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Effects of insulin on glucose metabolism by explants of mouse mammary gland maintained in organ culture
280
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25592 EFFECTS OF I N S U L I N ON GLUCOSE METABOLISM BY E X P L A N T S OF MOUSE MAMMARY GLAND M A I N T A I N E D IN ORGAN C U H ' U R E RICHARD L. MORETTI* AND S. ABRAHAM Department of Zoology, Cancer Research Genelics Laboratory, a~zd the Department ()f Physiology, University of California, Berkeley, Calif. (U.S.A.)
(Received January i7th, I966)
SUMMARY I. A technique was described for the isolation of the I14Cllactic acid, E'~C]fatty acid, and 14CO~ produced from [14C~glucose by explants of mouse mammary tissues in organ culture. 2. Insulin maintained the I'4Cjlactic acid, [14CJfatty acid and 14CO2 production from [l~Clglucose by explants of both prelactating and lactating mouse mammary tissues. 3. In the presence of insulin, the [14C]lactic acid production, 14CO2 production, and glucose uptake by explants of prelactating mouse m a m m a r y tissues continued throughout the 4-day culture period but there was no increase in recovery of El'C!fatty acid after 48 h in either the insulin-treated or control groups. 4-The I14C]fatty acid production, 14CO2 production and glucose uptake by explants of lactating mouse mammary gland was much greater than that of explants of prelactating mouse m a m m a r y gland. The [~4Cllactic acid production by lactating tissues was lower than that of prelactating tissue. 5. Insulin caused an increase in the [14Clfatty acid and 14CO2 production by explants of lactating gland but had no effect on [14Cllactic acid production. 6. The data indicate that insulin is required by explants of mammary tissues for the maintenance of glucose uptake and metabolism in the organ culture system.
INTRODUCTION It has been reported' 3 that the addition of insulin to an incubation medium containing slices of m a m m a r y gland from lactating rats results in an increased utilization of glucose and in its conversion to CO2 and fatty acids. When the slices were obtained from pregnant rats, however, insulin had little effect. The relatively short incubation periods of I 3 h which were used in experiments with mammary slices from pregnant rats may not have allowed sufficient time for the demonstration of an effect. When explants of mammary gland obtained from pregnant mice were incubated for periods of 1- 5 days in organ culture, the presence of insulin in tile medium resulted in a maintenance of glucose uptake. In the absence of insulin there was little glucose uptake after the first day of incubation 4. * Present address: Department of Life Sciences, University of California, Riverside, Calif. (u.s.A.). Biochim. Biophys. Acla, ~24 (1906) 280 28S
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
281
The present study compares the effects of insulin on glucose metabolism, in organ culture, on m a m m a r y tissues from pregnant and from lactating mice. In order to facilitate these studies, a new organ-culture chamber, designed to collect the respiratory CO s under sterile conditions, was developed. MATERIALS AND METHODS
Explants of prelactating lobules were selected from multiparous C3H/Crgl mice on the i 5 - I 8 t h day of pregnancy. Explants of lobules from lactating mice were selected from C3H/Crgl mice IO days post partum; each mouse was nursing 6 pups. The synthetic Medium 199 (ref. 5) containing I.O mg glucose per ml was used in all experiments. E14C]Glucose~ was added to Medium 199 in an amount which gave 2. lO 5 counts/min per ml. Amorphous insulin, containing 0.04 To Zn and assaying at 20 units per mg, was added to the medium to yield a concentration of 5.0 ~g/ml. The preparation of the tissues and media as well as the culturing procedures were performed as described previously 4, except for the use of the new culture chambers. The culture chambers (Fig. I) were constructed with ground glass flanges on the opposing upper and lower sections which, with the aid of silicone stopcock grease, could be joined to form a gas-tight seal. The lower section of each chamber contained one well 32 m m in diameter (Fig. IA), and 2 smaller wells IO m m in diameter (Fig. IB). The large well was designed to contain the incubation medium and the explants. One of the small wells was designed to contain the CO 2 absorbant and the other an O~ absorbant for experiments conducted under anaerobic conditions. All three wells had a depth of 20 m m in order to prevent spilling of their liquid contents. The upper section of each chamber was constructed with a I 6 - m m opening at its top which was designed to accommodate a self-sealing rubber serum bottle stopper.
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Fig. i. D r a w i n g of a culture c h a m b e r showing the two sections held t o g e t h e r with springs and the opening at the top sealed with a serum-bottle stopper. A, large well 32 m m in diameter and 20 m m deep for culture m e d i u m ; B, two small wells IO m m in diameter and 20 m m deep for CO 2 a b s o r b a n t and o t h e r reagents. The entire c h a m b e r was constructed of p y r e x glass b y Microchemical Specialties, Berkeley, Calif.
Biochim. Biophys. Acta, 124 (1966) 280-288
282
R.L.
