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Leptin and leptin receptor expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment^1,2

Growth Biology Laboratory, USDA-ARS, Beltsville, MD 20705

	Abstract

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The present study was performed to examine the response of leptin and leptin receptor (Rb) genes to porcine somatotropin (pST) stimuli in finishing pigs. Twelve crossbred barrows (Yorkshire x Landrace) were used in this study. Animals were individually fed a basal diet containing 18% CP, 1.2% lysine, and 3.5 Mcal of DE/kg ad libitum (as-fed basis). At 90 kg, six pigs were treated with daily injections of recombinant pST (10 mg) in sterile bicarbonate buffer, whereas the other six pigs were injected with sterile bicarbonate buffer (controls). With initiation of pST treatment, the quantity of feed offered was 85% of calculated ad libitum intake based on BW and adjusted every 3 d. Diet restriction was designed to correct for the effects of the known inhibition in feed intake because of pST treatment in swine. Animals were maintained on treatment for 2 wk. A blood sample was obtained from each pig on d 14 of treatment, 6 h after pST injection. Tissue samples were collected on d 15, frozen in liquid N₂, and stored at –80° C before analysis for mRNA abundance. Total RNA was amplified by reverse transcription (RT) PCR with subsequent quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. Samples included outer subcutaneous adipose tissue (OSQ), middle subcutaneous adipose tissue (MSQ), leaf fat (LF), liver, latissimus dorsi (LD), and biceps femoris (BF). Restricted feeding resulted in no change in BW of control pigs, whereas pST treatment increased BW by 6.9 ± 0.5 kg (P < 0.001). Treatment with pST produced a 12-fold increase in serum ST concentration relative to control pigs (P < 0.002). Serum leptin concentration was increased by 17% in swine treated with pST relative to control pigs (P < 0.011). Leptin mRNA abundance was increased in liver by pST treatment (P < 0.05). Administration of pST decreased leptin Rb (Ob-Rb) mRNA abundance by 27% in liver (P < 0.044) and by 49.5% in OSQ (P < 0.025) relative to controls. The present data suggest that pST does not affect leptin expression independent of dietary intake because the restricted feeding regimen used in the present study precluded detection of major change in leptin gene expression. Changes in Ob-Rb mRNA abundance by pST treatment indicate that ST or the metabolic adaptations to ST have a role in regulating Ob-Rb expression.

Key Words: Leptin • Leptin Receptor • Somatotropin • Swine

	Introduction

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Porcine somatotropin (pST) has been demonstrated in swine to have dramatic effects on adipose tissue accretion, metabolism, and gene expression (Etherton, 2004). The processes affected by pST are many of the same that are affected by the hormone leptin. Leptin is a hormone that is produced and secreted by adipose tissue with numerous effects on metabolism, immune response, reproduction, and feeding behavior (Barb et al., 2001).

The regulation of leptin expression has been well characterized in vitro; however, characterization of the in vivo regulation has been limited in the pig. Changes in leptin mRNA abundance have been correlated with adiposity (Leininger et al., 2000), whereas differences in tissue leptin mRNA abundance have been associated with differences in serum leptin concentrations between lean and genetically obese pigs (Ramsay et al., 1998). Very few studies have examined the role of specific hormones in the in vivo regulation of leptin mRNA abundance in the pig. In numerous species, ST has been demonstrated to alter leptin mRNA abundance in subcutaneous adipose tissue (Isozaki et al., 1999; Houseknecht et al., 2000) including swine (Spurlock et al., 1998). Nonetheless, the relationship of tissue leptin mRNA abundance to serum leptin has not been evaluated in swine in response to pST administration.

Leptin functions through a receptor (Rb) with multiple splice sites. The long form of the Rb is the only one to demonstrate signaling capability consistently. Leptin Rb (Ob-Rb) mRNA (long form) has been detected in a variety of porcine tissues, including adipose tissue (Lin et al., 2000). Regulation of the Ob-Rb has not been well characterized for any species. The present study was designed to examine the in vivo response of leptin and Ob-Rb genes to pST administration.

