Effect of Somatotropin, Insulin, and Glucocorticoid on Lipolysis in Chronic Cultures of Adipose Tissue from Lactating Cows1

Effect of Somatotropin, Insulin, and Glucocorticoid on Lipolysis in Chronic Cultures of Adipose Tissue from Lactating Cows1 D.P.D. LANNA2 and D. E. BAUMAN3 Department of Animal Science, Cornell University, Ithaca, NY 14853

tonic inhibition of lipolysis via changes in the inhibitory G-protein signaling system. ( Key words: somatotropin, adenosine, adipose lipolysis, glucocorticoids)

ABSTRACT In vitro effects of bovine somatotropin (bST) and insulin plus dexamethasone on lipolysis were evaluated using chronic cultures (48 h ) of adipose tissue from lactating cows. Treatments were control (culture medium alone), bST (100 ng/ml), insulin (100 ng/ml) plus dexamethasome (10 nM) , and insulin plus dexamethasone plus bST. Following the 48-h cultures, rates of lipolysis were measured in 3-h incubations with isoproterenol (10 mM) , adenosine deaminase (0.75 U/ml), and various concentrations of a nonhydrolyzable adenosine analog. The addition of bST to cultures did not alter basal or isoproterenolstimulated lipolysis. However, the ability of adenosine to inhibit rates of lipolysis was reduced by bST. When measured in the presence of maximal concentrations of adenosine analog, isoproterenol caused an increase in lipolysis above basal, which was twofold greater for explants cultured with insulin plus dexamethasone plus bST than for explants cultured with insulin plus dexamethasone. Dose-response curves for adenosine inhibition of isoproterenol-stimulated lipolysis demonstrated that chronic culture with bST decreased adipose tissue responsiveness and sensitivity to adenosine. Overall, results demonstrated that an in vitro chronic culture system can be used to examine factors that regulate lipolysis. The addition of insulin plus dexamethasone to chronic cultures better maintained the intracellular signaling system, including sensitivity and responsiveness to adenosine inhibition of lipolysis. Results also confirm that bST alters the antilipolytic response to adenosine. Thus, bST effects are in large part due to a relief in the

Abbreviation key: ADA = adenosine deaminase, DEX = dexamethasone, ED50 = effective dose that produces 50% of maximal response, Gi = inhibitory Gproteins, Gs = stimulatory G-proteins, HSL = hormone-sensitive lipase, INS = insulin, ISO = isoproterenol, PIA = (–)-N 6-(2-phenylisopropyl)adenosine, Rmax = maximal response, ST = somatotropin. INTRODUCTION The onset of lactation results in coordinated metabolic changes that accommodate the shift in nutrient use for milk synthesis while preserving animal wellbeing ( 2 ) . Two basic adaptations occur in adipose tissue to support lactation: a decrease in nutrient use for fat accretion and an increase in the ability to mobilize body fat reserves. Similarly, exogenous bST treatment of lactating cows can both decrease lipid synthesis and increase net lipid mobilization in adipose tissue; the relative magnitude of changes in these two processes is dependent on the nutritional status of the cow [see reviews (3, 11)]. In vivo treatment of lactating cows with bST increases lipolytic response to b-adrenergic challenge (21, 33), which also occurs with the onset of lactation (2, 22, 39). However, the increased response of adipose tissue to b-adrenergics with bST treatment appears to occur primarily via a relief from the inhibitory signaling cascade including the inhibition by adenosine (17, 20). In contrast, the onset of lactation results in an increased response of adipose cells to adenosine in rats and sheep [see review (39)]. Chronic cultures of adipose tissue from pigs and sheep have been successfully employed to investigate the effects of somatotropin ( ST) on both lipolysis and lipogenesis (12, 40). In general, results have been consistent with those of in vivo studies, thus providing an alternative system to study the cellular mechanisms by which ST modulates lipid metabolism

Received April 4, 1998. Accepted September 4, 1998. 1Supported in part by Cornell University Agricultural Experiment Station and USDA Competitive Research Grant 93-372069025. 2Supported by Coordenac ¸ a˜o de Aperfeic¸oamento de Pessoal de Nivel Superior (Brasilia, Brazil). Present address: Growth and Nutrition Laboratory, Departamento de Zootecnia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sa˜o Paulo, CP 09 Piracicaba, SP 13418-900. 3Address correspondence to Dale E. Bauman, Cornell University, 262 Morrison Hall, Ithaca, NY 14853-4801. 1999 J Dairy Sci 82:60–68

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The protocol and conduct of this study were approved by the Cornell University Institutional Animal Care and Use Committee. Six multiparous Holstein cows (614 ± 57 kg of BW; X ± SE) in midlactation (171 ± 37 d postpartum) were used as the source of adipose tissue. Cows were housed in an environmentally controlled room at 23°C with a light:dark cycle of 16:8 h. Cows were fed for ad libitum intake a total mixed diet that was formulated to provide energy and protein at approximately 110% of estimated requirements (26). In addition, they received 1 kg/d of long hay. At the time of the adipose tissue biopsies, cows averaged 25.4 kg/d of 4% FCM, and the net energy balance averaged near zero (+0.35 Mcal/d). Chemicals, antibiotics, and DEX used in the chronic cultures and the lipolysis incubations were obtained from Sigma Chemical Co. (St. Louis, MO). Bovine INS (lot 615-70N-80) was from Eli Lilly Co. (Indianapolis, IN), and recombinantly derived bST (sometribove; methionyl bST) was supplied by Monsanto Co. (St. Louis, MO).

vessels and connective tissues using a scalpel. The adipose tissue was then transferred to a Petri dish with culture medium. The culture medium consisted of medium 199 (pH 7.4; 37°C; with Earle’s salts, 0.1 g/L of L-glutamine, 25 mM HEPES, and 25 mM sodium bicarbonate) supplemented with 5.6 mM sodium acetate, 60 mg/ml of penicillin, 100 mg/ml of streptomycin, 10 mg/ml of neomycin, and 5 mg/ml of amphotericin. Individual explants, weighing 10 to 25 mg, were dissected with scissors. Explants (totaling approximately 50 mg) were rinsed twice with culture medium and transferred to polystyrene conical centrifuge tubes containing 10 ml of culture medium. The tubes were then incubated in an atmosphere of 95% O2 and 5% CO2 at 37°C for 48 h. To evaluate the effects of chronic exposure of adipose tissue to bST, two series of cultures were conducted. The first series involved explants from four individual cows; these explants were incubated with culture medium that contained no hormones (control) or bST (100 ng/ml). The second series involved tissue from four individual cows and chronic culture with INS (100 ng/ml) plus DEX (10 nM) in the absence or presence of bST (100 ng/ml). A total of six cows were biopsied to obtain sufficient tissue (two of the cows were represented in both series). Bovine INS was solubilized in 0.001N HCl at pH 3.0. Somatotropin was supplied as lyophilized protein and was solubilized with sterile 75 mM sodium bicarbonate solution (pH 9.0). Dexamethasone was dissolved in ethanol. An equivalent amount of ethanol was added to those incubations that did not contain DEX, and the final concentration of ethanol in the culture medium was 0.025% (vol/vol).

Chronic Cultures

Lipolysis Incubations

Adipose tissue was biopsied from the rump region as described by McNamara and Hillers (23). Tissue (approximately 5 g ) was placed in sterile transport buffer (pH 7.4, 37°C ) and transferred to the laboratory within 5 min after biopsy. The transport buffer contained 0.15 M sodium chloride, 25 mM HEPES, 60 mg/ml of penicillin, 100 mg/ml of streptomycin, 10 mg/ ml of neomycin, and 5 mg/ml of amphotericin. The streptomycin was solubilized in ethanol and added to culture medium so that final concentration of ethanol was less than 0.01% (vol/vol); the other antibiotics were solubilized in sterile water. Adipose tissue explants were prepared under a laminar flow hood and were cultured according to the methods of Robertson et al. ( 2 8 ) with some modifications. The biopsy tissue was rinsed with transport buffer, and adipose tissue was separated from blood

The rates of lipolysis were measured following chronic cultures according to Mersmann and Hu ( 2 4 ) with modifications. At the end of the 48-h culture period, explants were rinsed in Krebs-Ringer bicarbonate buffer (pH 7.4; 37°C ) and were blotted, weighed, and transferred to scintillation vials containing 2 ml of lipolysis medium. The lipolysis medium (pH 7.4; 37°C ) consisted of Krebs-Ringer bicarbonate buffer containing 25 mM HEPES, 1.27 mM calcium, 5.6 mM glucose, and 3% BSA (fraction V, essentially free of fatty acids). Lipolysis incubations ( 3 h ) were performed in triplicate under an atmosphere of 95% O2 to 5% CO2 in a shaking water bath, and lipolysis rates were based on the release of glycerol and NEFA. Glycerol concentrations were determined by the method of Boobis and Maughan ( 5 ) as modified by Sechen et al.

in adipose tissue. The objectives of the present study were to validate a chronic culture system to examine lipolysis in adipose tissue from lactating cows and to use this system to evaluate the effects of bST, insulin ( INS) , and dexamethasone ( DEX) . Of special interest were effects of chronic culture with bST on acute response to homeostatic signals that regulate rates of lipolysis. MATERIALS AND METHODS Cows and Materials

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(33). Fatty acid concentrations were measured using a commercial kit (NEFA-C; Wako Chemicals USA Inc., Dallas, TX) as detailed by McCutcheon and Bauman ( 2 1 ) and modified by Sechen et al. (33). Isoproterenol ( ISO; 10 mM) was used to examine lipolysis stimulated by b-adrenergics. Basal lipolytic rates and isoproterenol-stimulated lipolysis were also determined in the presence of adenosine deaminase ( ADA) . Adenosine deaminase (type A-1030) was dialyzed and was added to the lipolysis incubation medium at a final concentration of 0.75 U/ml. This concentration of ADA was based on preliminary doseresponse studies that established the ADA concentration needed to overcome inhibition of lipolysis by endogenous adenosine. The antilipolytic effects of adenosine were studied by addition of varying concentrations of (–)-N 6-(2-phenylisopropyl)-adenosine ( PIA) to incubation flasks containing ADA and ISO.

Dose-response curves for PIA were generated using the following logistic equation (14): y = d +

Statistical Analysis Statistical analyses were performed using the t test module for paired observations of the Minitab statistical package (25). For data not obtained from the same tissue biopsy, ANOVA were performed using the random effects of source of tissue (cow), treatments (bST), culture media (presence or absence of INS and DEX), and interactions. Journal of Dairy Science Vol. 82, No. 1, 1999

1 + (x/m) B

where y = response variable (percentage of inhibition of lipolytic rates), d = asymptotic value of y at infinity ( Rmax = response maximum), x = concentration of PIA, m = concentration of PIA that produced a halfmaximal effect ( ED50) , and B = standardized slope parameter. A program was written for SAS ( 3 2 ) to estimate these parameters by iteration. From the dose-response curves, estimates of Rmax and ED50 and the slope parameter were generated for each cow. These values were subjected to ANOVA as described previously. RESULTS

Hormone-Sensitive Lipase Hormone-sensitive lipase ( HSL) was assayed according to the methods of Fredrikson et al. ( 1 3 ) in the range in which activity was linear with regard to time and amount of enzyme. Following the chronic cultures, adipose explants were rinsed and were added to 3 ml of a buffer solution (30 mM Tris, 0.25 M sucrose, 1 mM glutathione, and 1 mM sodium EDTA; 4°C; pH 7.4). This mixture was homogenized by polytron (two 10-s bursts), and the homogenate was centrifuged at 90,000 × g for 60 min at 4°C. Infranatant was carefully pipetted and filtered through glass wool to avoid small fat droplets that can alter specific activity of the substrate. A triolein emulsion (pH 6.95) was prepared by sonication of 0.1 M phosphate buffer with 8 mM unlabeled triolein, 2 mg/ml of phosphatidylcholine, 4% of BSA, and 8 mCi/ml of 3Hlabeled triolein (New England Nuclear, Boston, MA) in a total volume of 4 ml (13). Incubation of 80 ml of homogenate with an equal volume of the phosphate buffer with substrates (final concentration, 4 mM triolein) was carried out for 1 h. Extraction of fatty acids was performed using the liquid-liquid partition system described by Belfrage and Vaughan ( 4 ) .

y – d

Basal Lipolysis and b-Adrenergic– Stimulated Lipolysis At the end of the 48-h culture, the rates of lipolysis were assessed in 3-h incubations by measurement of the release of NEFA and glycerol. The results were similar for both metabolites; therefore only results with NEFA are presented in Tables 1 and 2. In some

TABLE 1. Lipolysis rates (fatty acid release) of adipose tissue previously cultured (48 h ) in the presence or absence of bST. Culture medium1 Variable

