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Insulin is essential for the accumulation of rat casein messenger RNA (mRNA) in the presence of glucocorticoid and prolactin. The accumulation of certain mRNA's in other tissues has also been linked to insulin action. The present study shows that the accumulation effect on the 25,000 molecular weight rat casein mRNA does not reflect stabilization of the transcript by insulin. Rather, insulin is essential for its synthesis in the presence of glucocorticoid and prolactin.

During the conduct of a time-course study of peripheral blood T lymphocytes in BB rats, a hitherto unreported islet lesion was recognized in 8 of 17 glucose-tolerant rats which had been subjected to repetitive cardiac puncture in early life and sacrificed at 186-190 days of age. Some of the large islets contiguous to fibrous septa demonstrated variable degrees of obliterative sclerosis and reduction of islet cell mass, associated with evidence of remote peri- and intrainsular hemorrhage. In 3 rats, additional features included an active, proliferative replacement of islet substance by fibroblasts and histiocytes. These islet alterations are not characteristic of BB rats, nor have identical lesions been described in studies of experimental or human diabetes mellitus. Affected rats had sustained repetitive episodes of blood loss (from 15% to 28% of total blood volume each time) during the early phase of the study. It is possible, but not proved, that hypovolemic shock was induced in some of these animals. Ischemic necrosis of islets has been reported in premature and young infants succumbing to shock. It is posited that hypovolemic shock in the young animal may, as in the infant, result in islet cell ischemic necrosis, sufficient to produce, with time, the described islet morphologic features.

The developmentally and hormonally regulated expression of the mouse whey acidic protein (WAP) gene and a hybrid gene containing the WAP gene promoter and a cDNA for human tissue plasminogen activator (tPA) were studied in a line of transgenic mice. During mammary gland development from the mature virgin state to the seventh day of lactation, the relative concentration of WAP mRNA increased about 10(4)-fold, the increase being most pronounced between days 14 and 16 of gestation. In mammary gland organ culture from virgin and midpregnant animals, the concerted actions of insulin, hydrocortisone, and PRL were required to increase WAP mRNA levels. Steady state levels of transcripts from the WAP-tPA hybrid gene increased about 100-fold during pregnancy; this occurred mainly around day 10 of gestation. Insulin, hydrocortisone, and PRL were necessary to maintain the levels of WAP-tPA RNA in explants from virgin and pregnant animals, but could not further elevate it. The results suggest that the WAP gene promoter and upstream region contains some, but perhaps not all elements conferring developmental and hormone regulated expression of the mouse WAP gene.

Summary It was shown previously that induction of milk proteins in mouse mammary explants has an absolute requirement for glucocorticoid and spermidine, and that the latter can largely substitute for the hormone. Induction in the rabbit system was reported to be enhanced by, but not absolutely dependent on, glucocorticoids and to be independent of spermidine. It is demonstrated here that this enhancement is dependent on spermidine, although the polyamine cannot substitute for glucocorticoid. Moreover, it is also shown that the rat system represents an intermediate state: it has an absolute requirement for both glucocorticoid and spermidine, but the latter cannot mimic the steroid.

Placental lactogen and prolactin stimulate the rate of RNA synthesis by nuclei isolated from mammary epithelial cells. The effect is tissue specific. It is suggested that these protein hormones may perform some of their functions within the mammary cell.

Mammary explants from both pregnant and virgin rats cultured in medium containing insulin, hydrocortisone and prolactin show a progressive increase in alpha-lactalbumin activity. However, when cultured with insulin and hydrocortisone only, explants from pregnant, but not those from virgin rats show an induction of alpha-lactalbumin-like activity similar to that seen when prolactin is present. The activity induced in the absence of exogenous prolactin corresponds to newly synthesized alpha-lactalbumin molecules, since 1) the activity is suppressed completely by rabbit anti-rat alpha-lactalbumin serum and 2) culture in the presence of [3H]-amino acids generates [3H]-alpha-lactalbumin, identified by SDS-polyacrylamide gel electrophoresis. Mammary tissue from virgin rats can be rendered responsive to insulin and hydrocortisone in this regard by administration in vivo of either progesterone or estrogen, but not by desoxycorticosterone acetate. This ability to convert virgin rat mammary cells to a state in which they are independent of exogenous prolactin in terms of alpha-lactalbumin synthesis represents a heretofore unreported effect of the female sex steroids.

