Effect of Porcine Somatotropin on In Vivo Glucose Kinetics and Lipogenesis in Growing Pigs l J 2 r 3

F. R. Ih1nshea4, D. M. Hams, D. E. Ba~man5, R. D. Boyd, and A. W. Bell

Department of Animal Science, Cornell University, Ithaca, N Y 14853

ABSTRACT: Crossbred barrows were used for in vivo studies investigating hormonal regulation of lipogenesis. The first experiment examined an in vivo method for determining rates of lipogene- sis. Three barrows were infused with [U- Wlglucose and incorporation of radioactivity into triglycerides was determined in up to five biopsies of subcutaneous adipose tissue obtained over 7 h. Incorporation was linear after blood glucose specific radioactivity had reached a plateau and was constant over the entire infusion. For the second experiment, eight pigs (71 2.5 kg) were allocated to one of two treatments involving daily injections of excipient (control) or porcine somato- tropin (pST; 120 pg/kg of B W . On d 10, beginning 15 h after injection, glucose incorporation into

adipose tissue lipid was determined under both basal and hyperinsulinemic/euglycemic condi- tions. Basal glucose incorporation into lipid, par- ticularly fatty acids, was markedly reduced ( > 90%) during pST treatment. Although glucose incorporation was increased to a similar extent in both groups by hyperinsulinemia, the pST-treated pigs still exhibited markedly lower rates. Based on kinetic data, the decrease in lipid accretion of pST-treated pigs was primarily the result of a decrease in the rate of de novo synthesis. Further- more, the reductions in glucose incorporation into fatty acids, glucose irreversible loss rate, and feed intake that occur with pST treatment were quanti- tatively similar.

Key Words: Pigs, Somatotropin, Glucose, Lipogenesis, Insulin

Introduction

Porcine somatotropin (pS'l") treatment of grow- ing pigs causes dramatic decreases in adipose tissue lipid accretion by decreasing rates of lipogenesis and(or1 by increasing rates of lipolysis

'Work supported in part by Cornell Univ. Agric. Exp. Sta. and USDA Competitive Res. Grant X89-37265-4478.

2We acknowledge PitmanMoore Inc. Rerre Haute, IN) for donation of the porcine somatotropin and Eli Lilly (Greenfield, IN) for bovine insulin and porcine insulin. Authors wish to thank P. McNamara, W. English, R. Dickens, M. Harkins, D. Ceurter, M. A. McGuire, D. Dwyer, and R. Slepetis for technical support. The loan of the biopsy gun by Norman C. Steele of the USDA, Beltsville, MD is appreciated.

3Presented in part at the joint meeting of the ASAS and Am. Dairy Sci. Assoc., Lexington, KY, July 31 to August 4, 1989.

4Present address: Victorian Inst. of Anim. Sci., Werribee, Victoria 3030, Australia.

5To whom correspondence should be addressed. Received April 10, 1991. Accepted August 1, 1991.

J. Anim. Sci. 1992. 70:141-151

(Machlin, 1972; Boyd et al., 1986; Campbell et al., 1988, 1989; Evock et al., 1988). In vitro studies with adipose tissue from pST-treated pigs have shown decreased rates of lipogenesis (Walton et al., 1987; Mike1 et al., 1988; Harris et al., 1090; Magri et al., 1990). However, in vitro adipose tissue incubations are in a net degradative state and although they may provide good qualitative data, their ability to provide reliable quantitative estimates can be limited (Mersmann and Koong, 1984; Mersmann et al., 1984; Mersmann, 1986a),

In vivo methods have been employed by many groups to estimate rates of protein synthesis (Waterlow et al., 1978). Recently, Davey (19861 applied similar principles for in vivo measure- ment of lipid synthesis in adipose tissue of growing lambs. Our initial objective was to extend this work and to determine the efficacy of an in vivo technique for determining lipogenesis in swine. After it was validated, we used this technique for our main objective, which was to

141

142 DUNSHEA ET AL.

determine the effect of chronic pST treatment on adipose tissue lipogenesis in the growing pig. In addition, acute effects of hyperinsulinemia on lipogenesis were examined.

Materials and Methods

Animals and Treatments. Crossbred barrows were used in these studies. Details of the mainte- nance and housing of animals and diet composi- tion were provided in a companion paper (Dun- shea et al., 1992a). Pigs were fed a nutrient-dense diet every 4 h in quantities such that approxi- mately 150 g of feed was refused each day. Three pigs (approximately 72 kg BW) were used in a preliminary study (Exp. 1) to evaluate an in vivo method of measuring lipid synthesis. In a second study (Exp. 21, eight pigs were randomly allocated to one of two treatments @ST or control). Pigs commenced this study weighing 71 f 2.5 kg and received daily i.m. injections of pST (120 pghg of BW; lot UI-LGCOMP-1776/47-1, Pitman-Moore, Terre Haute, IN) or excipient (controll at 1800 (Dunshea et al., 1992a).

Infusions. Details of the methods of catheteriza- tion and maintenance of the blood catheters for these animals were described in a companion paper (Dunshea et al., 1992a).