MORETTI, S. ABRAHAM
Before use, each c h a m b e r was sealed a n d the two sections held firmly t o g e t h e r w i t h springs while t h e c h a m b e r s were sterilized with d r y h e a t at I0o ° for 4 h. The opening at t h e t o p was t e m p o r a r i l y covered with A1 foil. U n d e r sterile conditions the u p p e r section of each c h a m b e r was removed, I.O ml of m e d i u m i n t r o d u c e d into the large well, a n d a piece of lens p a p e r floated on the medium. A group of ~) e x p l a n t s weighing a t o t a l of 2.5 -3.5 m g was placed on the lens p a p e r a n d the u p p e r section of each c h a m b e r replaced a n d secured with springs. Each culture c h a m b e r was flushed with a m i x t u r e of 95 % O2 a n d 5 % CO~ which was first passed t h r o u g h distilled w a t e r a n d t h e n t h r o u g h an H A Millipore filter. The gas m i x t u r e was i n t r o d u c e d into each culture c h a m b e r t h r o u g h t h e opening on top. E a c h c h a m b e r was flushed for z5 sec a n d then sealed with a sterile r u b b e r serum b o t t l e stopper. The sealed c h a m b e r s were i n c u b a t e d at 3 7 - After incubation, o.5 ml of I M H y a m i n e in m e t h a n o l was injected, b y means of a long h y p o d e r m i c needle inserted t h r o u g h the serum b o t t l e stopper, into one of the side wells of each chamber, o.5 ml of 2.5 M HeSO4 was injected into t h e m e d i u m in each c h a m b e r to i n a c t i v a t e t h e e x p l a n t s a n d to release the r e s p i r a t o r y CO,,. The c h a m b e r s were then g e n t l y shaken for I h to insure t h a t all the CO,, was a b s o r b e d in the h y a m i n e solution. A f t e r shaking, the t o p section of each c h a m b e r was r e m o v e d a n d t h e h v a m i n e solution q u a n t i t a t i v e l y t r a n s f e r r e d to scintillation vials b y r e p e a t e d rinsings with totuenc. To each vial was a d d e d 48 m g of 2,5-diphenyloxazole in 2 ml of toluene, a n d enough a d d i t i o n a l toluene to m a k e a final volume of a b o u t IO ml. To d e t e r m i n e the [14C]fatty acid c o n t e n t of the e x p l a n t s each group was carefully b l o t t e d on filter paper, placed in o.I ml of 3o % K O H , a n d saponified at 9 ° for 4 h. T h e saponified m i x t u r e was first cooled in an ice b a t h , then a 3.o-ml portion of h e x a n e was added. This m i x t u r e was acidified with o.I ml of c o n c e n t r a t e d HCI and, after shaking, I . o - m l portions of t h e h e x a n e e x t r a c t were transferred to scintillation vials c o n t a i n i n g 4 8 m g of 2,5-diphenyloxazole in IO ml of toluene. To d e t e r m i n e the '.~14C]lactic acid c o n t e n t of the m e d i u m i.o-ml portions of the acidified m e d i a were c o n t i n u o u s l y e x t r a c t e d with ether for 24 h using a modified Palkin a u t o m a t i c e x t r a c t i o n a p p a r a t u s (constructed b y Microchemical Specialties, Berkeley, Calif.). The e x t r a c t s were collected in I.o ml of o.I5 M N a O H . The ether was e v a p o r a t e d and the N a O H solutions a d j u s t e d to a volume of I.o ml. For counting, o . I - m l samples from each flask were t r a n s f e r r e d to scintillation vials containing I o ml of 1:2 m i x t u r e of t o l u e n e : e t h y l e n e g l y c o l m o n o m e t h y l ether. A solution of 48 mg of 2,5-diphenyloxazole in 2.o ml of toluene was a d d e d to each vial a n d mixed. All samples were a s s a y e d for ~4(- a c t i v i t y in the P a c k a r d liquid scintillation spectrophotometer. Glucose u p t a k e was e s t i m a t e d on o.2-ml portions of acidic m e d i u m from each culture c h a m b e r b y a d d i t i o n of 2 ml of o.2 M N a O H ; I.o-ml portions were used for glucose d e t e r m i n a t i o n b v the glucose oxidase m e t h o d (Glucostat, W o r t h i n g t o n Biochemical Corporation, Freehold, N.J.). RESULTS
Effect of insulin on glucose metabolism of prelactating tissues E x p l a n t s of p r e l a c t a t i n g mouse m a m m a r y g l a n d were i n c u b a t e d in t h e presence a n d in t h e absence of insulin for periods from 3 h to 9 6 h. T h e glucose u p t a k e a n d
Biochim. Biophys. Acta, t24 (1960) 28o- 288
283
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
the [14C]lactic acid, [14C]fatty acid, and 14CO2 production from ~liC]glucose w a s determined. The results of the glucose uptake studies are summarized in Fig. 2A. No difference was noted between the control and insulin-treated groups incubated for 24 h or less. However, after 48 h of incubation the explants in the presence of insulin showed a higher glucose uptake than the explants in the absence of insulin. The [14Cllactic acid production (Fig. 2B) from [14Clglucose remained about the same in the control and insulin-treated groups incubated for 48 h or less, but explants incubated in the presence of insulin for 72 h or longer showed a higher [14C]lactic acid production than did those in the absence of insulin. G" o
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Fig. 2. Glucose uptake and [14C]lactic acid, Ex4C]fatty acid, and 14COz production from [14CJglucose, by organ cultures of prelactating mouse mammary gland incubated for periods indicated. [14C]Glucose, sufficient to yield 2. lO5 counts/min per ml was added to the medium in each experiment. Each point represents the mean of 6-8 experiments. • - - • , insulin present; O - - O, insulin absent.
The effect of insulin on [laC]fatty acid production by these explants is shown in Fig. 2C. Explants incubated in the presence of insulin showed an increase over the controls after only 3 h of incubation, in contrast to the longer incubation periods required to demonstrate the effects of insulin on glucose uptake and [14C]lactic acid production. There was a considerable variation in the lac activity of the fatty acid extracts from explants incubated for 48 h or longer in the presence of insulin. Usually the [14C]fatty acid recoveries from explants incubated for 72-96 h were less than that for explants incubated for 48 h but this decrease was not a significant one. Insulin had a marked effect on 14COz production by explants of prelactating mouse mammary gland (Fig. 2D). In the presence of insulin the 14CO2 production continued through the 4-day culture period, whereas in the absence of insulin the rate of 14CO2 production was greatly reduced after the first day.
Effect of insulin on glucose metabolism of lactating mouse mammary tissues Explants of lactating mouse mammary gland were incubated in the presence and in the absence of insulin for periods from 3 h to 96 h. The glucose uptake and Biochim. Biophys. ~4cta, 124 (1966) 28o-288
284
R.L.