17 beta-Estradiol (E2; 1 ng/ml) can significantly (P less than 0.05) augment lactose synthetase activity (85%) and casein synthesis (65%) in mammary gland explants (from midpregnant C3H/HeN mice) cultured in medium 199 containing insulin, cortisol, PRL, and T3. Both T3 (0.65 ng/ml) and a physiological concentration of PRL (50 ng/ml) are required for stimulation by E2; at higher PRL levels, the effects of E2 are obscured. Sex steroid specificity was supported by the observations that the E2 effects are blocked by the antiestrogen nafoxidine (5 micrograms/ml), and that 17 alpha-estradiol (5 ng/ml) was inactive; however, at 5 ng/ml, estrone and diethylstilbestrol were equipotent to E2. Testosterone (5 micrograms/ml) and progesterone (1 microgram/ml) were inhibitory, although progesterone did not suppress casein synthesis. Since E2 did not alter the amount of epithelial DNA, its effects represent an increase in biological activities per cell. E2, therefore, in conjunction with the classical lactogenic hormones and T3 can act directly on mammary tissue by promoting differentiation.

Insulin-like material with elevated insulin specific acitivity is released from insulin-Sepharose in the presence of bovine-serum albumin. The mechanism of release and the chemical nature of this insulin-like material are revealed by the finding that amine-Sepharose is O-Sepharose-N-substituted isourea. Nucleophilic attack by amino groups releases N-1-N-2-disubstituted guanidines. Correspondingly, it is shown that the super-active insulin-like material is an N-1-N-2-disubstituted guanidine in which insulin and bovine-serum albumin are the substituents.

A number of growth factors can maintain hormonally-responsive epithelium in murine mammary explants as well as insulin, but only insulin can promote the synthesis of casein and α-lactalbumin, in the presence of glucocorticoid and prolactin. Anti-insulin receptor serum can elicit these effects of insulin on milk protein gene expression. The anti-serum is unique in its ability to mimic the developmental role of insulin in murine mammary epithelium.

Mammary explants from pregnant rats showed a progressive increase in α-lactalbumin activity during culture with insulin, hydrocortisone and prolactin. Unexpectedly, culture with only insulin and hydrocortisone produced a similar rate of increase of α-lactalbumin-like activity, but this increase commenced about 24 hr later. The delay suggests that the enhanced activity effected by insulin and hydrocortisone is not a reflection of carry-over of endogenous mammotrophic hormones. Insulin plus hydrocortisone did not stimulate casein or fatty acid synthesis by pregnancy tissue, and did not enhance α-lactalbumin-like activity in virgin rat mammary explants. Enhancement of this activity by insulin plus hydrocortisone in pregnant tissue was constant over a wide range of glucocorticoid concentrations, but was inhibited by progesterone. Available evidence indicates that the active factor in extracts from insulin-hydrocortisone-explants is a heat-stable protein which is either α-lactalbumin itself, or another molecule with similar specifier properties.

In pursuit of a model system in which to determine whether or not exposure to progesterone is necessary for mammary epithelial cells to develop their differentiative potential, we explored hormone-dependent growth of the mammary epithelial rudiment in adult male mice. Initiation of the formation of ductal cells can be effected by administration of estradiol in the absence of endogenous progesterone and glucocorticoid using adrenalectomized-castrated animals. The resulting epithelium contains three times more lactose synthetase activity per epithelial cell than that in midpregnant mice. The blood spermidine level in these doubly operated animals was similar to the concentration of spermidine required to substitute effectively for glucocorticoid during mammary differentiation in vitro. It is suggested that spermidine can partially supplant glucocorticoid in vivo in milk protein synthesis. We also concluded that, unlike other secondary sex tissues, mammary cells do not require exposure to progesterone during their ontogeny in order to realize their differentiative potential. The positive role of this steroid in mammary development is apparently limited to its effect on the formation of alveolar structures.

Complexes between insulin, prolactin or placental lactogen and cyanogen bromide-activated Sepharose release hormone-like materials when treated with bovine serum albumin (BSA) (1). These materials have enhanced biological activities, and are, presumably, N 1 N 2 -disubstituted guanidines in which the hormones and BSA are the substituents. The present studies show that ammonium bicarbonate can substitute for BSA in the generation of the super-active hormones. Super-activity of the released, guanidinated hormones, therefore, can be manifested in the absence of the BSA substituent. The key derivatized amino acid residues have not yet been identified, but it appears that guanidination of lysine is either unnecessary or insufficient. Several operational considerations which are important for the demonstration of these enhanced activities are discussed.