Experiment 1 was conducted to determine whether the rate of incorporation of [U- 14Clglucose h e r s h a m , Arlington Heights, ILI into adipose tissue lipids was linear over an extended interval. A primed (120 pC3, continuous (1.5 pCi/min) infusion of W4Clglucose in saline solution was given via an intravenous catheter (jugular vein) for 4 h (two pigs) or 7.5 h (one pig) using a dual syringe pump (Vial Medical, USA, Panamedic Inc., Costa Mesa, CAI. Arterial Sam- ples (10 mL1 for glucose concentrations and specific radioactivity (SRA) were taken at 5,10, 15, 20, 30, 45, and 60 min after commencement of the infusion and at 30-min intervals thereafter. Blood samples were dispensed into glass tubes contain- ing Na heparin (60 U/mL fiial concentration) as an anticoagulant and stored on ice before deproteinization. Blood (.5 mL) was added to distilled water (2.5 mL) and vortexed. One mil- liliter each of .3 A4 Ba(OHI2 and 5% &SO4 were added, and the solution waa vortexed after each addition. Deproteinized blood and the remainder of the whole blood were then centrifuged for 15 min at 2,000 x g and 4°C to obtain deproteinized supernatant and plasma, respectively. These were stored at -60°C until analyses.

For Exp. 2, infusions (via jugular vein catheter) and blood sampling (via catheter in the lumbar aorta) commenced on the moming after the loth

daily injection (pST or excipient). A primed (80 pCi), continuous (1.0 pCi/minl infusion of [U- 14Clglucose was initiated at 0900 and continued for 4 h. Arterial blood samples (10 mL) for blood glucose concentrations and SRA were taken at 5, 10, 15,20, 30, 45, and 60 min after commencement of the infusion and at 30-min intervals thereafter. Blood samples were dispensed into glass tubes containing Na heparin (60 U/mLl and stored on ice. After rapid determination of arterial blood glucose concentrations (Model 27, Yellow Springs Instrument, Yellow Springs, OH), blood was lysed and deproteinized.

A second component of Exp. 2 was to induce hyperinsulinemia and examine the same kinetic variables. During hyperinsulinemia, blood glucose concentration was monitored and held constant (euglycemic clamp). This phase was initiated immediately after the 4-h infusion period outlined above. Initially, a priming dose of bovine insulin (50 mU/kg of BW; lot 615-70N-80, 26.6 U/mg, Eli Lilly and Co., Indianapolis, IN) was given intrave- nously (via jugular vein catheter), which was then followed by continuous infusion of insulin (6.0 rnU.min-l.kg of BW-') and (U-14Clglucose (2.5 pCi/min). A catheter extension for infusing unla- beled glucose (dextrose monohydrate, 500 g/L, Baxter, Deerfield, IL) at varying rates was also connected. Arterial blood samples (.75 mU were taken at 5-min intervals and immediately ana- lyzed for blood glucose concentration. Based on the change in concentration over each 5-min interval, the infusion rate of unlabeled glucose was varied to maintain euglycemia. Arterial blood samples for other determinations continued to be taken at 30-min intervals. After 4 h, continuous infusions of insulin and radioactive glucose were terminated. Thereafter, arterial blood glucose concentrations were monitored for an additional 2 h and infusion rate of unlabeled glucose was slowly decreased to ensure that pigs remained normoglycemic.

Insulin concentrations were determined by RIA using a commercial kit CAutopak Insulin RIA kit, produced by Micromedic Systems, Inc. and ob- tained from ICN Biomedicals, Horsham, PA). Porcine insulin aot #092W6,26.8 U/mg, Eli Lilly and Co., Indianapolis, IN1 was used to make the RIA standards.

Adipose Tissue Biopsy and Analyses. Adipose tissue biopsies were obtained under local anesthe- sia from an area between 4 and 12 cm distd to the last lumbar using a spring-loaded biopsy gun Prototypes Inc., Kensington, MD) (Steele et al., 1974). Biopsies were obtained from alternate sides of the midline and sites were immediately flushed with local anesthetic and sprayed with an an- tibacteriostat. Pigs were not overtly concerned

SOMATOTROPIN AND LIPOGENESIS IN PIGS 143

with the biopsy procedure and continued to eat throughout the experiments. For Exp. 1, biopsies (approximately 1 g of subcutaneous adipose tis- sue) were obtained at approximately 1, 1.5, and 4 h after commencement of the continuous infusion of radioactive glucose. In one of the pigs, two additional biopsies were obtained at 5.5 and 7 h. During Exp. 2, adipose tissue biopsies were taken at 1 and 4 h (tl and tz) for basal determinations and at 5 and 8 h (t3 and t4) for measurements during hyperinsulinemia.

A portion of the adipose tissue biopsy was cut into small pieces of approximately 100 mg and homogenized in 20 mL of a chloroform-methanol (2:l) solution and allowed to extract overnight (Folch et al., 1957). After washing to remove nonlipid material, a measured aliquot was re- moved and dried under a gentle stream of air at 40°C until it achieved a constant weight. The dried lipid was dissolved in 500 pL of heptane and 10 mL of scintillation cocktail and radioactivity was determined Model no. 2200CA Tricarb, Pack- ard Instrument, Downers Grove, IL). The dual label mode was used to discriminate between I4C label from glucose and 3H label from oleic acid infused in an earlier study (Dunshea et al., 199223). The remaining dissolved lipid was dried under nitrogen gas and then saponified in 4 mL of 5% KOH in methanol. After washing with heptane to remove nonsapodiable lipid (containing negligi- ble amounts of radioactivity), fatty acids were extracted into heptane after acidification. An aliquot (.5 FL) was immediately subjected to gas- liquid chromatography to determine fatty acid composition CDunshea et al., 1992b). The r e mainder was dried under a gentle stream of nitrogen before being reconstituted in heptane and scintillation cocktail and counted as described above. The aqueous methanol fraction from the saponification, which contained the glycerol moiety, was taken up in scintillation cocktail and radioactivity was determined.

Blood Glucose Specific Radioactivity. Radiola- beled glucose was isolated by column chromatog raphy using three columns connected in a series. The sequence of columns was acetate (Dowex 1, 200-400 mesh), hydrogen (Dowex 50, 100-200 mesh), and borate (Dowex 1, 4 parts 50-100 mesh to 1 part 100-200 mesh), which were prepared as outlined by Mills et al. (1981). Deproteinized blood (3.5-mL aliquot) was added to the first column and then washed twice with 4.0 mL of distilled water. The acetate and hydrogen columns were subse- quently removed and the borate column (contain- ing the glucose complex) was washed with a further 4.0 mL of distilled water. Isolated glucose was eluted from the borate column with 4 x 4 mI, of 1 M acetic acid and collected into a

20-mL scintillation vial. Eluate was dried at 50°C under a gentle stream of air, after which the residue was dissolved in 10 mL of methanol and dried again to remove borate crystals. This was repeated if borate crystals persisted. The isolated glucose was then reconstituted in 2.0 mL of distilled water. After glucose concentration was determined (100 pL in duplicate), the remaining solution was combined with scintillation cocktail and radioactivity was determined.

Statistics and Calculations. The rate of glucose incorporation into adipose tissue lipid (Rupid) was calculated from the increase in accumulation of 14C over time. If it is assumed that all radioac- tivity in adipose tissue lipid originated from the radioactive glucose in blood, then

where REpid is the rate of glucose incorporation into adipose tissue lipid t h g glucosel/[g lipidJ.min), SRAupid is the lipid specific radioac- tivity (dpm/mg lipid) in adipose tissue, and t2

SRAglucose is the cumulative area under the tl

blood glucose specific radioactivity (dpm.min/tmg of glucosel) vs time curve between biopsies taken at tl and t2. This area was determined by integration over the time interval of interest. Rates of glucose incorporation into triglyceride fatty acids (RTG-FA) and triglyceride glycerol &G glycerol) were determined similarly based on the accumulation of label into these respective trigly- ceride moieties.

During Exp. 2, apparent glucose irreversible loss rate was calculated by dividing the infusion rate of [U-14Clglucose by blood SRAglucose over the last 2 h during both the basal condition and the hyperinsulinemideuglycemic clamp. Plasma clearance of insulin during the hyperinsulinemic/ euglycemic clamp was determined by dividing the insulin infusion rate by the resultant increment in plasma insulin concentrations above basal.

Statistical analyses were based on a split-plot design with treatment (pSTl as the whole-plot factor and insulin (INS) basal vs hyperinsuline- mia) as the subplot factor (SAS, 1985). The model used in the analysis of variance included: pST, pig(pST1, INS, pST x INS, and pig(pSTl x INS as factors. The error terms used for the F-statistics were pig(pSTl for evaluating significance of pST treatment and pig[pSTl x INS for evaluating hyperinsulinemia and pST x INS interaction.

144 DUNSHEA BT AL.

10

5 -

4 K In

-

s W vr 0

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15 r o o i 30

0 /"O

/ /"

o p I 1 I I

0 120 240 360 480

TIME (rnin)

20

10

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=' Ln Ln c m

Ln n P

Figure 1. Blood glucose specific radioactivity (SRA) (0) and adipose tissue lipid SRA (0) during a primed (120 pCi), continuous (1.5 pCifmin) infusion of [U- 14C]glucose into a barrow during Exp. 1. Fitted regression line for adipose tissue lipid SRA vs time is Y - 1.34 + .0641 X; r2 - .98.

Results

Experiment 1. The use of an in vivo method to measure rates of lipid synthesis was evaluated in three animals. Data from the animal in which five biopsies were obtained over a 7-h period are presented in tabular and figure form. Blood SRAglumse rapidly declined after initiation of the primed, continuous infusion of CU-14Clglucose,

reaching a plateau by about 60 min (Figure 1). This confirmed preliminary calculations based on estimates of glucose irreversible loss rate and hence corresponded to the timing of the first adipose tissue biopsy. The Rmid was constant, as evidenced by the linearity of the increase in S U l i p i d during plateau blood SRAglucose (Figure 1). The 9 between adipose tissue s m f i p i d and time was $8 for this pig and .96 and .99 for the other two pigs used to examine linearity of incorporation.

Further confirmation of COnStant Rlipid across time is provided by the close agreement between estimates of Rhpid determined from each of the individual biopsies (Table 1). Based on these data it was decided that only two biopsies were necessary during the basal period or during hyperinsulinemia to obtain reliable estimates of Rhpid for the subsequent experiments.

Experiment 2. Concentrations of blood glucose and plasma insulin were chronically elevated in pST-treated animals (Table 2). On the other hand, glucose irreversible loss rate was decreased by 35% by pST treatment (Table 21, consistent with the magnitude of reduction in feed intake (3.2 vs 2.1 kg/d, P < .05). Infusion of exogenous insulin increased plasma insulin by approximately 20-fold in both treatment groups compared with basal concentrations observed in the control pigs (Table 2). Plasma insulin clearance was not altered by treatment (18.1 f .51 vs 19.9 f 1.05 mL-min-l.kg of BW-l, mean f SE for control and pST-treated pigs, respectively). During hyperin- sulinemia, euglycemia was maintained by simul- taneous infusion of glucose, the rate of which did

Table 1. Comparison of estimates of the rate of glucose incorporation into lipid from sequential adipose tissue biopsies obtained from a growing barrow during a continuous infusion of [U-14Cjglucosea

Blood

tl Adipose tissue c. S ~ * l U C , B , ,

Time of

biopsy, min A smfipidn tl %pia. tl tz dpmfmg of lipid dpm.min-pg of glucose-' pg*min-'.g of lipid-'

0 67 6.ob 5MC 1l.d 0 155 10.4 003 10.5 0 255 10.4 1,546 12.6 0 340 21.1 2,037 10.3 0 430 29.8 2,537 11.7

67 430 23.8 2,034 11.7 aResults from Exp. 1 in which a single barrow received a primed, continuous -ion of IU-

'*Clglucose into the jugular vein for 7.5 h. Adipose tissue biopsies bee Materials and Methods section) were obtained at 0, 67, 155, W, 340, and 430 min of the idusion period.

bChange in adipose tissue lipid specific radioactivity SFLAB ccumulative area under the blood glucose specific radioadvity [sFtAgbse) vs time curve

between tl and klucose incorporation into total lipid [REpid) calculated as outlined in Materials and Methods

section.

between tl and tz.

calculated by integration over the time interval.

SOMATOTROPIN AND LIPOGENESIS IN PIGS

15

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Q 10

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. c

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i

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not differ between control and pST-treated pigs (Table 2). Glucose irreversible loss rate was increased during ,hyperinsulinemia to an extent that approximated the infusion rate of exogenous glucose. As a consequence of the decreased basal

glucose irreversible loss rate during pST treat- ment, blood SRAgl~cose took longer than antici- pated to reach plateau (Figure 21. Therefore, only the last 00-min interval (four blood samples) of the basal infusion period was used to determine glucose irreversible loss rate. The rate at which [U-14Clglucose was infused during the hyperin- sulinemia period was calculated from the ratio between the estimate of basal glucose irreversible loss rate obtained from Exp. 1 and the glucose infusion rate needed to maintain euglycemia in these pigs in a related study conducted 2 d earlier (Dunshea et al., 1992b). As a consequence, a relatively constant blood SRAglucose was main- tained during hyperinsulinemia (Figure 2) and glucose irreversible loss rate was calculated over the last 2 h of infusion ffive blood samples).

Two approaches were examined to determine Rlipid, as well as %GFA and h'Gglycero1. In the first method (A), Rfipid was determined over the intervals tl to t2 and t3 to t4 for the basal and hyperinsulinemia periods, respectively. During these periods blood SRAglucose was essentially at plateau, so rate of incorporation of radioactive glucose was likely to be constant. The second technique (B) was considered worthy of evaluation

hyper-INS

Table 2. Effect of porcine somatotropin on arterial concentrations of blood glucose and plasma insulin and glucose irreversible loss rate (ILR)

in growing barrows during basal conditions and during a hyperinsulinemideuglycemic clampa

Basal Hyper-INSb Statistical Variable Control pST Control pST SE= signiGcanced

Blood glucose, mg/dL 75.0 93.7 73.9 90.4 2.21 pST*** Plasma insulin, pu/mL 17 72 350 377 28.4 INS*** Glucose ILR mg-min-'.kg of BW' 10.0 0.0 23.0 20.5 1.37 pST*, INS*** mg.min-'.kg of MBW' 31.0 20.0 07.8 61.0 3.91 pST*. INS***

Glucose infusion*, mi~.min-'-kn of B W ' - - 21.1 19.8 1.04 -

qreatment involved daily i.m. injections of porcine somatotropin (pST, 120 W/kg of B W or excipient (control). Studies were conducted from 15 to 19 h (basall and 19 to 23 h (hyperinsulinemid after the 10th daily injection of pST or excipient. Values are based on samples obtained over the last 90-min interval of the basal period and the last 2-h interval of hyperinsulinemia.

bHyperinsulinemia (hyper-INS) was achieved by continuously infusing bovine insulin (0.0 mu.min-'.kg of B W ~ .

'Estimated SE for the difference of a pair of treatment means at the same or different levels of insulin.

dStatistical sigdicance of pST, insulin (INS), or an interaction (pST x INS) indicated by *(P c .051 and ***W c .001). Other effects were nonsignificant at P z .lo.

%ate of glucose infusion required to maintain euglycemia during the period of hyperinsuline- mia.