MORETTI,
S. ABRAHAM
the [14Cjlactic acid, [14C]fatty acid and 14CO2 productions were determined. The results of the glucose uptake studies are summarized in Fig. 3A. As with the explants of prelactating tissue, insulin had no effect on the glucose uptake by explants of lactating gland incubated for 3 h. When the explants were incubated for 24 h or longer, those in the presence of insulin consumed more glucose than those in the absence of insulin. Explants of lactating gland, in the presence or in the absence of insulin, used more glucose than explants of prelactating gland under the same conditions (compare Figs. 2A and 3A). A
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Fig. 3. Glucose u p t a k e a n d []4C]lactic acid, [14C]fatty acid a n d 14C() 2 p r o d u c t i o n from glucose, u n i f o r m l y labeled w i t h 14C b y o r g a n c u l t u r e s of l a c t a t i n g m o u s e m a m m a r y g l a n d i n c u b a t e d for periods indicated. I14C]Glucose, sufficient to yield 2- lO 5 c o u n t s / r a i n was a d d e d to t h e m e d i u m in each e x p e r i m e n t . E a c h p o i n t r e p r e s e n t s t h e m e a n of 6 - 8 e x p e r i m e n t s . Q - - O , insulin p r e s e n t ; O - - O , insulin absent.
The presence of insulin had no effect on [14Cllactic acid production by explants of lactating gland (Fig. 3B). Throughout the entire culture period the amount of []4Cilactic acid produced by explants in the presence of insulin was not different from that produced by explants in the absence of insulin. After 4-days' incubation, the E14Cllactic acid produced by explants of lactating gland was much less than that produced by explants of prelactating gland (compare Figs. 2B and 3B). Insulin had a marked effect on [14Clfatty acid production by explants of lactating gland (Fig. 3C). In the presence of insulin the E14C]fatty acid content of the explants increased throughout the 4 days of incubation. In the absence of insulin there was no significant increase in the [t4C~fatty acid yields of the explants after the first day of incubation. Explants of lactating glands showed a greater E14Clfatty acid content than did explants of prelactating gland (compare Figs. 2C and 3C). As with [14C]fatty acid production insulin had a marked effect on 14CO,, production by explants of lactating gland (Fig. 3D). After 4-days' incubation the t~CO2 produced by explants in the presence of insulin was about twice that produced by Biochim. Biophys. Acta, i24 (1966) 280-288
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
285
explants in the absence of insulin. Explants of lactating gland, in the presence or absence of insulin, produced much more ~4C02 than explants of prelactating tissue (compare Fig. 2D and Fig. 3D). The amount of glucose initially present in the medium which was converted to CO s, f a t t y acid, and lactic acid by the explants was calculated (Figs. 4 and 5). Prelactating tissues, in the presence or absence of insulin, produced relatively large amounts of lactic acid from glucose whereas lactating tissues produced proportionately more CO s and f a t t y acid. In the case of both prelactating and lactating tissues almost all of the utilized glucose was recovered in the CO 2, f a t t y acid, and lactic acid after 4-days' incubation whereas after shorter incubation periods some of the glucose which had been removed from the medium was not represented in these three products. DISCUSSION Earlier reportsT, s concerning the morphology of explants of prelactating mouse m a m m a r y glands suggested that in the presence of mammotropin and other hormones insulin was not required for the maintenance of these tissues. Other reportsg, 1° indicated that insulin was important in the maintenance of prelactating mouse m a m m a r y tissues in organ culture. ELIAS AND RIVERAs found that only one preparation of mammotropin would effectively maintain explants of prelactating gland in the absence of insulin. BERN AND RIVERA9 found all other preparations of mammotropin employed required the presence of insulin in the incubation medium in order to be effective. LASFARGUES11 also found that insulin was required in order for mammotropin and other hormones to be effective in maintaining explants of prelactating m a m m a r y gland in organ culture. RIVERA12,13 employed insulin in conjunction with other hormones to obtain a secretory response in organ cultures of prelactating mouse m a m m a r y tissues. In other studies concerning adult m a m m a r y tissues in organ culture~4,15 insulin was routinely added to the incubation medium. Insulin has also been used in studies concerning the maintenance and growth of m a m m a r y explants from immature mice 16-~i. It would seem that structural maintenance and development, as well as the entire secretory response of m a m m a r y tissues in organ culture, requires the presence of insulin in the incubation medium. Insulin has also been reported to be effective in the maintenance of explants from organs other than the m a m m a r y gland 2s-~. SJDMAN2~ noted that explants of brown fat from rats had a much higher content of lipid after incubation when treated with insulin than did explants not exposed to insulin. Organ cultures of hemi-uteri from the rat 26 and ventral prostates from castrated mice 25 had a much higher protein content when incubated in the presence of insulin than did controls, incubated in the absence of insulin. TROWELL2S,29 found that insulin enhanced the survival of m a n y tissues in organ culture. It would therefore appear that insulin is often effective in maintaining explants in organ culture but it is not clear what the action of insulin is in this in vitro system. It has been suggested in the case of the m a m m a r y gland, that insulin m a y act synergistically with ovarian hormones a° or that it m a y cause the explants to take up the other hormones present in the medium n, perhaps b y pinocytosis, so that the hormones could then exert their influence. This present study and the report b y 1V[ORETTIAND DEOME 4 show that glucose uptake and metabolism are maintained b y Biochim. Biophys. Acta, 124 (1966) 280-288