	Materials and Methods

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Twelve crossbred barrows (Yorkshire x Landrace) were individually penned in environmentally controlled housing at 70 kg. Animals were individually fed a basal diet ad libitum containing 18% CP, 1.2% lysine, and 3.5 Mcal of DE/kg (Table 1).

With initiation of pST treatment, the feed was offered at 85% of calculated ad libitum intake (ARC, 1981) based on BW and adjusted every 3 d. Animals were maintained on treatment for 2 wk. A blood sample was obtained from each pig on d 14 of treatment at 1500. Animals were euthanized on d 15 at 0800.

Somatotropin has been demonstrated to dramatically shift metabolic variables to promote muscle accretion and decrease adipose accumulation. In association with this shift, animals experience a decrease in feed intake but an increase in feed efficiency (Etherton, 2004). In the present study, feed restriction was used to specifically exclude any differences in feed intake caused by pST treatment as contributing to changes in leptin or Ob-Rb mRNA abundance. Feed withdrawal has been demonstrated to decrease leptin mRNA abundance in swine (Spurlock et al., 1998; Salfen et al., 2003), whereas feed restriction to 80% of ME intake decreased serum leptin concentrations by approximately 37% in gilts (Louveau et al., 2000). Thus, exclusion of feed intake as a component of the pST response would permit examination of the data in terms of a specific response to the pST treatment.

Various tissues were acquired following euthanasia by electrical stunning and exsanguination according to procedures approved by the Institutional Area Animal Use and Care Committee. Dorsal subcutaneous adipose tissue samples were collected from between the second and fourth thoracic vertebrae, and subsequently, outer (OSQ) and middle (MSQ) layers were separated, diced, and frozen in liquid N₂. In addition, samples of liver, leaf (perirenal) fat (LF), latissimus dorsi (LD), and biceps femoris (BF) muscle were collected, diced, and frozen in liquid N₂. Outer and MSQ were separated for analysis because of their known differences in metabolic activity, whereas LF has a different metabolic profile from subcutaneous adipose tissue (Anderson et al., 1972; Rule et al., 1989; Budd et al., 1994). The LD and BF were selected, as they represent large muscles in economically important regions that differ in their response to pST (Ono et al., 1995).

Hormone Analyses
Concentrations of pST were determined by a homologous RIA (Linco Research, Inc., Chesterfield, MO). Intra-assay CV was 6.03% for pST. Serum leptin was assayed using a heterologous RIA kit (Linco Research, Inc.). Human leptin standards were replaced with recombinant porcine leptin standards. Recombinant porcine leptin was obtained from A. Gertler (Rehovot, Israel; Raver et al., 2000). Cross-reactivity for the recombinant porcine leptin in the multi-species leptin RIA was 58%. Intra-assay CV was 4.94%, and the inter-assay CV was 10.3%. The minimal detectable concentration was 0.1 ng/tube, recovery of added leptin was > 93% across the range of 1 to 20 ng of leptin added.

Gene Expression Analyses by Reverse Transcription PCR
Total RNA was isolated using TRI reagent according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO). Integrity of RNA was assessed via agarose gel electrophoresis, and RNA concentration and purity were determined spectrophotometrically using 260 and 280 nm absorbance measurements. Reverse transcription (RT) 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 of dNTP, and 100 ng of random hexamer primers. Polymerase chain reaction was performed in 25 µL containing 20 mmol/L of Tris-HCl (pH 8.4), 50 mmol/L of KCl, 1.0 µL of the RT reaction, 1.0 U of Platinum Taq DNA polymerase (Hot Start; Invitrogen/Life Technologies), 0.2 mmol/L of dNTP, 2.0 mmol/L of Mg++ (Invitrogen/Life Technologies), 10 pmol each of the leptin and Ob-Rb-specific primers, and 10 pmol of an appropriate mixture of primers and competimers specific for 18S rRNA (QuantumRNA Universal 18S Internal Standard; Ambion, Inc., Austin, TX). Thermal cycling protocol was as follows: 1 cycle at 94° C for 2 min, followed by 30 cycles at 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 following primers were used for generating 348 base-pair PCR products corresponding to a portion of the pig leptin coding sequence: 5'- TGACACCAAAAC CCTCATCA -3' (forward), 5'- GCCACCACCTCTGTG GAGTA -3' (reverse). The primers for the long form Ob-Rb were used to generate a 396-basepair product: 5'- C AGTGACATTTGGCCCTCTT -3' (forward), 5'- AGGC CTGGGTTTCTATCTCC -3' (reverse). The leptin and Ob-Rb amplicons were excised from an agarose gel, re-amplified, and run through a GenElute PCR clean-up kit (Sigma-Aldrich). The amplicons were subsequently sequenced to confirm identity using automated fluorescent DNA sequencing (ABI 310; Perkin-Elmer Applied Biosystems, Foster City, CA).

Capillary Electrophoresis with Laser-Induced Fluorescence Detection
Aliquots (2 µL) of RT-PCR samples were diluted 1:100 with deionized water before capillary electrophoresis with laser-induced fluorescence detection (CE/LIF). A detailed description and validation of the CE/LIF technique used in this study for quantitative analysis of gene expression was reported previously (Richards and Poch, 2002). Briefly, a P/ACE MDQ series capillary electrophoresis instrument (Beckman Coulter, Fullerton, CA) equipped with an argon ion LIF detector was used. Capillaries were 75 µm i.d. x 32 cm µSIL-DNA (Agilent Technologies, Folsom, CA). EnhanCE dye (Beckman Coulter) was added to the DNA separation buffer (Sigma-Aldrich) to a final concentration of 0.5 µg/mL. Samples were loaded by electrokinetic injection at 3.5 kV for 3.5 s and run in reverse polarity at 8.1 kV for 5 min. Integrated peak area for the PCR products separated by CE was calculated using P/ACE MDQ software (Beckman Coulter).

Quantification of Messenger Ribonucleic Acid Abundance
Relative mRNA abundance was determined as the ratio of integrated peak area for each individual gene PCR product relative to that of a coamplified 18S internal standard (QuantumRNA Universal 18S Internal Standard; Ambion, Inc). Values are presented as the mean ± SEM of four to six individual determinations.

Statistical Analyses
Data were analyzed by one-way ANOVA using SigmaStat software (SPSS Science, Chicago, IL). Mean separation was done using Student-Newman-Keuls test, and means were defined as significantly different at P < 0.05.

	Results

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Cumulative feed intake did not differ (P = 0.240) between the two groups (38.58 ± 0.22 kg vs. 38.74 ± 0.15 kg for control vs. pST, respectively). Restricted feeding resulted in no change (P = 0.394) in BW of control pigs, but treatment with recombinant pST for 14 d resulted in an average increase (P < 0.001) in BW of 6.9 ± 0.5 kg relative to control pigs. Treatment with pST resulted in a 12-fold increase (P < 0.002) in serum ST relative to control pigs (Figure 1). Serum leptin concentration was increased (P < 0.011) by 17% in swine treated with pST relative to control pigs (Figure 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Serum porcine ST (pST; solid bars) and leptin (hatched bars) concentrations in feed-restricted swine following 14 d of pST administration (10 mg/d). Data (ng/mL) are expressed as means ± SEM. The asterisk indicates that the mean pST concentration differs from that of control (buffer-injected) swine, P < 0.002 (n = 6). The star indicates that the mean leptin concentration differs from that of the control (buffer-injected) swine, P < 0.011 (n = 6).

View larger version (18K): [in this window] [in a new window]   Figure 2. Relative leptin mRNA abundance in tissues from feed-restricted swine following 14 d of porcine ST (pST) administration (10 mg/d) followed by extraction for total RNA and subsequent reverse transcription PCR analysis for leptin mRNA abundance. Data are expressed as the mean ratios ± SEM of specific leptin mRNA:18S mRNA for six pigs in each group. The asterisk indicates that the mean differs from that of corresponding control (buffer-injected) swine, P < 0.05 (n = 6). Means that do not have a common letter differ, P < 0.05 (n = 6). Liv = liver, OSQ = outer subcutaneous adipose tissue, MSQ = middle subcutaneous adipose tissue, LF = leaf fat, LD = latissimus dorsi, and BF = biceps femoris.

