EFFECT OF I N S U L I N ON TOTAL PROTEIN S Y N T H E S I S IN PIG A D I P O C Y T E S Dennis R. Campion ~'2, Larry L. Benyshek 3, Robert R. Kraeling 1,3 and James O. Reagan 2

INTRODUCTION

The studies of Mersmann and coworkers (1974, 1975b, 1976) have demonstrated that pig adipose tissue or adipocytes respond to a variety of lipolytie agents. However, pig adipose tissue is generally unresponsive to insulin. In young pigs, i n s u l i n e x h i b i t s no a n t i l i p o l y t i c a c t i v i t y (Mersmann et al., 1975c), and little or no lipogenic activity (O'Hea and Leveille, 1970; Steele et al., 1974). The action of insulin on protein metabolism

~Animal Physiology Research Unit, Richard B. Russell Agricultural Research Center, United States Department of Agriculture, Science and Education Administration, Agricultural Research, Athens, GA 30604. 2Department of Food Science, University of Georgia, Athens, GA 30602. 3Department of Animal Science, University of Georgia, Athens, GA 30602.

in pig adipocytes, however, was not previously investigated. The general lack of influence of insulin on lipid metabolism in pig adipose tissue does not, apriori, suggest that protein synthesis in this cell would also be unresponsive. Indeed, insulin can influence protein synthesis quite apart from its influence on lipid metabolism (Wool, 1975). Furthermore, in view of the considerable variation among species in hormonal regulation of glucose metabolism in adipose tissue (Rudman and DiGirolamo, 1967), it is interesting that the i n f l u e n c e of i n s u l i n on a d i p o c y t e p r o t e i n metabolism has been investigated only in species in which the adipose tissue shows a strong lipogenic response to insulin. Cyclic nucleotides and divalent cations are associated with cellular regulation of protein metabolism. Both have been implicated in the mechanism of action of insulin (Minemura et aL, 1970; Jacobs and Krahl, 1973). In the present study, the effect of a single dose of insulin, and of various regulators of the cellular cyclic adenosine monophosphate (cAMP) level, and of divalent cations on total protein synthesis in isolated pig adipocytes was investigated.

MATERIAL AND METHODS

Crossbred pigs (Yorkshire, Hampshire and Duroc) were castrated and weaned at approximately 28 days of age. At 45 days of age a biopsy of backfat was taken from non-fasted animals in the mid-dorsal area of the cervical region. Adipocytes were isolated by the procedure of Rodbell (1964) as modified by Mersmann et al. (1973, 1976). The adipose tissue was placed in .9% NaC1 at room temperature and slices were prepared using a Stadie-Riggs microtome 4. From these slices, 1.5 g portions were removed to 25 ml polyethylene Erlenmeyer flasks that contained 4.5

4Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 853 JOURNAL OF ANIMAL SCIENCE, Vol. 48, No. 4 (1979)

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SUMMARY

Adipocytes were isolated from the backfat of young, domestic pigs. Incubation of the adipocytes in the presence of insulin stimulated protein synthesis 16 to 36% when compared to control levels. Theophylline and ACTH, alone or in combination, and dBcAMP neither changed the control rate of protein synthesis nor prevented the normal insulin response. Chelation of Ca 2§ in the buffer prevented the stimulation of protein synthesis by insulin. Removal of both Ca 2§ and Mg 2§ significantly reduced the basal rate of protein synthesis. These data support the hypothesis that cAMP does not act as second messenger for insulin, and that Ca 2§ is involved in the mechanism of action of insulin. These in vitro observations suggest that in vivo the pig adipocyte increases uptake and incorporation of amino acids into protein in response to elevation of circulating levels of insulin. ( K e y Words: Insulin, C a l c i u m , M a g n e s i u m , Adrenocorticotrophic Hormone, Theophylline, Dibutyrylcyclic-Adenosine Monophosphate.)

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CAMPION ET AL.

ml of Krebs-Ringer bicarbonate buffer (Umbreit et al., 1957) with ca 2+ at one-half concentration, 2