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EFFECTS OF INSULIN ON GLUCOSE METABOLISM
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explants in organ culture t o a much greater degree in the presence of insulin as the only added hormone. It should be evident that without adequate nutrition the cells of the explants would soon degenerate. From the results reported here it seems that insulin, b y whatever its mechanism of action, serves to maintain the uptake and utilization of the glucose in the medium and thereby to insure survival of the cells. These surviving cells, then, are able to respond to stimuli such as other hormones and otherwise to function more or less as they normally should. Explants of lactating gland showed a higher glucose uptake and [14C~fatty acid and 14C02 production from [14Clglucose than did explants of prelactating gland. These results are in agreement with the results of ABRAHAM AND CHAIKOFF1 and MCLEAN ~, who reported that the [14Clfatty acid and 14CO~ productions from [14C!glucose by slices of lactating rat m a m m a r y gland were much greater than that of slices of prelactating m a m m a r y gland. These workers found, independently, that insulin caused an increase in [~4Clfatty acid and 1~C0 2 production in slices of lactating rat m a m m a r y gland incubated for periods up to 3 h. However, insulin had no effect on slices of prelactating rat gland. The results reported herein show that insulin had an effect on 14C02 and [14Clfatty acid production by explants of mouse prelactating tissue incubated for 3 h. These differences in results m a y be explained by a difference in response to insulin between the two species or to a difference between the tissue-slice technique and the organ-culture system. In the presence of insulin the 14C02production from [14CJglucose and glucose uptake by explants of both prelactating and lactating gland continued throughout the 4 days of incubation. The [14C]fatty acid recovered from the explants of lactating gland increased throughout the 4 days of incubation, whereas in explants of prelactating gland there was no increase after the second day. It is not clear why, in the presence of insulin but no other hormone, the recovery of f a t t y acids did not increase in prelactating tissues after a 2-day incubation period but did continue to increase in lactating tissues for the entire 4-day incubation period. Although the organ culture system has been used extensively in morphological or histological studies, it has only infrequently been used in metabolic studies. MORETTI AND DEOME 4 found that explants of prelactating mouse m a m m a r y gland, incubated for periods up to 5 days required the presence of insulin for continued glucose uptake whereas explants of mouse m a m m a r y adenocarcinomas utilized glucose throughout the 5-day incubation period in the absence of insulin. Insulin increased protein synthesis in organ cultures of rat hemi-uteri ~6 and in ventral prostate of mice 25. This present investigation shows that the organ-culture system is adaptable to metabolic studies and m a y be advantageous because the incubation period can be a few hours or extended for several days. Effects which are not evident after a few hours incubation m a y become apparent after longer incubation periods. The organ-culture system m a y thus become a useful addition to the study of metabolism in vitro. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Professors K. B. DEOME and H. A. BERN for their counsel in regard to this study. Aided by U.S. Public Health Service grants CA o5 388 and 2 T I CA 5045 . Amorphous insulin was generously supplied by Dr. O. K. BEHRENS, Lilly Research Laboratories, Indianapolis, Ind. Biochim. Biophys. dcta, 124 (1966) 280-288
288
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REFERENCES I "2 3 4 5 0 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 -22 23 24 25 26 27 28 29 3°
ABRAHAM AND i. L. CHAIKOFF, J. Biol. Chem., 234 (1959) 2246. H. BALMAIN, S. J. VOLLEY AND I{. V. GLASCOCK, Biochem. J., 56 (1954) 234. MCLEAN, Biochim. Biophys. Acta, 37 (196°) 296. L. MORETTI AND i(. B. DEOME, J. Natl. Cancer Inst., 29 (I962) 321. v. MORGAN, M. J. MORTON AND t~. C. PARKER, Proc. Soc. Exptl. Biol. Med., 73 (195o) r. ABRAHAM, E. V~,;. PUTMAN AND V~T. Z. HASSID, Arch. Biochem. Biophys., 41 (1952) 61. J" ELIAS, Proc. Soc. Exptl. Biol. }Vled., IOI (1959) 5oo. J. J. ELIAS AND E. ~ . RIVERA, Cancer Res., 19 (1959) 505. H. A. BERN AND E. ~ . RIVERA, Proc. Am. Assoc. Cancer Res., 3 (196o) 94. E. M. RIVERA AND H. A. BERN, Endocrinology, 69 (1961) 34 o. E. Y. LASEARGUES, Exptl. Cell Res., 28 (1962) 531. E. M. RIVERA, Proc. Soc. Exptl. Biol. Med., 116 (1964) 568. E. M. RIVERA, Endocrinology, 74 (1964) 853E. B. BARNAXVELL, Ph.D. Thesis, University of California, 1964. E. Y. LASFARGUES AND ]). G. FELDMAN, Cancer Res., 23 (1963) 191. J. J. ELIAS, Anat. Record, 139 (1961) 224. J. J. ELIAS, Exptl. Cell Res., 27 (1962) 6Ol. R. R. [CHINOSE AND S. NANOI, Science, 145 (1964) 3631. J. KOZlOROWSKA, Acta Med. Polona, 3 (1962) 237. F. J. A. PROP, Exptl. Cell Res., 20 (196o) 256. F. J. A. PROP, Expll. Cell Res., 24 (1961) 629. F. J. A. PROP, Palhol. Biol. Semaine Hop., 9 (1961) 640. E. M. RIVERA, Proc. Soc. Expll. Biol. Med., 114 (I963) 735E. M. RIVERA, J. Endoerinol., 3 ° (1964) 33. S. S. COLAME AND A. J. LOSTROH, Endocrinology, 75 (1964) 451A. J. LOSTROH, Exptl. Cell Res., 32 (1963) 327 . R. L. SIDMAN, Anat. Record, 124 (1956) 723 . O. A. TROWELL, Exptl. Cell Res., 9 (1955) 258. O. A. TROWELL, Exptl. Cell Res., 16 (1959) 118. J. KOZlOROWAKA, Acla V~lio Intern. Co~tra Cancrum, 18 (1962) 211. S. J. P. R. JS. J.
