation was stimulated similarly by pig or mouse leptin at 1,000 ng/mL (an approximately 30% increase in 3H-thymidine incorporation). These data suggest that leptin does not have a specific mitogenic effect on the preadipocyte, but functions as a general mitogen. Machinal-Quelin et al. (2002)Go reported that 10 nM murine leptin (160 ng/mL) could increase 3H-thymidine incorporation by 44 ± 7%, which is comparable to the rates observed in the present study, although the concentration used was approximately 60% lower.

No attempt was made to segregate the different cell populations in that study. The ability of leptin to function as a general mitogen in adipose tissue is not surprising, as it has been shown previously to stimulate proliferation of hemapoietic cells (Gainsford et al., 1996Go), chondrocytes (Nakajima et al., 2003Go), embryonic chick myoblasts (Lamosova and Zeman, 2001Go), and endothelial cells (Sierra-Honigmann et al., 1998Go). The high concentrations of leptin necessary to produce the mitogenic response in the present study were pharmacological, which raises the question of physiological relevance. Without a standardized bioassay, one cannot ascertain the relative quality of a leptin preparation. Therefore, we cannot be certain of the relative potency of our pig leptin preparation relative to that of Machinal-Quelin et al. (2002)Go. Irrespective of this quandary, the present study demonstrates that the porcine preadipocyte is responsive to leptin as shown by its mitogenic effects in vitro.

Leptin has been shown to induce apoptosis in rodent adipose tissue (Gullicksen et al., 2003Go). The majority of studies have demonstrated this action in rodents following central administration of leptin. Recently, however, Della-Fera et al. (2003)Go reported that peripheral leptin administration may stimulate apoptosis in mouse adipocytes, implying action at the adipocyte and a potential decrease in preadipocyte proliferation. Yet in the present study, leptin promoted the proliferation of the preadipocyte. It would seem that these two processes are in opposition. This may be due to the use of pig adipose tissue in the present study, whereas apoptosis has only been detected in rodent adipose tissue. However, the two processes may be functioning as counterregulatory events, depending on the metabolic state of the animal and consequent hormonal milieu. Apoptosis results in the death of enlarged, aged adipocytes, thereby providing physical space for recruitment of new cells through the proliferation and differentiation of preadipocytes. The results of the present study suggest that leptin may promote the proliferation of the preadipocytes to enhance the pool of potential adipocytes for the replacement of adipocytes undergoing programmed cell death.


   Footnotes
 
1 Mention of a trade name, vendor, proprietary product or specific equipment is not a guarantee or a warranty by the USDA and does not imply an approval to the exclusion of other products or vendors that also may be suitable. Back

2 The author thanks M. Stoll for her assistance with the cell culture and enzyme analysis. Back

3 Correspondence: BARC-East, Bldg. 200, Rm. 207 (phone: 301-504-5958; fax: 301-504-8623; e-mail: tramsay{at}anri.barc.usda.gov).

Received for publication August 9, 2004. Accepted for publication March 21, 2005.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 


Aprath-Husmann, I., K. Rohrig, H. Gottschling-Zeller, T. Skurk, D. Scriba, M. Birgel, and H. Hauner. 2001. Effects of leptin on the differentiation and metabolism of the human adipocyte. Int. J. Obes. 25:1465–1470.

Barb, C. R., X. Yan, M. J. Azain, R. R. Kraeling, G. B. Rampacek, and T. G. Ramsay. 1998. Recombinant porcine leptin reduces feed intake and stimulates growth hormone secretion in swine. Domest. Anim. Endocrinol. 15:77–86.[Medline]

Bjoerbaek, C., J. K. Elmquist, J. D. Frantz, S. E. Shoelson, and J. S. Flier. 1998. Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol. Cell 1:619–625.[Medline]

Bornstein, S. R., M. Abu-Asab, A. Glasow, G. Path, H. Hauner, M. Tsokos, G. P. Chrousos, and W. A. Sherbaum. 2000. Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture. Diabetes 49:532–538.[Abstract]

Chen, X., J. Lin, D. B. Hausman, R. J. Martin, R. G. Dean, and G. J. Hausman. 2000. Alterations in fetal adipose tissue leptin expression correlate with the development of adipose tissue. Biol. Neonate 78:41–47.[Medline]

Considine, R., and J. F. Caro. 1997. Leptin and the regulation of body weight. Int. J. Biochem. Cell Biol. 29:1255–1272.[Medline]

Della-Fera, M. A., C. Li, and C. A. Baile. 2003. Resistance to IP leptin-induced adipose apoptosis caused by high-fat diet in mice. Biochem. Biophys. Res. Commun. 303:1053–1057.[Medline]

Endo, T. A., M. Masuhara, M. Yokouchi, R. Suzuki, H. Sakamoto, K. Mitsui, A. Matsumoto, S. Tanimura, M. Ohtsubo, H. Misawa, T. Miyazaki, N. Leonor, T. Taniguchi, T. Fujita, Y. Kanakura, S. Komiya, and A. Yoshimura. 1997. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921–924.[Medline]

Frick, F., J. Oscarsson, K. Vikman-Adolfsson, M. Ottosson, N. Yoshida, and S. Eden. 2000 Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 278:E729–E737.[Abstract/Free Full Text]

Gainsford, T., T. A. Willson, D. Metcalf, E. Handman, C. McFarlane, A. Ng, N. A. Nicola, W. S. Alexander, and D. J. Hilton. 1996. Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells. Proc. Natl. Acad. Sci. USA 93:14564–14568.[Abstract/Free Full Text]

Guerre-Millo, M. 2004. Adipose tissue and adipokines: For better or worse. Diabetes Metab. 30:13–19.[Medline]

Gullicksen, P. S., M. A. Della-Fera, and C. A. Baile. 2003. Leptin-induced adipose apoptosis: Implications for body weight regulation. Apoptosis 8:327–335.[Medline]

Hauner, H., G. Entenmann, M. Wabitsch, D. Gaillard, G. Ailhaud, R. Negrel, and E. F. Pfeiffer. 1989. Promoting effect of glucocorticoids on the differentiation of human adipocyte precursors cells cultures in a chemically defined medium. J. Clin. Invest. 84:1663–1670.