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mg/ml of glucose, 4% fraction V bovine serum albumin (BSA) and 15 mg of crude collagenase w h i c h was o b t a i n e d f r o m W o r t h i n g t o n Biochemical Co. The flasks were gassed with 95% 02-5% CO2 and incubated for 2 hr at 37C in a Dubnoff metabolic shaker set at 90 strokes per minute. Under standard conditions, the cells were washed three times and suspended in a similar m e d i u m ( l a c k i n g collagenase), except 4.5% essentially fatty acid-free BSA was used. The BSA was dialyzed before use. The cells isolated from 1 g of adipose tissue were suspended to a volume of 4 milliliters. Total lipid was extracted from a sample of tissue and from an aliquot of the suspended cells that had been stored at - 6 2 C by the procedure of Folch et aL (1957). The triglycerides content was determined in duplicate aliquots of the extract after saponification and after conversion of glycerol to formaldehyde (Sigma Chemical Co., 1974). T r o i l e i n was used as the s t a n d a r d . Adipocyte size and number were estimated by the p r o c e d u r e of D i G i r o l a m o e t al. (1971). In determination of adipocyte number, triglyceride density was assumed equal to that of triolein (p = 915 mg/cm3). Diameters of 300 adipocytes per animal were measured from black and white photographic contact prints of the suspended cells. A contact was developed from a photograph of a Zeiss stage micrometer. The photographic contact of the adipocytes was overlaid with the contact of the micrometer and determinations were aided by viewing the contacts through a stereoscope. A frequency distribution of adipocyte diameters was recorded with the midpoints of each successive class at 10-#m intervals. By this procedure, all the adipocytes fell into l of 12 classes. The mean adipocyte volume was calculated by the formula X(Vi. F0/300, where Vi is the volume of the classes based on the diameter at the midpoint and F~ is the number of cells in the classes. The final assay volume for determination of the incorporation of amino acid into protein was .6 milliliters. The final nanomolar concentration of L-amino acids was proportioned at one-tenth the concentration reported in pig serum (Richardson et al., 1965; Bengtsson, 1971): alanine, 44; arginine, 14; aspartic acid, 9; cystine, 1.5; glutamic acid, 23; glycine, 67; histidine, 12; isoleucine, 13; leucine, 20; lysine, 19; methionine, 2; hydroxyproline, 30; p h e n y l a l a n i n e , 10; proline, 34; serine, 16; threonine, 13; tryptophan, 5; tryosine, 11; and valine, 19 nanomolar. Each assay tube contained approximately .3 ~Ci of L-leucine [14C(U)] and 1.5 • 105 adipocytes. Assays were done in triplicate

at 37C for 2 hr under 95% 0_,-5% CO2 in a Dubnoff metabolic shaker at 90 strokes per minutes. The incorporation of [~4C] leucine into protein was determined during preliminary trials to be linear with time under standard conditions in the presence or absence of insulin. Three-. l ml aliquots of the incubation mixture were deposited on Whatman filter discs and incorporation of [14C] leucine into t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e protein was determined accordingto the procedure of Mans and Novelli (1961) as modified by Miller and Beigelman (1967). The dried discs holding the isolated protein were put into glass scintillation vials to which were added 10 ml of a toluene-based scintillation fluid. The samples were counted in a Searle Isocap/300 scintillation counter. Counter efficiency for 14C was 90%. The average count for three unincubated tubes was subtracted from each sample count to correct for unincorporated counts trapped on the filter. The corrected count for each sample was then divided by the number of adipocytes contained in the samples. The results are expressed as cpm/102 adipocytes. It was assumed that an increase in incorporation of[~4C] leucine into t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e material was indicative of an increase in protein synthesis. Three separate experiments were conducted. Within each experiment the adipocytes from each animal were tested across all treatments and both levels of insulin. In experiment one, cells were washed and suspended according to standard conditions and incubated with no further additions (control) or in the presence of 100 m U / m l adrenocorticotrophic hormone (ACTH), 5 mM N6,02'-dibutyryl adenosine 3',5'-cyclic monophosphate (dBcAMP),I mM Theophylline or l mM T h e o p h y l l i n e plus 100 m U / m l A C T H . In experiment two, the standard conditions for washing and suspending medium were varied in that the Krebs-Ringer bicarbonate solution was prepared without Ca 2+ and Mg 2§ This allowed the addition of several concentrations and combinations of Ca 2§ and m g 2+ to the assay medium. In experiment three, the adipocytes were first washed and suspended by standard conditions and several aliquots were removed for assay (control). Then the remaining cells were washed and suspended in buffer prepared without Ca 2§and Mg 2§ Assays were conducted immediately in the presence or absence of either l mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethyleneglycol bis (fl-aminoethyl) N,N'-tetraacetic acid (EGTA). For each set of conditions within the three e x p e r i m e n t s , assays were c o n d u c t e d in the

PROTEIN SYNTHESIS IN PIG ADIPOCYTES

RESULTS AND DISCUSSION The mean adipocyte diameter for the 16 pigs in this study was 40.9+ 1. ! #m and the mean volume was 6.15• 10-8_+.39• l0 -~ cm 3. These values are within the range of mean values reported by Mersmann et al. (1975a) for 23- to 60-day-old pigs. A normal frequency distribution of adipocyte diameters resulted from pooling of the diameter data from all the pigs used in the three experiments (figure I) which confirms the work of others (Anderson and Kauffman, 1973; Hood and Allen, 1977; Etherton, 1978). There was considerable variation in the rate of 2O

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(P>.05). In rat adipocytes, insulin increased protein synthesis from 30 to 100% above control levels (Miller and Beigelman, 1967; Jarett et al., 1972; Jacobs and Krahl, 1973; Sakai et al., 1973). In our experiments with pig adipocytes, insulin stimulated protein synthesis from 16 to 36%, and in

Is

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.05). Therefore, theophylline and ACTH, alone or in combination, and dBcAMP did not significantly change the amount of []4C] leucine incorporated into protein when compared to the amount incorporated into control cells under standard conditions (table l). But addition of insulin to the incubation medium significantly (P

Effect of insulin on total protein synthesis in pig adipocytes.

EFFECT OF I N S U L I N ON TOTAL PROTEIN S Y N T H E S I S IN PIG A D I P O C Y T E S Dennis R. Campion ~'2, Larry L. Benyshek 3, Robert R. Kraeling 1...
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