Control

bST

Lipolysis3 Basal 3861 4429 Basal + ADA 7268 7427 ISO + ADA 12,764 13,055 ISO + ADA + PIA 7318 9243 (ISO + ADA + PIA) – Basal 3457 4818 Hormone-sensitive lipase4 27.0 33.4

SEM

P2

285 464 612 441 571 5.5

NS NS NS 0.05 NS NS

1Adipose tissue was cultured for 48 h in culture medium 199 with no addition of exogenous hormones (control) or with 100 ng/ ml of bST. 2Probability of treatment effects (NS = P > 0.10); n = 4 cultures from individual cows. 3Following the 48-h culture, tissue was incubated for 3 h to measure lipolytic rates under different conditions. Additions included adenosine deaminase (ADA), isoproterenol (ISO), and 100 nM (–)-N 6-(2-phenylisopropyl)-adenosine (PIA). Lipolytic rates are expressed as nanomoles of NEFA released from each gram of adipose tissue in 3 h. 4At the end of the 48-h cultures, explants were homogenized, and hormone-sensitive lipase was measured in a 1-h incubation. Rates are expressed as nanomoles of fatty acids released from each milligram of infranatant protein in 1 h.

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SOMATOTROPIN AND ADIPOSE LIPOLYSIS TABLE 2. Lipolysis rates (fatty acid release) of adipose tissue previously cultured (48 h ) with insulin (INS) and dexamethasone (DEX) in the presence or absence of bST. Culture medium1 Variable

INS + DEX

INS + DEX + bST

SEM

P2

Lipolysis3 Basal Basal + ADA ISO + ADA ISO + ADA + PIA (ISO + ADA + PIA) – Basal Hormone-sensitive lipase4

2093 7094 13,691 3117 1024 34.8

3533 8879 13,407 5550 2017 42.4

386 587 515 441 217 6.1

0.08 NS NS 0.03 0.05 NS

1Adipose tissue was cultured for 48 h in culture medium 199 with INS and DEX in the presence or absence of 100 ng/ml of bST. 2Statistical probability of treatment effects (NS = P > 0.10); n = 4 cultures from individual cows. 3Following the 48-h culture, tissue was incubated for 3 h to measure lipolytic rates under different conditions. Additions included adenosine deaminase (ADA), isoproterenol (ISO), and 100 nM (–)-N 6(2-phenylisopropyl)-adenosine (PIA). Lipolytic rates are expressed as nanomoles of NEFA released from each gram of adipose tissue in 3 h. 4At the end of the 48-h cultures, explants were homogenized, and hormone-sensitive lipase was measured in a 1-h incubation. Rates are expressed as nanomoles of fatty acids released from each milligram of infranatant protein in 1 h.

cases, incubations included ADA to eliminate adenosine, an inhibitor of lipolysis. Tissue incubations contain variable amounts of endogenous adenosine, which ADA metabolizes to inosine, a compound that has no effect on lipolysis. The addition of ADA increased the basal rates of lipolysis by a mean of 78% for explants cultured with no hormones ( P < 0.05; Table 1 ) and by 195% for explants cultured with INS plus DEX ( P < 0.01; Table 2). The presence of bST in the chronic culture medium had no effect on the magnitude of increase when ADA was included in the lipolysis medium ( P > 0.1). To examine the effect of b-adrenergic stimulation, we used ISO, a specific b-receptor agonist, at a concentration found to give maximal lipolytic rates in preliminary dose-response studies (data not presented). Incubations also included ADA to eliminate the confounding effects of adenosine. The rates of NEFA release (Tables 1 and 2 ) and glycerol release (data not presented) were markedly stimulated by ISO. Chronic culture in the presence of INS plus DEX caused a modest increase in maximal lipolysis rates over that observed for explants cultured in the absence of exogenous hormones based on NEFA release (Tables 1 and 2; P < 0.10) and glycerol release (data not shown; P < 0.05). However, chronic culture with bST did not alter the response to ISO, regardless of whether the cultures were in the presence or absence of INS plus DEX (Tables 1 and 2). In agreement with these observations, the activity of HSL was not altered in chronic cultures containing bST (Tables 1 and 2).