Hausman, D. B., M. DiGirolamo, T. J. Bartness, G. J. Hausman, and R. J. Martin. 2001. The biology of white adipocyte proliferation. Obes. Rev. 2:239–254.[Medline]

Hube, F., and H. Hauner. 1999. The role of TNF-alpha in human adipose tissue: Prevention of weight gain at the expense of insulin resistance? Horm. Metab. Res. 31:626–631.

Hwang, C. S., T. M. Loftus, S. Mandrup, and M. D. Lane. 1997. Adipocyte differentiation and leptin expression. Annu. Rev. Cell Dev. Biol. 13:231–259.[Medline]

Kim, S., and N. Moustaid-Moussa. 2000. Secretory, endocrine and autocrine/paracrine function of the adipocyte. J. Nutr. 130:3110S–3115S.[Abstract/Free Full Text]

Lamosova, D., and M. Zeman. 2001. Effect of leptin and insulin on chick embryonic muscle cells and hepatocytes. Physiol. Res. 50:183–189.[Medline]

Lin, J., C. R. Barb, R. L. Matteri, R. R. Kraeling, X. Chen, R. J. Meinersmann, and G. B. Rampacek. 2000. Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig. Domest. Anim. Endocrinol. 19:53–61.[Medline]

Machinal-Quelin, F., M. N. Dieudonne, M. C. Leneveu, R. Pecquery, and Y. Guidicelli. 2002. Proadipogenic effect of leptin on rat preadipocytes in vitro: Activation of MAK and STAT3 signaling pathways. Am. J. Physiol. 282:C853–C863.

Nakajima, R., H. Inada, T. Koike, and T. Yamano. 2003. Effects of leptin to cultured growth plate chondrocytes. Horm. Res. 60:91–98.[Medline]

Nilsson-Ehle, P., and M. C. Schotz. 1976. A stable, radioactive substrate emulsion for assay of lipoprotein lipase. J. Lipid Res. 17:536–541.[Abstract]

Ramsay, T. G. 2001. Porcine leptin alters insulin inhibition of lipolysis in porcine adipocytes in vitro. J. Anim. Sci. 79:653–657.[Abstract/Free Full Text]

Ramsay, T. G. 2003. Porcine leptin inhibits lipogenesis in porcine adipocytes. J. Anim. Sci. 81:3008–3017.[Abstract/Free Full Text]

Ramsay, T. G. 2004. Porcine leptin alters isolated adipocyte glucose and fatty acid metabolism. Domest. Anim. Endocrinol. 26:11–21.[Medline]

Ramsay, T. G., G. J. Hausman, and R. J. Martin. 1987. Preadipocyte proliferation and differentiation in response to hormone supplementation of decapitated fetal pig sera. J. Anim. Sci. 64:735–744.

Ramsay, T. G., C. Morrison, and X. Yan. 1998. The obesity gene in swine: Sequence and expression of porcine leptin. J. Anim. Sci. 76:484–490.[Abstract/Free Full Text]

Ramsay, T. G., M. E. White, and C. K. Wolverton. 1989a. Glucocorticoids and the differentiation of porcine preadipocytes. J. Anim. Sci. 67:2222–2229.

Ramsay, T. G., M. E. White, and C. K. Wolverton. 1989b. Insulin-like growth factor 1 induction of differentiation of porcine preadipocytes. J. Anim. Sci. 67:2452–2459.

Ramsay, T. G., C. K. Wolverton, G. J. Hausman, R. R. Kraeling, and R. J. Martin. 1989c. Alterations in adipogenic and mitogenic activity of porcine serum in response to hypophysectomy. Endocrinology 124:2268–2276.[Abstract]

Sierra-Honigmann, M. R., A. K. Nath, C. Murakami, G. Garcia-Cardena, A. Papapetropoulos, W. C. Sessa, L. A. Madge, J. S. Schechner, M. B. Schwabb, P. J. Polverini, and J. R. Flores-Riveros. 1998. Biological action of leptin as an angiogenic factor. Science 281:1683–1686.[Abstract/Free Full Text]

Thomas, T., F. Gori, S. Khosla, M. D. Jensen., B. Burguera, and B. L. Riggs. 1999. Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 140:1630–1638.[Abstract/Free Full Text]

Wang, Z., Y. T. Zhou, T. Kakuma, Y. Lee, S. P. Kalra, P. S. Kalra, W. Pan, and R. H. Unger. 2000. Leptin resistance of adipocytes in obesity: Role of suppressors of cytokine signaling. Biochem. Biophys. Res. Commun. 277:20–26.[Medline]

Wise, L. S., and H. Green. 1978. Studies of lipoprotein lipase during the adipose conversion of 3T3 cells. Cell 13:233–242.[Medline]

Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M. Friedman. 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432.[Medline]


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
B. Wagoner, D. B. Hausman, and R. B. S. Harris
Direct and indirect effects of leptin on preadipocyte proliferation and differentiation
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1557 - R1564.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ramsay, T. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ramsay, T. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS