IntroductionTreatment of rats with the vitamin B12 analogue hydroxy-cobalaminic-lactaml (HCCL) impairs methylmalonyl-CoA mutase function and leads to methylmalonic aciduria due to intracellular accumulation of propionyl and methylmalonyl-CoA.Since accumulation of these acyl-CoAs disrupts normal cellular regulation, the present investigation characterized metabolism in hepatocytes and liver mitochondria from rats treated subcutaneously with HCCL or saline (control) by osmotic minipump. Consistent with decreased methylmalonyl-CoA mutase activity, 14C02 production from 1-"4C-propionate (1 mM) was decreased by 76% and 82% after 2-3 wk and 5-6 wk of HCCL treatment, respectively. In contrast, after 5-6 wk of HCCL treatment, "4CO2 production from 1-"4C-pyruvate (10 mM) and 1-'4C-palmitate (0. Vitamin B 12 (cobalamin) deficiency leads to decreased activity of the cobalamin-requiring enzymes methylmalonyl-CoA mutase and methionine synthetase (1). Decreased activity of methylmalonyl-CoA mutase results in accumulation of methylmalonyl-and propionyl-CoA, and methylmalonic aciduria (2-4). This defect is analogous to the metabolic defect in patients with hereditary methylmalonic acidurias. The vitamin B 12-deficient rat has provided a useful animal model for the human methylmalonic acidurias and allowed the investigation of hepatic metabolism in the presence of this specific, metabolic insult (4-6). Dietary vitamin B 12 deficiency is difficult to achieve, and the resulting metabolic defect shows a high variability between treated animals (7, 8). An alternative methodology to induce decreased activity of methylmalonyl-CoA mutase is treatment with hydroxy-cobalamin [c-lactam] (HCCL),' a synthetic vitamin B 12 analogue (4-6). Rats treated with HCCL develop decreased hepatic vitamin B 12 levels (4), decreased methylmalonyl-CoA mutase and methionine synthetase activity (6), and decreased propionate metabolism with an increased renal excretion of methylmalonic acid (4-6).Increased hepatocellular propionyl and methylmalonylCoA concentrations inhibit several metabolic activities, including gluconeogenesis (9, 10), urea synthesis (9, 11) fatty acid oxidation (11, 12) and pyruvate decarboxylation (13, 14). In contrast, in rats with chronic accumulation of propionyl and methylmalonyl-CoA induced by dietary vitamin B 12 deficiency, gluconeogenetic and ketogenetic responses to fasting were intact in vivo (4), suggesting compensatory mechanisms that mitigate the toxic effects of accumulated acyl-CoAs. One compensatory mechanism in rats with dietary and functional vitamin B 12 deficiency is increased hepatic total CoA concentration (4, 15).To further study hepatic metabolism under conditions of impaired methylmalonyl-CoA mutase activity, hepatic mitochondrial function in HCCL-and saline-treated rats was characterized. Mitochondrial function was quantified by measurement of fuel oxidation rates in isolated hepatocytes and mitochondria. In addition, activities of mitochondrial and extramitochondrial enzymes were determined in isolat...

Rat milk contains at least three major caseins with apparent molecular weights of 41,000 (alpha-casein), 25,000 (beta-casein), and 22,000 (gamma-casein) (estimated in 10% sodium dodecyl sulfate-polyacrylamide gels). These three caseins and alpha-lactalbumin, a major whey protein, were purified from rat milk. The purified caseins and alpha-lactalbumin were used to immunize BALB/c mice, and spleen cells from these mice were hybridized with cells of the mouse myeloma SP-2/0 cell-line. We have isolated a small library of hybridoma cell-lines secreting monoclonal antibodies specific for each of the major caseins and alpha-lactalbumin from rat milk. Antibodies were tested for immunoreactivity with each of the purified milk proteins and with total rat milk proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some heterogeneity in apparent molecular weight was observed for purified alpha-casein, gamma-casein, and alpha-lactalbumin. Monoclonal antibodies against alpha-casein, gamma-casein, and alpha-lactalbumin recognized all of the molecular weight forms of the antigen for which they were specific. Each monoclonal antibody was specific for one of the caseins or alpha-lactalbumin and did not react with the other caseins or alpha-lactalbumin, suggesting that there is limited structural homology among these proteins. All of the monoclonal antibodies against the rat caseins reacted with components of mouse milk, and the monoclonal antibodies against rat gamma-casein reacted with a component of human milk of apparent molecular weight 27,000. No interspecies reactivity was observed with the antibodies against rat alpha-lactalbumin. These monoclonal antibodies are being used to develop sensitive assays for each of these major rat milk proteins.