Biochim. Biophys. Acta, 124 (1966) 280--288
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25592 EFFECTS OF I N S U L I N ON GLUCOSE METABOLISM BY E X P L A N T S OF MOUSE MAMMARY GLAND M A I N T A I N E D IN ORGAN C U H ' U R E RICHARD L. MORETTI* AND S. ABRAHAM Department of Zoology, Cancer Research Genelics Laboratory, a~zd the Department ()f Physiology, University of California, Berkeley, Calif. (U.S.A.)
(Received January i7th, I966)
SUMMARY I. A technique was described for the isolation of the I14Cllactic acid, E'~C]fatty acid, and 14CO~ produced from [14C~glucose by explants of mouse mammary tissues in organ culture. 2. Insulin maintained the I'4Cjlactic acid, [14CJfatty acid and 14CO2 production from [l~Clglucose by explants of both prelactating and lactating mouse mammary tissues. 3. In the presence of insulin, the [14C]lactic acid production, 14CO2 production, and glucose uptake by explants of prelactating mouse m a m m a r y tissues continued throughout the 4-day culture period but there was no increase in recovery of El'C!fatty acid after 48 h in either the insulin-treated or control groups. 4-The I14C]fatty acid production, 14CO2 production and glucose uptake by explants of lactating mouse mammary gland was much greater than that of explants of prelactating mouse m a m m a r y gland. The [~4Cllactic acid production by lactating tissues was lower than that of prelactating tissue. 5. Insulin caused an increase in the [14Clfatty acid and 14CO2 production by explants of lactating gland but had no effect on [14Cllactic acid production. 6. The data indicate that insulin is required by explants of mammary tissues for the maintenance of glucose uptake and metabolism in the organ culture system.
INTRODUCTION It has been reported' 3 that the addition of insulin to an incubation medium containing slices of m a m m a r y gland from lactating rats results in an increased utilization of glucose and in its conversion to CO2 and fatty acids. When the slices were obtained from pregnant rats, however, insulin had little effect. The relatively short incubation periods of I 3 h which were used in experiments with mammary slices from pregnant rats may not have allowed sufficient time for the demonstration of an effect. When explants of mammary gland obtained from pregnant mice were incubated for periods of 1- 5 days in organ culture, the presence of insulin in tile medium resulted in a maintenance of glucose uptake. In the absence of insulin there was little glucose uptake after the first day of incubation 4. * Present address: Department of Life Sciences, University of California, Riverside, Calif. (u.s.A.). Biochim. Biophys. Acla, ~24 (1906) 280 28S
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
281
The present study compares the effects of insulin on glucose metabolism, in organ culture, on m a m m a r y tissues from pregnant and from lactating mice. In order to facilitate these studies, a new organ-culture chamber, designed to collect the respiratory CO s under sterile conditions, was developed. MATERIALS AND METHODS
Explants of prelactating lobules were selected from multiparous C3H/Crgl mice on the i 5 - I 8 t h day of pregnancy. Explants of lobules from lactating mice were selected from C3H/Crgl mice IO days post partum; each mouse was nursing 6 pups. The synthetic Medium 199 (ref. 5) containing I.O mg glucose per ml was used in all experiments. E14C]Glucose~ was added to Medium 199 in an amount which gave 2. lO 5 counts/min per ml. Amorphous insulin, containing 0.04 To Zn and assaying at 20 units per mg, was added to the medium to yield a concentration of 5.0 ~g/ml. The preparation of the tissues and media as well as the culturing procedures were performed as described previously 4, except for the use of the new culture chambers. The culture chambers (Fig. I) were constructed with ground glass flanges on the opposing upper and lower sections which, with the aid of silicone stopcock grease, could be joined to form a gas-tight seal. The lower section of each chamber contained one well 32 m m in diameter (Fig. IA), and 2 smaller wells IO m m in diameter (Fig. IB). The large well was designed to contain the incubation medium and the explants. One of the small wells was designed to contain the CO 2 absorbant and the other an O~ absorbant for experiments conducted under anaerobic conditions. All three wells had a depth of 20 m m in order to prevent spilling of their liquid contents. The upper section of each chamber was constructed with a I 6 - m m opening at its top which was designed to accommodate a self-sealing rubber serum bottle stopper.
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Fig. i. D r a w i n g of a culture c h a m b e r showing the two sections held t o g e t h e r with springs and the opening at the top sealed with a serum-bottle stopper. A, large well 32 m m in diameter and 20 m m deep for culture m e d i u m ; B, two small wells IO m m in diameter and 20 m m deep for CO 2 a b s o r b a n t and o t h e r reagents. The entire c h a m b e r was constructed of p y r e x glass b y Microchemical Specialties, Berkeley, Calif.
Biochim. Biophys. Acta, 124 (1966) 280-288
282
R.L.
MORETTI, S. ABRAHAM
Before use, each c h a m b e r was sealed a n d the two sections held firmly t o g e t h e r w i t h springs while t h e c h a m b e r s were sterilized with d r y h e a t at I0o ° for 4 h. The opening at t h e t o p was t e m p o r a r i l y covered with A1 foil. U n d e r sterile conditions the u p p e r section of each c h a m b e r was removed, I.O ml of m e d i u m i n t r o d u c e d into the large well, a n d a piece of lens p a p e r floated on the medium. A group of ~) e x p l a n t s weighing a t o t a l of 2.5 -3.5 m g was placed on the lens p a p e r a n d the u p p e r section of each c h a m b e r replaced a n d secured with springs. Each culture c h a m b e r was flushed with a m i x t u r e of 95 % O2 a n d 5 % CO~ which was first passed t h r o u g h distilled w a t e r a n d t h e n t h r o u g h an H A Millipore filter. The gas m i x t u r e was i n t r o d u c e d into each culture c h a m b e r t h r o u g h t h e opening on top. E a c h c h a m b e r was flushed for z5 sec a n d then sealed with a sterile r u b b e r serum b o t t l e stopper. The sealed c h a m b e r s were i n c u b a t e d at 3 7 - After incubation, o.5 ml of I M H y a m i n e in m e t h a n o l was injected, b y means of a long h y p o d e r m i c needle inserted t h r o u g h the serum b o t t l e stopper, into one of the side wells of each chamber, o.5 ml of 2.5 M HeSO4 was injected into t h e m e d i u m in each c h a m b e r to i n a c t i v a t e t h e e x p l a n t s a n d to release the r e s p i r a t o r y CO,,. The c h a m b e r s were then g e n t l y shaken for I h to insure t h a t all the CO,, was a b s o r b e d in the h y a m i n e solution. A f t e r shaking, the t o p section of each c h a m b e r was r e m o v e d a n d t h e h v a m i n e solution q u a n t i t a t i v e l y t r a n s f e r r e d to scintillation vials b y r e p e a t e d rinsings with totuenc. To each vial was a d d e d 48 m g of 2,5-diphenyloxazole in 2 ml of toluene, a n d enough a d d i t i o n a l toluene to m a k e a final volume of a b o u t IO ml. To d e t e r m i n e the [14C]fatty acid c o n t e n t of the e x p l a n t s each group was carefully b l o t t e d on filter paper, placed in o.I ml of 3o % K O H , a n d saponified at 9 ° for 4 h. T h e saponified m i x t u r e was first cooled in an ice b a t h , then a 3.o-ml portion of h e x a n e was added. This m i x t u r e was acidified with o.I ml of c o n c e n t r a t e d HCI and, after shaking, I . o - m l portions of t h e h e x a n e e x t r a c t were transferred to scintillation vials c o n t a i n i n g 4 8 m g of 2,5-diphenyloxazole in IO ml of toluene. To d e t e r m i n e the '.~14C]lactic acid c o n t e n t of the m e d i u m i.o-ml portions of the acidified m e d i a were c o n t i n u o u s l y e x t r a c t e d with ether for 24 h using a modified Palkin a u t o m a t i c e x t r a c t i o n a p p a r a t u s (constructed b y Microchemical Specialties, Berkeley, Calif.). The e x t r a c t s were collected in I.o ml of o.I5 M N a O H . The ether was e v a p o r a t e d and the N a O H solutions a d j u s t e d to a volume of I.o ml. For counting, o . I - m l samples from each flask were t r a n s f e r r e d to scintillation vials containing I o ml of 1:2 m i x t u r e of t o l u e n e : e t h y l e n e g l y c o l m o n o m e t h y l ether. A solution of 48 mg of 2,5-diphenyloxazole in 2.o ml of toluene was a d d e d to each vial a n d mixed. All samples were a s s a y e d for ~4(- a c t i v i t y in the P a c k a r d liquid scintillation spectrophotometer. Glucose u p t a k e was e s t i m a t e d on o.2-ml portions of acidic m e d i u m from each culture c h a m b e r b y a d d i t i o n of 2 ml of o.2 M N a O H ; I.o-ml portions were used for glucose d e t e r m i n a t i o n b v the glucose oxidase m e t h o d (Glucostat, W o r t h i n g t o n Biochemical Corporation, Freehold, N.J.). RESULTS
Effect of insulin on glucose metabolism of prelactating tissues E x p l a n t s of p r e l a c t a t i n g mouse m a m m a r y g l a n d were i n c u b a t e d in t h e presence a n d in t h e absence of insulin for periods from 3 h to 9 6 h. T h e glucose u p t a k e a n d
Biochim. Biophys. Acta, t24 (1960) 28o- 288
283
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
the [14C]lactic acid, [14C]fatty acid, and 14CO2 production from ~liC]glucose w a s determined. The results of the glucose uptake studies are summarized in Fig. 2A. No difference was noted between the control and insulin-treated groups incubated for 24 h or less. However, after 48 h of incubation the explants in the presence of insulin showed a higher glucose uptake than the explants in the absence of insulin. The [14Cllactic acid production (Fig. 2B) from [14Clglucose remained about the same in the control and insulin-treated groups incubated for 48 h or less, but explants incubated in the presence of insulin for 72 h or longer showed a higher [14C]lactic acid production than did those in the absence of insulin. G" o
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Fig. 2. Glucose uptake and [14C]lactic acid, Ex4C]fatty acid, and 14COz production from [14CJglucose, by organ cultures of prelactating mouse mammary gland incubated for periods indicated. [14C]Glucose, sufficient to yield 2. lO5 counts/min per ml was added to the medium in each experiment. Each point represents the mean of 6-8 experiments. • - - • , insulin present; O - - O, insulin absent.
The effect of insulin on [laC]fatty acid production by these explants is shown in Fig. 2C. Explants incubated in the presence of insulin showed an increase over the controls after only 3 h of incubation, in contrast to the longer incubation periods required to demonstrate the effects of insulin on glucose uptake and [14C]lactic acid production. There was a considerable variation in the lac activity of the fatty acid extracts from explants incubated for 48 h or longer in the presence of insulin. Usually the [14C]fatty acid recoveries from explants incubated for 72-96 h were less than that for explants incubated for 48 h but this decrease was not a significant one. Insulin had a marked effect on 14COz production by explants of prelactating mouse mammary gland (Fig. 2D). In the presence of insulin the 14CO2 production continued through the 4-day culture period, whereas in the absence of insulin the rate of 14CO2 production was greatly reduced after the first day.
Effect of insulin on glucose metabolism of lactating mouse mammary tissues Explants of lactating mouse mammary gland were incubated in the presence and in the absence of insulin for periods from 3 h to 96 h. The glucose uptake and Biochim. Biophys. ~4cta, 124 (1966) 28o-288
284
R.L.