Lipolytic Response to Adenosine Effects of adenosine on the inhibition of badrenergic–stimulated lipolysis were of special interest. To evaluate effects, we added ADA to remove endogenous adenosine together with the addition of PIA. This adenosine analog is not hydrolyzed by ADA; however it is able to bind to adenosine A1 receptors and to elicit all the adipose tissue responses that are observed with adenosine (16). The PIA concentration (100 nM) was shown to give maximal inhibition of lipolysis in preliminary studies (data not presented). When lipolysis rates were measured in incubations containing ADA and ISO, PIA decreased rates under all culture conditions (Tables 1 and 2), but the ability of PIA to inhibit lipolysis was greater when adipose explants were cultured with INS plus DEX ( P < 0.05). However, the magnitude of the PIA inhibition of lipolysis was less than that when bST was included in the chronic cultures for both the control medium (Table 1 ) and the medium containing INS plus DEX (Table 2). The difference between basal rates of lipolysis and ISO-stimulated rates of lipolysis, obtained in the presence of high concentrations of PIA (100 nM) , is an indication of the increase caused by ISO under the condition of maximal inhibition of lipolysis by adenosine. These increases caused by ISO in tissue cultured in the absence of exogenous hormones or with bST were 3457 and 4818 nmol/3 h·g of tissue, respectively (NS; Table 1). Thus, there was a 39% increase caused by bST when ISO-stimulated lipolysis was measured in the presence of PIA but no effect of bST Journal of Dairy Science Vol. 82, No. 1, 1999

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of exogenous hormones ( P < 0.05; Tables 1 and 2; Figure 1). Analysis of the dose-response curves demonstrated that responsiveness of adipose tissue was greater when tissue was chronically cultured with INS plus DEX than when cultures did not include hormones ( P < 0.05; Tables 3 and 4). Sensitivity was also increased by the addition of INS plus DEX to the chronic culture medium, although significance was observed only for NEFA release ( P < 0.1; Tables 3 and 4). DISCUSSION Effects of ST on Lipolysis Figure 1. Effect of culture of adipose tissue with bST on inhibition of maximally stimulated lipolysis by (–)-N 6-(2phenylisopropyl)-adenosine (PIA). Adipose tissue was cultured for 48 h with no additions (open symbols) or with the addition of insulin plus dexamethasone (filled symbols) in the presence (triangles) or absence (circles) of bST. After culture, explants were incubated for 3 h in the presence of isoproterenol, adenosine deaminase, and various concentrations of PIA as indicated.

when PIA was not present. The same increases were 1024 and 2017 nmol/3 h·g of tissue for explants cultured with INS plus DEX and INS plus DEX plus bST, respectively, which represents a 97% change in the response to ISO ( P < 0.05; Table 2). The effects of varying PIA concentrations on rates of ISO-stimulated lipolysis were also investigated. Analysis of the dose-response curves indicated that the addition of bST to the chronic cultures reduced the ability of adenosine (PIA) to inhibit lipolysis (Figure 1). The standardized slope parameter of the PIA dose-response curve was altered by bST addition, regardless of whether comparisons involved explants cultured without exogenous hormones ( P < 0.05) or with INS plus DEX ( P < 0.01) (Figure 1). In explants cultured without hormones, the addition of bST did not alter sensitivity (ED 50) or responsiveness ( R max) to PIA based on glycerol release but significantly decreased the Rmax based on NEFA release ( P < 0.05; Table 3). When the response to bST was studied in chronic cultures containing INS plus DEX, responsiveness was significantly decreased for both glycerol and NEFA release ( P < 0.05; Table 4), and the numerical reduction in sensitivity for both glycerol and NEFA release tended to be significant for NEFA ( P < 0.10; Table 4). The ability of PIA to inhibit lipolysis was increased in explants chronically cultured with INS plus DEX over that observed in explants cultured in the absence Journal of Dairy Science Vol. 82, No. 1, 1999

The regulation of lipolysis involves cAMP and a signal transduction system that includes stimulatory G proteins ( Gs) and inhibitory G proteins ( Gi) . In the present study, the inclusion of bST in chronic cultures of adipose explants in the presence or absence of INS plus DEX did not alter the rates of lipolysis under basal conditions or when stimulated by ISO, a b-adrenergic agonist (Tables 1 and 2). The rates of lipolysis in the presence of ISO plus ADA should represent maximal lipolytic capacity, and our results indicate that ST does not change maximal rates. These results are in agreement with previous work involving lactating cows treated with bST (17,

TABLE 3. Effect of culture of adipose tissue in the presence or absence of bST on inhibition of lipolysis by the adenosine analog (–)-N 6-(2-phenylisopropyl)-adenosine (PIA). Culture medium1 Variable2 Glycerol release4 Rmax, % of Maximum ED50, nM NEFA Release Rmax, % of Maximum ED50, nM