146 DUNSHEA ET AL.

because it required fewer biopsies. In this method Rfipid was determined over the intervals t o to tz and t2 to t4 for basal and hyperinsulinemia, respectively. Over these intervals, blood SRAglu- case was in a greater state of flux, particularly in the interval immediately after the IU-14Clglucose priming dose and initiation of continuous infusion and again immediately after insulin prime and commencement of the continuous infusion of insulin. Nevertheless, the relationship between

Rfipid and SRAglucMe remains the same and, if

the latter can be accurately determined, a single biopsy should be sufficient for each state. A critical assumption is that Rfipid is constant. Although this was demonstrated in the basal state (Exp. 1, Figure 11, it is likely that the non- steady state that occurred at the commencement of the hyperinsulinemia period invalidates this assumption. However, if the period of non-steady state is brief compared to the total duration of the infusion, then its influence on the final estimate of Rlipid is minimized. Indeed, the two approaches gave similar estimates of incorporation (Table 3). ValUeS Of Rfipid, &GFA, and &Gglycerol estimated by method A averaged 108 f 4.1, 109 f 4.8, and 97 f 7.2% (mean f SE across periods and treatments) of estimates obtained with method B, respectively. Regressions comparing the two methods had slopes of 1-00, 1.00, and 1.19 and 1.2 values of .98, .98, and 3 4 , respectively.

t2

t l

Somatotropin treatment resulted in dramatic reductions in basal Rfipid (Table 3). This is clearly demonstrated in Figure 3, in which cumulative glucose incorporation is shown for each of the serial biopsies. The predominant effect waa on RTGFA, which was decreased over 12-fold whereas RTG@ycerol was decreased just over fivefold. Insu- lin infusion increased Rfipid. and &-GFA by a similar absolute amount m both treatment jgoups. Nevertheless, insulin-stimulated rates of glucose incorporation into lipid were still much lower in pST-treated animals.

Despite the dramatic decrease in WGFA in the pST-treated pigs, the fatty acid composition of adipose tissue triglycerides remained unchanged (Table 41. The major fatty acid was oleic acid, which constituted approximately 45% of all fatty acids.

Discussion

An in vivo approach using pulse labeling with radioactive glucose or acetate has been used to investigate lipogenesis in growing pigs and other species (O'Hea and Leveille, 1969; Ingle et al., 1972). Based on the recovery of label at slaughter a few minutes after injection, it was demonstrated that adipose tissue was the major site of de novo lipid synthesis in pigs and ruminants. However, this type of in vivo approach does not allow dynamic measurements of the rates of lipogene- sis.

Table 3. Effect of porcine somatotropin on in vivo measurements of the rate of glucose incorporation into lipids in adipose tissue in growing barrows during basal conditions and during a hyperinsulinemicleuglycemic clampa

Basd Hyper-INSd Statistical Variableb MethodC Control pST Control pST SEe significance'

pg.~&~-' . g of lipid-l

Rlipid A 15.9 1.5 24.4 8 2 3.40 pST*, INS** B 18.5 1.1 23.3 7.0 3.16 pST*, INS**

%GFA A 14.2 1.1 21.5 8.7 2.9 1 pST*, INS** B 14.7 .8 20.7 5.8 2.74 pST**, INS**

%Gglycerol A 1.7 .3 2.8 1.5 .89 INS* B 1.8 .3 2.6 1.2 .50 pSTt, INS*

%eatment involved M y i.m. injections of porcine somatotropin @ST, 120 pgkg of B W or excipient (control). Studies were conducted from 15 to 19 h (basal) and 19 to 23 h Qlyperinsulinemia) after the loth daily injection of pST or excipient. Values are based on samples obtained over the last 00-min intervd of the basal period and the last 2-h interval of hyperinsulinemia.

b ~ t e s of glucose incorporation in adipose tissue were calculated for lipid @@id), triglyceride- fatty acids ~ G F A ) and triglycerideglycerol ~Gglpcerol ) .

CTechniques for calculation of glucose incorporabon rates (methods A and B1 are outlined in Materials and Methods section.

dHyperinsulinemia (Hyper-INS1 was achieved by continuously inpuSing bovine insulin (6.0 rnU-min-l-kg of BW'I.

?Estimated SE for the difference of a pair of treatment means at the same or different levels of insulin.

'Statistical significance of pST, insulin (INS), or an interaction @ST x INS) indicated by 'LP < .lo),* IP < .051, and **W < .01). Other effects were nonsignificant at P > .IO.

SOMATOTROPIN AND LIPOGENESIS IN PIGS 147

Table 4. Effect of porcine somatotropin on the molar percentages of fatty acids of triglycerides

from adipose tissue of growing barrowsa

Fatty Statistical acid Control DST SE s w i c a n c e b

- molar percentage - 18:O 29.0 29.7 .57 NS 18:l 2.3 2.3 .14 NS 18:O 12.9 12.9 .24 NS 18:l 46.5 44.5 .57 NS 18:2 9.3 10.5 .96 NS

%eatment involved daily i.m. injections of porcine somato- tropin (pST, 120 pg/kg of B W or excipient (controll. Adipose tissue was obtained by biopsy from the subcutaneous lumbar re 'on on the loth d of treatment.

'Statistical significance of pST, insulin (INS), or an interac- tion (pST x INS). Differences were nonsignificant (NS) at P > .lo.