Mammary cells from normal tissue of virginal, pregnant, and lactating rats have been adapted to long term monolayer culture in plastic culture dishes with retention of hormone-responsive functional activity. The addition of PRL, insulin, and corticosterone resulted in an increase in the proportion of epithelial cells. the development of intracellular lipid droplets, and the ordered aggregations of these cells. In the presence of these hormones, the milk protein, alpha-lactalbumin (a-LA), was secreted into the growth medium at rates of 20-100 ng/mg cellular protein . 24 h. A double antibody RIA for a-LA capable of measuring 0.1 ng a-LA/100 microliter growth medium was developed in our laboratory for these studies. Both intracellular and extracellular a-LA fell below detectability within 2-3 weeks after hormone withdrawal. Intracellular a-LA accounted for less than 3% of the total a-LA accumulated in each culture in 24 h. the production rate of cells continuously given hormones increased 4- to 7-fold over a period of several months in culture, and their output was greater than 100-fold above that of cells not given hormones. These cells were obtained by overnight digestion and dispersion of tissue using selected batches of collagenase in the presence of 5% fetal calf serum. Plating densities of at least 3 X 10(4) cells/cm2 in Minimum Essential Medium supplemented with 14% fetal calf serum were required for optimal functional activity. Despite several months without added hormones, these cultures can retain their hormone responsiveness, since subsequent hormone addition resulted in detectable a-LA production beginning within 7-14 days. Our studies demonstrate for the first time that normal mammary cells can be maintained in a functional hormone-responsive state for extended periods in primary cell culture. These long term cell cultures provide a system with which the effects of these and other hormones on milk production and cell differentiation can be assessed under conditions which minimize the influence of the prior in vivo hormonal millieu. (Endocrinology 108: 573, 1981)

The concentrations of PRL, corticosterone, and insulin required by long term cultures of normal rat mammary cells to produce alpha-lactalbumin (alpha LA) and the 25,000 mol wt beta-casein were evaluated with a variety of hormone ratios and concentrations. For these studies a double antibody RIA for beta-casein capable of measuring 0.5 ng beta-casein/100 microliter growth media was developed and used along with our previously reported RIA for alpha LA. PRL was active at physiological levels (0.05-0.15 micrograms/ml) and quantitatively stimulated beta-casein more than alpha LA, whereas physiological levels of corticosterone (0.05-0.15 micrograms/ml) quantitatively stimulated alpha LA more than beta-casein. The concentration of corticosterone greatly altered the magnitude of the cells' response to insulin and PRL for alpha LA output by cells from either virginal or midpregnant rats. Insulin also enhanced production of these milk proteins, but very little effect was measured in the physiological range. alpha LA was increased more by insulin than by PRL, and beta-casein was enhanced more by insulin than by corticosterone. Cells from midpregnant rats required less insulin to stimulate beta-casein production than to stimulate alpha LA. Cells from virginal rats required a supraphysiological insulin level to stimulate both beta-casein and alpha LA under these conditions. These cells generally require 5-6 weeks to achieve a steady-state rate of milk protein output. The complexities of our observations help explain some of the conflicting reports in the literature concerning which hormone is of prime importance for quantitatively increasing the synthesis of a particular milk protein, particularly since high hormone levels are often employed and time in culture varies considerably among reports. We conclude that lower levels of all these hormones can and should be used in vitro. Our messenger RNA (mRNA) studies using cloned complementary DNA probes for two rat casein mRNAs show that cells grown for 2 months with hormones contain significant amounts of both alpha- and beta-casein mRNAs. Simultaneous quantification of beta-casein mRNA levels and rates of beta-casein protein production in these long term cell cultures indicated that a substantial portion of their beta-casein protein production is regulated by the amount of its mRNA. This could be controlled by mRNA synthesis and/or mRNA degradation.(ABSTRACT TRUNCATED AT 400 WORDS)