In terms of the rate of accumulation of α-aminoisobutyric acid, mammary epithelial cells from mature virgin mice respond rapidly to insulin covalently bound to sepharose particles, but do not respond initially to soluble insulin in vitro . The response to insulin-sepharose is prevented if the cells are first exposed to insulin. These observations may provide new insight into the dynamics of insulin action.

Mammary gland tissue from immature mice [1] , adult virgin mice [2] and pregnant mice [3] can be stimulated to differentiate in vitro. Additions of insulin, glucocorticoid and prolactin to the synthetic culture medium are required for the complex sequence of cellular events which culminates in the synthesis of secretory proteins such as casein [3]. The interplay of these hormones during development of mouse mammary epithelial cells, in vitro, is the subject of this discussion, with particular emphasis on the roles of the steroids.

Mammary epithelial cell differentiation, as effected by the interplay of insulin, corticosteroid(s), and prolactin, has been further studied in vitro. Emphasis has been placed on the synthesis of casein in relation to the fabrication and function of the rough endoplasmic reticulum (RER). Minimal hormone requirements for the accumulation of the RER are insulin and corticosteroid. This is based on determinations of membrane-linked NADH-cytochrome c reductase (EC 1.6.99.3) and of RNA content of isolated RER. Accumulation of the enzyme is affected very little by insulin, modestly increased by hydrocortisone, and greatly enhanced by the combination of the two hormones. RNA content of RER is virtually unaltered by hydrocortisone, but is increased by insulin. This effect of insulin is, however, enhanced by the presence of hydrocortisone. Addition of hydrocortisone to an insulin-containing postmitotic system causes redistribution of free ribosomes to the membrane-bound variety without a net increase in ribosomes. In the absence of the steroid, insulin promotes development of an unstable RER which also is deficient in NADH-cytochrome c reductase. Prolactin does not affect the membrane-linked enzyme. It does, however, stimulate the rate of RNA synthesis and increases the RNA content of the membrane system. In the absence of RER, prolactin also increases the rate of RNA synthesis, but this effect is not sustained. Three lines of evidence strongly suggest that casein formation by these epithelial cells is dependent upon the presence of the RER that is induced by insulin and hydrocortisone. (a) There is close agreement between casein synthesis and RER formation as a function of hydrocortisone concentration. (b) RER formation precedes casein synthesis. (c) Casein formation requires sustained stimulation of RNA synthesis by prolactin, which, in turn, is dependent on the presence of RER.

Angiotensin II, given subcutaneously in pressor doses to rats three times a day, causes a prompt increase in the size and granulation of the juxtaglomerular cells. After one week of angiotensin injections, there is some loss of granules from these cells. Concurrently, degenerative changes develop in the intrarenal arteries. These changes may result from vasoconstriction in the vessels proximal to the afferent arterioles. Epinephrine given in an amount similar in pressor effect to angiotensin II did not produce these changes. Vascular lesions were not seen in the heart, lung, brain, aorta, liver, and spleen of animals given angiotensin over the two-week injection period. This study provides additional evidence that the vessels of the kidney are uniquely sensitive to angiotensin.

Prolactin causes the emergence of casein synthesis by mammary epithelial cells formed in vitro in the presence of insulin and hydrocortisone. Prolactin similarly exerts a stimulatory effect on the synthesis of α-lactalbumin and β-lactoglobulin.

Studies of the biosynthesis and metabolism of L-fucose in man have been performed. The 14C labeling pattern of L-fucose in human milk subsequent to the intravenous administration of [6-14C]glucose is compatible with the conversion of the intact hexose carbon chain of D-glucose to L-fucose. These results parallel those observed previously in studies of the bacterial synthesis of L-fucose. [I-14C]fucose administered intravenously to a human subject was found to be rapidly and extensively converted to 14CO2.

Any attempt to separate the metabolism of carbohydrates from that of fats or of amino acids is at best arbitrary. Many metabolic pathways are now known to be shared in common between various groups of tissue constituents, many intermediates may be derived from non-carbohydrate as well as carbohydrate precursors. In the composition of the present report the authors have limited the subject matter to the metabolic reactions of those compounds which the organic chemist would call "sugars." On this basis, and also on the basis that it is as much a part of fatty acid or amino acid metabolism as of carbohydrate metabolism, discussion of the tricarboxylic acid cycle has not been included. An attempt has been made to review the current state of our knowledge of the origins of the common sugars, their catabolic fates, their metabolic transformations and the devices employed in their biologic storage.