In vivo methods have been extensively used to obtain protein synthetic rates Waterlow et al., 1978). Recently, Davey (1986) applied these princi- ples to measure rates of triglyceride-glycerol synthesis in sheep. This involved [U-14Clglucose infusion for up to 24 h and obtaining serial biopsies of subcutaneous adipose tissue through- out the infusion interval. Davey (1986) found that when blood SRAglucose was at plateau, the rate of incorporation of label into the triglyceride- glycerol of adipose tissue was linear. However, no priming dose of [U-14Clglucose was used and plateau blood SRAglucme was not achieved until 2 to 4 h after commencement of the infusion. Although a plateau in blood SRAg1ucose is not essential provided that the area under the blood SRAglu,e vs time curve can be integrated, we thought that in foundation studies it was desira- ble to achieve plateau in blood SRAg1Uc-e as soon as possible. Hence, we used a primed, continuous infusion of [U-14Clglucose to label the precursor pool in growing pigs. We observed that the increase in SRAUpid of subcutaneous adipose tissue was linear with time when blood SRAglucos, was at plateau (Figure 1). Furthermore, very similar estimates for rates of lipid synthesis were derived from adipose tissue biopsies obtained at various times intervals during the infusion of radioactive glucose (Tables 1 and 3).

A consideration in the interpretation of results from studies involving in vitro incubations of adipose tissue is that the lipogenic rates fre- quently underestimate observed lipid accretion rates in Vivo. For instance, basal Rfipid for the control pigs (80 nmol-min-' - g of lipid-l, Table 31 is greater than that typically observed during in vitro incubation of adipose tissue from pigs of similar weight (approximately 14 to 30 nmol.min-l.g of lipid-l; Mike1 et al., 1988; Harris

et al., 1990). In a study designed to optimize conditions for in vitro investigations of lipid metabolism with adipose tissue from young swine, which exhibit very high rates of lipid accretion,

achieved when concentrations of medium compo- nents were optimized (Mersmann and Hu, 1987). In our laboratory, there was about a threefold difference between RKpid rates determined in vivo (Table 3) and in vitro (Harris et al., 1990) in the same animal.

Effects of insulin on rates of lipogenesis have been extensively investigated using in vitro sys- tems. Whereas insulin is a potent stimulator of lipogenic rates in rat adipocyte preparations, effects on porcine adipose tissue have been equivocal. Many researchers working with por- cine adipose tissue have failed to observe any effect of insulin on rates of glucose incorporation into lipids, whereas others have observed mar-

Rfipid O f Up to 70 n m O l . m . h - ' * g of lipid-' was

or modest increases (see discussion and

l 2 r basal hyper-INS

0 120 240 360 480

TIME (min)

Figure 3. In vivo rates of glucose incorporation into lipid by subcutaneous adipose tissue during continuous infusion of [U-14C]glucose into barrows treated with a daily injection of excipient (0, n - 4) or porcine somatotropin (pST, 120 pgkg of BW) (0, n - 4). Measurements were made during the basal period (0 to 240 min) and during a hyperinsulinemic/euglycernic clamp (240 to 480 min). Rates of continuous infusion of labeled glucose were 1.0 and 2.5 pCi/min during basal and hyperinsulinernia conditions, respectively . Hyperinsulinemia (hyper-INS) was induced by a primed (50 mU/kg of BW), continuous (6.0 mU.min-l.kg of BW-I) infusion of insulin, and euglycemia was maintained by infusion of exogenous glucose. Bars on data points depict SE.

148 DUNSHEA ET AL.

references in Walton and Etherton, 1986 and Mersmann and Hu, 19871. A portion of the differences relate to variation in preparations of BSA, which are routinely included in the incuba- tion media. However, this does not explain the lack of effects observed in some studies in which BSA was not a component of the incubation medium (Walton and Etherton, 1986; Mersmann and Hu, 1987). Our study represents the first in vivo investigation and conclusively demonstrates that rates of lipogenesis in adipose tissue of growing pigs are acutely stimulated by insulin. Circulating insulin concentrations were elevated about 20-fold (Table 21 and the rate of glucose incorporation into lipids were increased by ap- proximately 50% during the hyperinsulinemid euglycemic clamp in control pigs (Table 3, Figure 31. Other work has shown that this insulin dose is sufficient to ensure maximum rates of whole-body glucose utilization in growing pigs during hyperinsulinemideuglycemic clamp studies (Wray-Cahen et al., 19901. By examining the effect of insulin during a euglycemic clamp, we avoided the confounding counter-regulatory changes that occur when blood glucose falls.

Treatment with somatotropin resulted in a dramatic decrease in basal rates of glucose incorporation into lipid (Table 31. The decrease is of a similar magnitude to the decreased rates of lipid accretion for pST-treated pigs observed with slaughter-balance experiments (Boyd et al., 1986; Campbell et al., 1988, 19891. Workers using in vitro measurements have also observed qualitatively similar decreases in the lipogenic capacity of adipose tissue obtained from pST-treated pigs (Walton et al., 1987; Mike1 et al., 1988; Harris et al., 1990; Magri et al., 19901. In addition, the activities of key enzymes involved in de novo fatty acid synthesis were decreased to a similar extent in adipose tissue obtained from pST-treated grow- ing pigs (Harris et al., 1990; Magri et al., 19901.

A particularly pertinent finding from the pres- ent study was that de novo fatty acid synthesis (as estimated from ~ G F A ) was reduced to a much greater extent than triglyceride synthesis (&G glycerol1 (Table 31. One consequence of reduced de novo fatty acid synthesis in pST-treated pigs could be a greater proportionate incorporation of preformed fatty acids of dietary origin into newly synthesized adipose tissue triglycerides. Despite this possibility, there was no change in the fatty acid composition of adipose tissue triglycerides with pST treatment (Table 41. Upon reflection, this is not surprising, because dietary fatty acids could only contribute a maximum of 5% toward total body triglyceride during the 10 d of treat- ment and would therefore have a minimal impact on the fatty acid composition.