Control

bST

SEM

P3

71 0.84

76 0.76

7 0.11

NS NS

57 0.66

74 0.76

4 0.33

0.05 NS

1Adipose tissue was cultured for 48 h in culture medium 199 with no addition of exogenous hormones (control) or with 100 ng/ ml of bST. 2Following the 48-h culture, tissue was incubated for 3 h in the presence of adenosine deaminase, isoproterenol, and varying doses of PIA. Lipolysis rates were determined by glycerol and NEFA release. 3Statistical probability of treatment effects (NS = P > 0.10); n = 4 cultures from individual cows. 4Dose-response parameters for the inhibition of lipolysis were estimated by nonlinear curve fitting of curves generated for each cow. Responsiveness ( R max) is expressed as a percentage of maximal rates of lipolysis (PIA dose = 0); sensitivity (ED 50) represents the PIA dose at which 50% of maximal inhibition occurs.

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SOMATOTROPIN AND ADIPOSE LIPOLYSIS TABLE 4. Effect of culture of adipose tissue with insulin (INS) and (DEX) in the presence or absence of somatotropin on inhibition of lipolysis by the adenosine analog (–)-N 6-(2-phenylisopropyl)adenosine (PIA). Culture medium1 Variable2 Glycerol release4 Rmax, % Maximum ED50, nM NEFA Release Rmax, % Maximum ED50, nM

INS + DEX

INS + DEX + bST

SEM

P3

35 0.57

60 0.76

6 0.13

0.05 NS

21 0.36

45 0.82

7 0.14

0.05 0.10

1Adipose tissue was cultured for 48 h in culture medium 199 with INS plus DEX in the presence or absence of 100 ng/ml of bST. 2Following the 48-h culture, tissue was incubated for 3 h in the presence of adenosine deaminase, isoproterenol, and varying doses of PIA. Lipolysis rates were determined by glycerol and NEFA release. 3Statistical probability of treatment effects (NS = P > 0.10); n = 4 cultures from individual cows. 4Dose-response parameters for the inhibition of lipolysis were estimated by nonlinear curve fitting of curves generated for each cow. Responsiveness ( R max) is expressed as a percentage of maximal rates of lipolysis (PIA dose = 0); sensitivity (ED 50) represents the PIA dose at which 50% of maximal inhibition occurs.

20) and lactating rats injected with exogenous ST or antibodies to ST (1, 36). However, Watt et al. ( 4 0 ) conducted chronic cultures of sheep adipose tissue and observed that ST caused a modest increase in ISO-stimulated rates of lipolysis; ADA was not included in the medium that was used to measure rates of lipolysis, and the difference from our results probably reflects variation in endogenous adenosine. As is discussed subsequently, in vitro studies of lipolysis mechanisms have to address endogenous adenosine concentrations. Consistent with the lack of bST effects on basal rates of lipolysis, we also failed to observe changes in HSL activity (Tables 1 and 2). Thus, it would appear that alterations in the activity of HSL do not play a major role in the response of adipose tissue to bST, although modest changes have been observed in an adipose cell line (3T3-F442A) that was chronically cultures with ST ( 8 ) and with bST treatment of lactating cows (20). However, the subcellular distribution and the interaction of HSL with the lipid droplet may also be regulated (10, 38), and these aspects were not examined in the present study. The inclusion of bST in the chronic cultures of adipose tissue reduced the antilipolytic response to PIA, an adenosine analog (Tables 1 and 2). Adenosine effects are via the Gi system, and examination of the dose-response relationship demonstrated that bST altered both the sensitivity and responsiveness to adenosine (Tables 3 and 4). Similar results were observed with adipose tissue obtained from cows that had been treated with bST (17, 20). Two other effectors, the E-series of prostaglandins and a2-adrenergic