MORETTI,
S. ABRAHAM
the [14Cjlactic acid, [14C]fatty acid and 14CO2 productions were determined. The results of the glucose uptake studies are summarized in Fig. 3A. As with the explants of prelactating tissue, insulin had no effect on the glucose uptake by explants of lactating gland incubated for 3 h. When the explants were incubated for 24 h or longer, those in the presence of insulin consumed more glucose than those in the absence of insulin. Explants of lactating gland, in the presence or in the absence of insulin, used more glucose than explants of prelactating gland under the same conditions (compare Figs. 2A and 3A). A
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Fig. 3. Glucose u p t a k e a n d []4C]lactic acid, [14C]fatty acid a n d 14C() 2 p r o d u c t i o n from glucose, u n i f o r m l y labeled w i t h 14C b y o r g a n c u l t u r e s of l a c t a t i n g m o u s e m a m m a r y g l a n d i n c u b a t e d for periods indicated. I14C]Glucose, sufficient to yield 2- lO 5 c o u n t s / r a i n was a d d e d to t h e m e d i u m in each e x p e r i m e n t . E a c h p o i n t r e p r e s e n t s t h e m e a n of 6 - 8 e x p e r i m e n t s . Q - - O , insulin p r e s e n t ; O - - O , insulin absent.
The presence of insulin had no effect on [14Cllactic acid production by explants of lactating gland (Fig. 3B). Throughout the entire culture period the amount of []4Cilactic acid produced by explants in the presence of insulin was not different from that produced by explants in the absence of insulin. After 4-days' incubation, the E14Cllactic acid produced by explants of lactating gland was much less than that produced by explants of prelactating gland (compare Figs. 2B and 3B). Insulin had a marked effect on [14Clfatty acid production by explants of lactating gland (Fig. 3C). In the presence of insulin the E14C]fatty acid content of the explants increased throughout the 4 days of incubation. In the absence of insulin there was no significant increase in the [t4C~fatty acid yields of the explants after the first day of incubation. Explants of lactating glands showed a greater E14Clfatty acid content than did explants of prelactating gland (compare Figs. 2C and 3C). As with [14C]fatty acid production insulin had a marked effect on 14CO,, production by explants of lactating gland (Fig. 3D). After 4-days' incubation the t~CO2 produced by explants in the presence of insulin was about twice that produced by Biochim. Biophys. Acta, i24 (1966) 280-288
EFFECTS OF INSULIN ON GLUCOSE METABOLISM
285
explants in the absence of insulin. Explants of lactating gland, in the presence or absence of insulin, produced much more ~4C02 than explants of prelactating tissue (compare Fig. 2D and Fig. 3D). The amount of glucose initially present in the medium which was converted to CO s, f a t t y acid, and lactic acid by the explants was calculated (Figs. 4 and 5). Prelactating tissues, in the presence or absence of insulin, produced relatively large amounts of lactic acid from glucose whereas lactating tissues produced proportionately more CO s and f a t t y acid. In the case of both prelactating and lactating tissues almost all of the utilized glucose was recovered in the CO 2, f a t t y acid, and lactic acid after 4-days' incubation whereas after shorter incubation periods some of the glucose which had been removed from the medium was not represented in these three products. DISCUSSION Earlier reportsT, s concerning the morphology of explants of prelactating mouse m a m m a r y glands suggested that in the presence of mammotropin and other hormones insulin was not required for the maintenance of these tissues. Other reportsg, 1° indicated that insulin was important in the maintenance of prelactating mouse m a m m a r y tissues in organ culture. ELIAS AND RIVERAs found that only one preparation of mammotropin would effectively maintain explants of prelactating gland in the absence of insulin. BERN AND RIVERA9 found all other preparations of mammotropin employed required the presence of insulin in the incubation medium in order to be effective. LASFARGUES11 also found that insulin was required in order for mammotropin and other hormones to be effective in maintaining explants of prelactating m a m m a r y gland in organ culture. RIVERA12,13 employed insulin in conjunction with other hormones to obtain a secretory response in organ cultures of prelactating mouse m a m m a r y tissues. In other studies concerning adult m a m m a r y tissues in organ culture~4,15 insulin was routinely added to the incubation medium. Insulin has also been used in studies concerning the maintenance and growth of m a m m a r y explants from immature mice 16-~i. It would seem that structural maintenance and development, as well as the entire secretory response of m a m m a r y tissues in organ culture, requires the presence of insulin in the incubation medium. Insulin has also been reported to be effective in the maintenance of explants from organs other than the m a m m a r y gland 2s-~. SJDMAN2~ noted that explants of brown fat from rats had a much higher content of lipid after incubation when treated with insulin than did explants not exposed to insulin. Organ cultures of hemi-uteri from the rat 26 and ventral prostates from castrated mice 25 had a much higher protein content when incubated in the presence of insulin than did controls, incubated in the absence of insulin. TROWELL2S,29 found that insulin enhanced the survival of m a n y tissues in organ culture. It would therefore appear that insulin is often effective in maintaining explants in organ culture but it is not clear what the action of insulin is in this in vitro system. It has been suggested in the case of the m a m m a r y gland, that insulin m a y act synergistically with ovarian hormones a° or that it m a y cause the explants to take up the other hormones present in the medium n, perhaps b y pinocytosis, so that the hormones could then exert their influence. This present study and the report b y 1V[ORETTIAND DEOME 4 show that glucose uptake and metabolism are maintained b y Biochim. Biophys. Acta, 124 (1966) 280-288