We also investigated the effects of insulin on rates of glucose incorporation into lipids in pST- treated pigs (Table 31. Although hyperinsulinemia dramatically increased Rfipid, RTGFA, and FI,TG glycerol in pST-treated pigs, there were no quan- titative differences in the incremental responses between the control and pST treatment groups. Thus, the differences in rates that existed during the basal state were maintained during hyperin- sulinemia. Qualitatively similar effects of insulin were found for glucose transport and lipogenesis in adipose tissue obtained from control and pST- treated pigs (Magri et al., 19901.

The level of insulin used in this experiment would elicit a maximal response (Rmaxl in rates of whole-body use of glucose (Wray-Cahen et al., 19901. Other studies have also observed that the Rmax to insulin is not altered in pST-treated pigs. An in vivo study produced no difference in Rmax for insulin's effect on rates of whole-body utiliza- tion of glucose (Wray-Cahen et al., 19901 and an in vitro study showed no difference in Rmax for insulin's effect on adipose tissue rates of lipogene- sis, although the absolute rate was still lower in adipose tissue from pST-treated pigs (Walton et al., 19871. In contrast, both of these studies showed that pST treatment did reduce insulin sensitivity so that the dose of insulin required to get 50% of Rmax was markedly increased. Although investi- gated to a more limited extent, the same seems to occur in growing cattle. Basal glucose uptake by the hindlimb was reduced in bST-treated steers, despite markedly elevated plasma insulin concen- trations (Boisclair et al., 1989; Eisemann, 19891, suggesting that sensitivity to insulin had been altered. In contrast, responsiveness seemed unaf- fected because glucose uptake by the hindlimb did not differ between treatments when insulin was maintained at very high levels during a hyperinsulinemideuglycemic clamp (Boisclair et al., 19891.

Our findings that basal REpid was reduced but plasma insulin concentrations were increased over fourfold during pST treatment (Tables 2 and 3) demonstrate a reduction in adipose tissue sensitivity to insulin. Plasma insulin clearance was not altered by pST treatment, which is consistent with the lack of a pST effect on insulin binding in adipose tissue Ma@ et al., 1990). Therefore, it is likely that the elevated plasma insulin concentrations in pST-treated pigs is the result of increased insulin production.

Based on the kinetic data, we have constructed a balance sheet for the fate of glucose in control and pST-treated pigs (Table 51. Glucose irreversi- ble loss rate was 35% lower in pST-treated pigs and is consistent with the 34% decrease in energy intake. Values for the true rate of glucose irre-

SOMATOTROPIN AND LIPOGENESIS IN PIGS 149

versible loss rate may be underestimated because hepatic extraction of absorbed glucose occurs in the fed pig. This glucose would not have an adequate opportunity to mix with the total glu- cose pool, leading to an overestimation of blood SFLAglucose and an underestimation of glucose irreversible loss rate (Freeman et al., 1970). This can be a substantial problem in meal-fed animals, especially during a fast (Anderson and Northrop, 1974; Simoes-Nunes et al., 19891, when hepatic uptake of glucose would occur in the absorptive period followed by hepatic output of glucose during the postabsorptive period. However, this should not be a complication in our study because animals were fed at 4-h intervals and allowed an ad libitum intake.

The major impact of pST treatment on the fate of glucose waa a reduction in glucose utilization by adipose tissue (Table 5). This reduction was quantitatively similar to the reduction in glucose irreversible loss rate and energy intake. In con- trast, the rate of glucose utilization by nonadipose tissue was almost identical for control and pST- treated pigs Uable 5). Thus, results indicate that pST causes a reduction in the use of nutrients by adipose tissue, which in turn would necessitate a reduction in feed intake and lead to a reduction in glucose irreversible loss rate.

We have also constructed a balance sheet for lipid accretion in pigs treated with excipient or pST (Table 6). To do this we have extrapolated our estimates of lipogenic rates from biopsies of a

Table 5. Balance sheet for the fate of glucose in the growing barrowa

Variable Control PST

Wholebody

Adipose tissue

- mg/min -

Glucose ILRb 848 528

Lipid synthesisC 220 19 Glucose oxidationd 136 18

Nonadipose tissue Glucose utilizatione 492 401

9 e a t m e n t involved daily i.m. injections of porcine somato- tropin (pST, 120 Kgkg of BW) or excipient (controD. Data based on basal results obtained on the loth d of treatment. AU rates are adjusted to an animal that is 80 kg BW.

bGlucose irreversible loss rate IDLR) derived from the glu- cose kinetic data reported in Table 2.

'Derived from the estimates of Ru id (Table 3, method A) and the amount of chemical lipid founcf at slaughter (14 h after ces ation of the glucose kinetic study).