View larger version (21K): [in this window] [in a new window]   Figure 3. Relative long-form leptin receptor (Ob-Rb) mRNA abundance in tissues from feed-restricted swine following 14 d of porcine ST (pST) administration (10 mg/d) followed by extraction for total RNA and subsequent reverse transcription PCR analysis for leptin mRNA abundance. Data are expressed as the mean ratios ± SEM of specific Ob-Rb mRNA:18S mRNA for six pigs in each group. The asterisks indicate that the means are different than that of control (buffer-injected) swine, P < 0.05 (n = 6). Means that do not have a common letter differ, P < 0.05 (n = 6). Liv = liver, OSQ = outer subcutaneous adipose tissue, MSQ = middle subcutaneous adipose tissue, LF = leaf fat, LD = latissimus dorsi, and BF = biceps femoris.

	Discussion

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Previous studies in humans and rodents have demonstrated that somatotropin concentrations can be correlated with increased (Iglesias et al., 2002 ), decreased (Eden-Engstrom et al., 2003; Malmlof and Johansen, 2003), or normal (Boni-Schnetzler et al., 1999; Bolanowski et al., 2002; Schulz et al., 2002) serum leptin concentrations. The serum leptin response to pST may depend on the status of carcass adipose mass, serum insulin, serum glucocorticoid, renal state, pre-experimental ST concentration, timing of collection, or other factors.

The observed shift in serum leptin with pST treatment could not be related to any change in adipose tissue leptin mRNA abundance. Previous research in swine has demonstrated that serum leptin is elevated when adipose tissue mRNA abundance is elevated within obese pigs relative to lean pigs (Ramsay et al., 1998). Nonetheless, results of the present study suggest that leptin mRNA abundance in adipose tissue does not necessarily correlate with changes in serum leptin following pST treatment.

Spurlock et al. (1998) reported that pST administration can decrease leptin mRNA abundance by 50% in adipose tissue of feed-restricted pigs, suggesting that pST-treated pigs have less stimulus for satiety and are thus hungrier; this would indicate that pST-treated swine would consume greater quantities of feed when re-fed. In contrast, pST-treated swine consume less feed than control swine in most studies (Etherton, 2004). Equalizing the intake of control animals with pST-treated swine in the present experiment eliminated the suppression in leptin mRNA abundance. The additive suppressive effect of pST with the feed withdrawal response on leptin, as reported by Spurlock et al. (1998) might be due to the high concentration of free fatty acids in that study. Free fatty acids have been demonstrated to down-regulate leptin expression (Rentsch and Chiesi, 1996; Shintani et al., 2000; Cammisotto et al., 2003). Free fatty acid concentrations in pST-treated pigs in the present study were 17% (data not presented) of the concentration in the feed-restricted, pST-treated pigs in the Spurlock et al. (1998) study. The extremely high concentration of free fatty acids in the feed-restricted, pST-treated swine may account for the pST inhibition of leptin mRNA abundance in the study of Spurlock et al. (1998) vs. the absence of a difference in the restricted-fed pigs in the present study.

Leptin mRNA abundance was greatest in subcutaneous adipose tissue, with no differences between middle and outer layers in our feed-restricted swine. Leptin mRNA abundance has been correlated with adipocyte size (Zhang et al., 2002; Guo et al., 2004), but OSQ and MSQ have been reported to have similar cellularity (Anderson et al., 1972). Thus, similar abundance of leptin mRNA would be predicted for OSQ and MSQ. The leptin mRNA abundance in LF was approximately 50% of that in subcutaneous adipose tissue. Previous work with human adipose tissue has demonstrated that the adipocytes from internal sites of adipose accretion have lower leptin mRNA abundance than subcutaneous adipose tissue (Montague et al., 1997).

Under our experimental conditions, leptin mRNA was detectable in skeletal muscle with abundance approaching that in LF. Leptin mRNA has previously been detected in skeletal muscle (Wang et al., 1998). The primary source of this leptin mRNA is most likely the intramuscular adipose tissue, which was not dissected from the muscle in the present study.