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EFFECTS OF INSULIN ON GLUCOSE METABOLISM
287
explants in organ culture t o a much greater degree in the presence of insulin as the only added hormone. It should be evident that without adequate nutrition the cells of the explants would soon degenerate. From the results reported here it seems that insulin, b y whatever its mechanism of action, serves to maintain the uptake and utilization of the glucose in the medium and thereby to insure survival of the cells. These surviving cells, then, are able to respond to stimuli such as other hormones and otherwise to function more or less as they normally should. Explants of lactating gland showed a higher glucose uptake and [14C~fatty acid and 14C02 production from [14Clglucose than did explants of prelactating gland. These results are in agreement with the results of ABRAHAM AND CHAIKOFF1 and MCLEAN ~, who reported that the [14Clfatty acid and 14CO~ productions from [14C!glucose by slices of lactating rat m a m m a r y gland were much greater than that of slices of prelactating m a m m a r y gland. These workers found, independently, that insulin caused an increase in [~4Clfatty acid and 1~C0 2 production in slices of lactating rat m a m m a r y gland incubated for periods up to 3 h. However, insulin had no effect on slices of prelactating rat gland. The results reported herein show that insulin had an effect on 14C02 and [14Clfatty acid production by explants of mouse prelactating tissue incubated for 3 h. These differences in results m a y be explained by a difference in response to insulin between the two species or to a difference between the tissue-slice technique and the organ-culture system. In the presence of insulin the 14C02production from [14CJglucose and glucose uptake by explants of both prelactating and lactating gland continued throughout the 4 days of incubation. The [14C]fatty acid recovered from the explants of lactating gland increased throughout the 4 days of incubation, whereas in explants of prelactating gland there was no increase after the second day. It is not clear why, in the presence of insulin but no other hormone, the recovery of f a t t y acids did not increase in prelactating tissues after a 2-day incubation period but did continue to increase in lactating tissues for the entire 4-day incubation period. Although the organ culture system has been used extensively in morphological or histological studies, it has only infrequently been used in metabolic studies. MORETTI AND DEOME 4 found that explants of prelactating mouse m a m m a r y gland, incubated for periods up to 5 days required the presence of insulin for continued glucose uptake whereas explants of mouse m a m m a r y adenocarcinomas utilized glucose throughout the 5-day incubation period in the absence of insulin. Insulin increased protein synthesis in organ cultures of rat hemi-uteri ~6 and in ventral prostate of mice 25. This present investigation shows that the organ-culture system is adaptable to metabolic studies and m a y be advantageous because the incubation period can be a few hours or extended for several days. Effects which are not evident after a few hours incubation m a y become apparent after longer incubation periods. The organ-culture system m a y thus become a useful addition to the study of metabolism in vitro. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Professors K. B. DEOME and H. A. BERN for their counsel in regard to this study. Aided by U.S. Public Health Service grants CA o5 388 and 2 T I CA 5045 . Amorphous insulin was generously supplied by Dr. O. K. BEHRENS, Lilly Research Laboratories, Indianapolis, Ind. Biochim. Biophys. dcta, 124 (1966) 280-288
288
R. L. MORETTI, S. ABRAHAIvl
REFERENCES I "2 3 4 5 0 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 -22 23 24 25 26 27 28 29 3°
ABRAHAM AND i. L. CHAIKOFF, J. Biol. Chem., 234 (1959) 2246. H. BALMAIN, S. J. VOLLEY AND I{. V. GLASCOCK, Biochem. J., 56 (1954) 234. MCLEAN, Biochim. Biophys. Acta, 37 (196°) 296. L. MORETTI AND i(. B. DEOME, J. Natl. Cancer Inst., 29 (I962) 321. v. MORGAN, M. J. MORTON AND t~. C. PARKER, Proc. Soc. Exptl. Biol. Med., 73 (195o) r. ABRAHAM, E. V~,;. PUTMAN AND V~T. Z. HASSID, Arch. Biochem. Biophys., 41 (1952) 61. J" ELIAS, Proc. Soc. Exptl. Biol. }Vled., IOI (1959) 5oo. J. J. ELIAS AND E. ~ . RIVERA, Cancer Res., 19 (1959) 505. H. A. BERN AND E. ~ . RIVERA, Proc. Am. Assoc. Cancer Res., 3 (196o) 94. E. M. RIVERA AND H. A. BERN, Endocrinology, 69 (1961) 34 o. E. Y. LASEARGUES, Exptl. Cell Res., 28 (1962) 531. E. M. RIVERA, Proc. Soc. Exptl. Biol. Med., 116 (1964) 568. E. M. RIVERA, Endocrinology, 74 (1964) 853E. B. BARNAXVELL, Ph.D. Thesis, University of California, 1964. E. Y. LASFARGUES AND ]). G. FELDMAN, Cancer Res., 23 (1963) 191. J. J. ELIAS, Anat. Record, 139 (1961) 224. J. J. ELIAS, Exptl. Cell Res., 27 (1962) 6Ol. R. R. [CHINOSE AND S. NANOI, Science, 145 (1964) 3631. J. KOZlOROWSKA, Acta Med. Polona, 3 (1962) 237. F. J. A. PROP, Exptl. Cell Res., 20 (196o) 256. F. J. A. PROP, Expll. Cell Res., 24 (1961) 629. F. J. A. PROP, Palhol. Biol. Semaine Hop., 9 (1961) 640. E. M. RIVERA, Proc. Soc. Expll. Biol. Med., 114 (I963) 735E. M. RIVERA, J. Endoerinol., 3 ° (1964) 33. S. S. COLAME AND A. J. LOSTROH, Endocrinology, 75 (1964) 451A. J. LOSTROH, Exptl. Cell Res., 32 (1963) 327 . R. L. SIDMAN, Anat. Record, 124 (1956) 723 . O. A. TROWELL, Exptl. Cell Res., 9 (1955) 258. O. A. TROWELL, Exptl. Cell Res., 16 (1959) 118. J. KOZlOROWAKA, Acla V~lio Intern. Co~tra Cancrum, 18 (1962) 211. S. J. P. R. JS. J.
Biochim. Biophys. Acta, 124 (1966) 280--288