HUMAN erythrocytes have a life-span in the circulation of approximately 120 days1. They are incapable of cell division and consequently leave no progeny. These characteristics make them attractive subjects for the study of biochemical changes incident to normal maturation and senescence, assuming it were possible to separate them into groups of old, middle-aged, and young cells without the use of artificial haematopoietic stimuli2. This communication presents a method of red cell fractionation which complies with these criteria.

Publisher Summary Multiple hormone dependencies provide the investigator with opportunities for chemical dissection that facilitate analysis of sequential events occurring during development. This, in turn, aids the delineation of the action of individual hormones. This chapter discusses some of the hormone-mediated processes that occur during development of the mouse mammary gland in vitro. During the first three postnatal weeks, the cells in the epithelial cord undergo little, if any, proliferation. In the course of the next month or two, presumably under the influence of larger amounts of estrogen and progesterone during puberty, the epithelial cells multiply so as to form ducts and primitive alveoli along the connective tissue septa. With the attainment of sexual maturity at the age of 2–3 months, mammary epithelial-cell proliferation virtually ceases. During pregnancy and the early postpartum period, epithelial growth resumes so that the lactating gland contains ducts and alveoli in profusion. Functional and cytological innovations also take place at this time. Within a few weeks after the end of lactation, the gland reassumes the histological and functional profile characteristic of the tissue in the mature nonpregnant animal. The cycle is repeated with the onset of another pregnancy. In all the studies described in the chapter, an organ-culture method was used. Expiants were supported on siliconized lens paper that floated on Medium 199 containing penicillin and various combinations of crystalline beef insulin, hydrocortisone, and ovine prolactin.

Differentiation of mouse mammary gland in vitro requires insulin, hydrocortisone, and prolactin. The epithelial cells must first divide in order to synthesize casein in response to these hormones. Insulin is required for the initiation of DNA synthesis and is also necessary during G 1 phase (after mitosis). Prolactin can elicit the overt differentiative responses after mitosis. Activity of hydrocortisone precedes that of prolactin, that is, after mitosis it is not capable of eliciting the differentiative response.

IT has recently been reported that two patients who had the characteristic syndrome of congenital galactosaemia as infants, and who lack gal-1-P uridyl transferase in their red blood cells, are nevertheless able to oxidize galactose-1-14C to 14CO2 in vivo in a near-normal fashion1. It is therefore apparent that some tissues within these individuals must be capable of metabolizing galactose. Further studies on one of these subjects (T. B.), a 30-year-old Negro who was the first case of galactosaemia reported in the American literature2, have inquired into possible tissue differences with respect to the presence or absence of the metabolic derangement typical of galactosaemia.

Galactose-1-C(14) oxidation was studied in eight individuals with congenital galactosemia. Although two of these subjects fulfilled the usual diagnostic criteria for this disorder, they oxidized the intravenously administered sugar to C(14)O(2) in a nearly normal fashion.

Rabbits pretreated with aldosterone exhibited lessened response to the pressor agent angiotensin. This observation may help to explain the absence of hypertension in certain instances in which both the formation of angiotensin and the secretion of aldosterone are above normal.

Albino rabbits and dogs received injections of heterologous, homologous and autologous antigens both with and without Freund adjuvant. The animals were studied as long as 17 months. Thirteen rabbits received crude calf thymus. Four rabbits were given homologous bone and joint suspension. Three dogs were injected with saline emulsions of their own tail bone and cartilage. There was no clinical or laboratory evidence of anti-desoxyribosenucleic acid antibodies, systemic lupus erythematosus or rheumatoid arthritis. Conilios albin e canes recipeva injectiones de antigenos heterologe, homologe, e autologe con e sin adjuvante Freund. Le animates esseva studiate durante periodos usque a 17 menses. Dece-tres conilios recipeva crude thymo de vitello. Quatro conilios recipeva homologe suspension de osso e articulation. Tres canes recipeva emulsiones salin preparate ab lor proprie osso e cartilagine caudal. Nulle evidentia clinic o laboratorial esseva trovate del presentia de anticorpore anti acido disoxyribonucleic, de systemic lupo erythematose, o de arthritis rheumatoide.

Studies on the induction of casein synthesis in competent daughter cells formed in mammary gland explants have revealed a new action of insulin. Treatment with prolactin, in the absence of insulin, is responsible for the formation of some polypeptides which are destined to become phosphoproteins, or for the formation of a unique protein-kinase, or both. In any case, insulin is required for participation of the prolactin-induced factor(s) in a protein-kinase reaction.