'Estimates of glucose oxidation by adipose tissue based on the ratio between Rfipfd and glucose oxidation rates deter- mined with in vitro incubations using adipose tissue obtained from the same site at slaughter (Harris et al., 1990).

eDerived from the difference in rates of glucose utilization between whole-body and adipose tissue.

single fat depot (subcutaneous adipose tissue from rump; Table 3) to the whole body and included results from related studies on rates of lipolysis and mobilization (Dunshea et al., 1992b). In doing so the following assumptions were made: 1) all chemically extractable lipid at slaughter was obtained from adipose tissue with the same metabolic activity for de novo lipogenesis as that from the biopsy site, 21 blood glucose is the sole precursor for de novo fatty acid and triglyceride- glycerol synthesis, 3) mobilized fatty acids and glycerol were estimated from the average plasma concentrations derived from the temporal pattern on d 7 of treatment aXlnshea et al., 1992a) and regression equations relating plasma nonester-

Table 6. Balance sheet for lipid accretion in adipose tissue of a growing barrowa

Variable Control DST

Fatty acids De novo synthesisb Synthesis from preformedC Mobilizedd Recycled*

Synthesizedf Mobilizedg

Glycerol

Total

134 8 49 16

-36 -70 33 28

16 2 -9 -5

19 1 -25 -

Observedh 242 42

%eatment involved daily i.m. injections of porcine somato- tropin (pST, 120 pg/kg of BW) or excipient (control). AU rates are adjusted to an animal that is 80 kg BW.

bDerived from estimates of RTG~A (Table 3. method A), and the amount of chemical lipid found at slaughter 14 h after cessation of this study (20.5 and 16.6% lipid in controls and pST- treated pigs, respectively).

'Derived using dietary composition and feed intake values (Dunshea et al., 1992a) and the assumptions that 1) 00% of dietary lipid is absorbed as fatty acids and 2) absorbed fatty acids are incorporated into adipose tissue lipid in a proportion similar to what was found for [9,101n)-3HJoleic acid at slaughter (Dunshea et al., 1992b).

dBased on the average plasma noneaterified fatty acid (NE- FA) concentration on d 7 of treatment (Dunshea et al., l992aJ and the regression equation relating plasma NEFA concentra tion and NEFA entry rate CDunshea et al., 1992b).

?Ektimates of the extent to which mobilized NEFA are rein- corporated into adipose tissue lipid based on recovery of in- b e d [O,1o(nl3Hloleic acid found for each treatment group at slaughter (Dunshea et d., 1992b).

'Derived from estimates of RrLyTglycerol (Table 3, method A) and the average chemical lipid found for each treatment group at slaughter.

%wed on the average plasma glycerol concentration of d 7 of treatment (Dunshes et al, 1992a) and the regression equa- tion relating plasma glycerol concentration and glycerol entry rate (Dunshea et al., 1992b).

hcalculated wing lipid accretion rates observed in pigs of the same genotype fed a similar diet and treated with the same dose of somatotropin over m interval besinning at 50 kg of BW and ending at 100 kg of BW (Boyd et d., 1986) combined with the average percentage of carbon in adipose tissue triglycer- ides as derived from fatty acid composition data (Table 4).

DUNSHEA ET AL. 150

ified fatty acids and glycerol concentrations to their respective entry rates (Dunshea et al., 1992131, and 4) incorporation of mobilized and dietary preformed fatty acids into adipose tissue lipid was the same as the recovery observed at slaughter for intravenously infused 19,100- 3Hloleic acid [Dunshea et al., 1992133.

A decrease in de novo synthesis of triglycerides is clearly the major component accounting for the reduced rates of lipid accretion that occur with pST treatment (Table 6). Considering the multi- tude of different variables and assumptions used to calculate the lipid accretion balance (see footnotes for Table 61, the totals are quite reasona- ble. It seems that the previously cited criteria were essentially satisfied, although overall there was an underestimation of the predicted rates of lipid accretion compared with those observed in similar pigs (Boyd et al., 1980). The major source of error is undoubtedly the assumption that all adipose tissue in the body has the same metabolic activity as that from the biopsy site. Adipose tissue depots develop at different rates through- out the body and lipogenic rates also differ CAnderson et al., 1972; Etherton et al., 1981; Mersmann, 1986b1. Estimates of the quantity of mobilized fatty acids and glycerol do not have that complication because they are based on whole-body kinetic measurements. Even so, the extent of incorporation of dietary fatty acids and the recycling of mobilized fatty acids may be underestimated from the incorporation of [ Q , l O M - 3Hloleic acid infused 3 d before slaughter (Dun- shea et al., lQQ2b). If recently formed triglycerides are preferentially hydrolyzed (Ekstedt and Olivecrona, 19701, then the extent of incorporation of labeled fatty acids would be underestimated, particularly in the pST-treated pig, which under- goes a temporal pattern of lipolysis and mobiliza- tion of fatty acids (Dunshea et al., 1992a). Rates of lipid accretion would also be underestimated due to the incorporation of lipogenic substrates other than glucose, but we would expect this to be of minor importance. Despite these uncertainties and qualifications, the external consistency of our results is impressive, especially when considered in terms of changes induced by pST. Clearly, the decreases in lipid accretion that occur with pST treatment are predominately due to decrease in glucose uptake and rates of de novo lipogenesis in adipose tissue.

Implications

We demonstrated the efficacy of an in vivo technique for determining rates of lipid synthesis in adipose tissue from the growing pig and that

exogenous infusion of insulin increased rates by approximately 50%. This approach was used to identify the mechanisms by which porcine somat- otropin treatment decreases lipid accretion. Clearly lipogenesis, and in particular de novo fatty acid synthesis, was decreased during por- cine somatotropin treatment of growing swine. Although lipid mobilization from adipose tissue was modestly increased, by far the greatest proportion of the decreased lipid accretion with porcine somatotropin treatment was due to the decrease in rates of de novo lipogenesis.

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