agonists also have antilipolytic effects via the Gi system. Somatotropin treatment caused a decrease in the antilipolytic response to prostaglandin E and a decrease in the production of prostaglandin E2 in subcutaneous adipose tissue of sheep ( 9 ) . A similar decrease in antilipolytic response to a2-adrenergic effectors was observed when adipose tissue of sheep was cultured with ST (40). Thus, bST affects lipolysis in adipose tissue through effects on the Gi system. Mechanisms whereby bST reduces the ability of adenosine to inhibit lipolysis have been investigated. Binding affinity and adenosine receptor number were unchanged by bST treatment (9, 18, 40). Furthermore, ST treatment did not alter the abundance of the a, b, or d subunits of the Gi proteins (9, 17). However, several posttranslational modifications of Gi proteins are possible ( 6 ) ; studies show the functionality of the Gi proteins (17, 31), assessed by ADP-ribosylation, and the ability of the a-subunit of Gi to interact with adenyl cyclase ( 3 4 ) were altered with ST treatment. Thus, the mechanism by which bST alters the lipolytic response of adipose tissue includes an altered ability of Gi to affect adenyl cyclase. In vivo studies (21, 33) with lactating dairy cows have demonstrated that bST treatment results in an enhanced lipolytic response to an in vivo challenge with epinephrine. Catecholamines affect lipolysis rates mainly through the Gs system, which appears to be paradoxical to results discussed previously for ISO. However, studies (17, 18, 20) with lactating cows have shown that b-adrenergic receptors in adipose tissue are unaltered by bST treatment and that there Journal of Dairy Science Vol. 82, No. 1, 1999

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were no effects on Gs proteins or other downstream responses. The apparent explanation for this paradox is that rates of lipolysis are a balance between the effects of the Gi system and the Gs system. The enhanced response to epinephrine observed in vivo with bST treatment is in large part due to relief in the tonic inhibition of lipolysis via changes in the Gi signaling system (11, 20). Effects of INS and Glucocorticoids Chronic cultures of adipose tissue in serum-free medium have been used to study the homeorhetic effects of ST on lipid metabolism. In several studies, INS, glucocorticoids, or both were added to maintain anabolic rates. In the present study, the combined effect of the addition of INS and DEX to chronic culture media was investigated. These additions were based on the concept that bST effects are mediated through a relief in the inhibition of lipolysis through Gi and alteration of responses to an adenosine analog, expression of Gi proteins, and functionality of Gi proteins by glucocorticoids ( 3 7 ) and diabetes (27). Sensitivity and responsiveness to the PIA inhibition of lipolysis in adipose tissue chronically cultured in the absence of exogenous hormones (Table 3 ) were lower than those found for fresh adipose tissue obtained by biopsy of lactating cows (20). However, the addition of INS plus DEX to cultures maintained adipose tissue responsiveness and sensitivity to PIA (Table 4 ) at values comparable with values obtained with fresh tissue. In effect, the ED50 values were slightly higher than the binding affinity value for A1 adenosine receptors of lactating cows (18). These results suggest that adipose tissue cultured in the presence of INS plus DEX maintained a more typical response to PIA and, thus, may be a more suitable system by which to study metabolic adaptations in adipose tissue, especially when alterations in G protein function are expected. The ability to maintain response to PIA was not observed in explants cultured with INS alone (data not presented). These limited results are consistent with previous data from Vernon et al. ( 3 7 ) in which response to PIA was decreased when chronic cultures of sheep adipose contained only INS, but response was maintained in chronic cultures that included only glucocorticoids. In these experiments, response to a2adrenergic stimulation was increased (beyond initial fresh tissue values) by DEX, but INS had contrasting effects (37). Because PIA and a2-adrenergic signals converge, it appears that chronic treatment with Journal of Dairy Science Vol. 82, No. 1, 1999