Liver leptin mRNA abundance was least in the tissues examined. Leptin mRNA has been previously detected in mammalian hepatic tissue (Otte et al., 2004), but it was primarily restricted to the stellate cell population (Potter et al., 1998). Somatotropin has been previously demonstrated to up-regulate leptin mRNA abundance in chicken liver (Ashwell et al., 1999); however, leptin mRNA abundance in the pig liver is extremely low and the biological significance of this up-regulation is unknown.

Little is understood of the regulation of the Ob-Rb in peripheral tissues for any species. The majority of research on the porcine Ob-Rb has focused simply on detection of the Rb (Czaja et al., 2002) or on its regulation at the pituitary gland and hypothalamus (Lin et al., 2003). Previous studies using adipose tissue explants have demonstrated an inhibitory effect of pST on total Ob-Rb mRNA abundance in MSQ explants in vitro (Ramsay and Richards, 2004). This inhibitory effect could not be replicated in MSQ by in vivo administration of pST. This result may be a consequence of interaction with the hormonal milieu in vivo relative to the controlled environment of tissue culture or it may be a consequence of differences in the regulation of the long form of the Ob-Rb relative to the total population of Ob-Rb. Differential regulation of Ob-Rb isoforms has been previously demonstrated in rat hypothalamus (Denis et al., 2003) and testis (Tena-Sempere et al., 2001).

In vivo treatment with pST resulted in lower abundance of long-form Ob-Rb mRNA in liver and OSQ relative to tissues from control animals consuming similar quantities of feed. Any shift in Ob-Rb expression in the liver could be of significance, as the liver has a significant role in regulating energy metabolism. Although leptin has not yet been demonstrated to alter hepatic metabolism in the pig (Fernandez-Figares et al., 2004; Raman et al., 2004), leptin has been shown to inhibit dexamethasone-induced hepatic IGF-1 mRNA abundance in vitro (Ajuwon et al., 2003).

Leptin Rb mRNA abundance was decreased by pST to a greater extent in OSQ than in the liver. Changes in the relative expression of the Ob-Rb by in vivo pST treatment would imply altered sensitivity to leptin and perhaps a decreased capacity to inhibit lipogenesis and promote lipolytic activity of the porcine adipocyte (Ramsay, 2000, 2003a). These same actions of leptin may be superseded by similar actions of pST on the pig adipocyte (Etherton, 2004). We hypothesize that a decrease in leptin binding and internalization may contribute to the elevation in the serum leptin concentration following pST treatment.

Abundance of the Ob-Rb mRNA was as great in skeletal muscles as liver, with less abundance in adipose tissues. Previous studies have detected the presence of Ob-Rb mRNA in muscle cells (Berti and Gammeltoft, 1999; Fruhbeck et al., 1999) and the ability of leptin to alter glucose, fatty acid, and amino acid metabolism to promote muscle anabolism (Ceddia et al., 1998; Berti and Gammeltoft, 1999; Ramsay, 2003b). Expression of the Ob-Rb in skeletal muscle and the known metabolic effects of leptin on muscle from studies in other species imply that leptin may function in pig skeletal muscle to alter metabolic activity.

Leptin is a hormone produced by pig adipose tissue that can affect feeding behavior, animal health, and reproduction. Studies in pigs have demonstrated that leptin can decrease feed intake. This study attempted to determine whether expression of leptin and its Rb can be manipulated by hormonal mechanisms in finishing swine by using pST treatment. The data indicate that circulating leptin concentrations are stimulated by pST when feed intake is equivalent. Second, expression of the Ob-Rb gene also is responsive to hormonal stimuli in finishing swine. In contrast, the leptin gene does not seem responsive to somatotropin treatment when the effects of pST to decrease feed intake are taken in account. These results suggest the potential to manipulate the efficiency of feeding behavior in finishing animals by altering expression of genes associated with feed intake and metabolism.

	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.

² The authors thank M. Stoll for her assistance with the RNA extractions and reverse transcription PCR. The authors also thank the swine herd staff, USDA-Beltsville, for their work in animal care and processing.

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

Received for publication December 22, 2004. Accepted for publication June 10, 2005.

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