glucocorticoids up-regulate an element of the signal transduction system downstream from these effectors. A logical possibility is the Gi system. Chronic incubation with INS plus DEX increased the rates of lipolysis in response to ISO over control explants (Tables 1 and 2). Similar increases in ISOstimulated lipolysis were observed after prolonged exposure to physiological concentrations of glucocorticoids in adipose tissue from rats ( 1 5 ) and sheep (37). The mechanisms by which glucocorticoids appear to alter the lipolytic cascade are not known, but, at least in some cell lines, glucocorticoids increase both numbers of b-adrenergic receptors and the amount of the as subunits of the Gs system (19, 29). These results differ from the previously discussed effects of ST in which expression of Gi and Gs proteins were not altered. Glucocorticoids increase elements of both the Gi and Gs systems that regulate cAMP concentrations, which appears paradoxical. However, similar adaptations have been observed with the onset of lactation; concomitant increases have occurred in b-adrenergic receptor numbers, lipolytic response to epinephrine, antilipolytic response to adenosine analog [see reviews (2, 39)], and increased amounts of Gi and Gs proteins in adipose tissue from rats ( 3 0 ) and Gi proteins from sheep (35). Adaptations in both stimulatory and inhibitory elements that regulate lipolysis may be advantageous with the onset of lactation because mammals can more efficiently orchestrate the use of body reserves to meet the metabolic demands of lactation. Regulation of adipose tissue lipolysis has been shown to involve alterations in G protein expression and function in a number of physiological states and situations including onset of lactation, chronic underfeeding, exercise, hyperthyroidism, and diabetes (7, 17, 27, 39). In the present study, these adaptations were demonstrated for bST addition in chronic cultures of adipose tissue and were shown to depend in part on other hormones added to the medium. Clearly, the chronic culture technique offers an opportunity to evaluate treatments that may alter functions of G proteins and their mechanisms of action. CONCLUSIONS Lipolysis is regulated by the cAMP cascade, involving a signal transduction system that includes stimulatory and inhibitory elements. The addition of ST to chronic cultures did not increase lipolysis stimulated by both ISO and ADA. However, addition of ST to the culture medium decreased the antilipo-

SOMATOTROPIN AND ADIPOSE LIPOLYSIS

lytic response to an adenosine analog. Similar changes in adipose tissue were previously reported with in vivo administration of bST to lactating cows and demonstrated that ST allows for enhanced rates of lipolysis primarily by a relief from the inhibition mediated by receptors coupled to the inhibitory G protein system. Results of the present study demonstrated that the addition of INS plus DEX to the culture medium better maintained the intracellular signaling systems as evidenced by lipolytic and antilipolytic responses to b-adrenergic analogs and adenosine analogs, respectively. Overall, results demonstrate that the chronic culture system with adipose tissue explants can be used to investigate aspects of the regulation of lipolysis in the bovine. ACKNOWLEDGMENTS Authors gratefully acknowledge the assistance of Debbie Dwyer, Bill English, and Dottie Ceurter. REFERENCES 1 Barber, M. C., R. A. Clegg, E. Finley, R. G. Vernon, and D. J. Flint. 1992. The role of growth hormone, prolactin and insulinlike growth factors in the regulation of rat mammary gland and adipose tissue metabolism during lactation. J. Endocrinol. 135: 195–202. 2 Bauman, D. E., and J. M. Elliot. 1983. Control of nutrient partitioning in lactating ruminants. Pages 437–468 in Biochemistry of Lactation. T. B. Mepham, ed. Elsevier Sci. Publ. BV, Amsterdam, The Netherlands. 3 Bauman, D. E., and R. G. Vernon. 1993. Effects of exogenous bovine somatotropin on lactation. Annu. Rev. Nutr. 13:437–461. 4 Belfrage, P., and M. Vaughan. 1969. Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J. Lipid Res. 10:341–343. 5 Boobis, L. H., and R. J. Maughan. 1983. A simple one-step enzymatic fluorometric method for the determination of glycerol in 20 microliters of plasma. Clin. Chim. Acta 132:173–179. 6 Busconi, L., and B. M. Denker. 1997. Analysis of the N-terminal binding domain of Goa. Biochem. J. 328:23–31. 7 Carey, G. B., and K. A. Skidmore. 1994. Exercise attenuates the anti-lipolytic effect of adenosine in adipocytes isolated from miniature swine. Int. J. Obesity 18:155–160. 8 Dietz, J., and J. Schwartz. 1991. Growth hormone alters lipolysis and hormone-sensitive lipase activity in 3T3-F442A adipocytes. Metabolism 40:800–806. 9 Doris, R. A., G. E. Thompson, E. Finley, E. Kilgour, M. D. Houslay, and R. G. Vernon. 1996. Chronic effects of somatotropin treatment on response of subcutaneous adipose tissue lipolysis to acutely acting factors in vivo and in vitro. J. Anim. Sci. 74:562–568. 10 Egan, J. J., A. S. Greenberg, M. Chang, S. A. Wek, M. C. Moos, Jr., and C. Londos. 1992. Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. Proc. Natl. Acad. Sci. USA 89: 8537–8541. 11 Etherton, T. D., and D. E. Bauman. 1998. The biology of somatotropin in growth and lactation of domestic animals. Physiol. Rev. 78:745–761. 12 Etherton, T. D., C. M. Evock, and R. S. Kensinger. 1987. Native and recombinant bovine growth hormone antagonize insulin

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