Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.     

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin,glucocorticoid, fatty acids and cordycepin

• 1.1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture.
• 2.2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture.
• 3.3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10⁻⁹M concentration, and a small stimulating effect was also observed with 10⁻⁶M dexamethasone.
• 4.4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity.
• 5.5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium.
• 6.6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

     

Lipoprotein lipase of cultured mesenchymal rat heart cells. IV. Modulation of enzyme activity by VLDL added to the culture medium.

Lipoprotein lipase activity was studied in rat heart cell cultures grown in the presence of 20% fetal calf and horse serum and a medium concentration of triacylglycerol of 0.03 mg/ml. After 6--8 days, when the enzyme activity had reached high levels, the cells were incubated for 24 h in a medium containing 20% serum derived from fasted or fed rats. No change in enzyme activity occurred in the presence of fasted rat serum, but a 50% fall was observed with fed rat serium. When the complete culture medium was supplemented with rat plasma VLDL (0.075--0.75 mg triacylglycerol) a pronounced decrease in lipoprotein lipase activity occurred after 3--5 h of incubation. Similar extent of enzyme fall was observed also in the presence of triacylglycerol-rich lipoproteins isolated from rat plasma after feeding of safflower oil or lard, even though the fatty acid composition of the triacylgylcerol varied markedly. As the addition of VLDL to the culture medium resulted in a lesser fall of heparin releasable than residual activity it seems that there was no direct inhibition of surface bound enzyme activity and that the transport of the enzyme to the cell surface was not affected. These data indicate that addition of VLDL to the culture medium resulted in a fall in enzyme synthesis, while total protein synthesis as determined by incorporation of [3H]leucine, remained unchanged. This inhibition could be reproduced by increasing free fatty acid concentration of the medium, however addition of excess albumin to VLDL-containing medium did not prevent the fall in enzyme activity. The present results obtained with cultured rat hearts cells suggest that in vivo plasma levels of triacylglycerol-rich lipoproteins could modulate the lipoproteins could modulate the lipoprotein lipase activity of the heart.     

Regulation of lipoprotein lipase activity and mRNA content in rat epididymal adipose tissue in vitro by recombinant tumour necrosis factor.

Tumour necrosis factor (TNF) has previously been shown to decrease lipoprotein lipase (LPL) activity and mRNA levels in 3T3-L1 cells and in adipose tissue from rats and guinea pigs when injected in vivo, but not to alter LPL activity in human adipocytes incubated in vitro. The effect of recombinant human TNF on LPL activity and mRNA levels in rat epididymal adipose tissue incubated in vitro was examined. LPL activity and mRNA levels fell in adipose tissue taken from fed rats and incubated in Krebs-Henseleit bicarbonate medium with glucose. The addition of insulin and dexamethasone prevented these falls. TNF (400 ng/ml) produced a fall of approx. 50% in LPL activity after 2 h of incubation and of approx. 30% in LPL mRNA levels after 3 h. TNF did not decrease LPL activity in isolated adipocytes. These results demonstrate that rat adipose tissue incubated in vitro is responsive to TNF whereas isolated adipocytes are not.     

Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.     

An association between glutamine synthetase activity and adipocyte differentiation in cultured 3T3-L1 cells

Confluent 3T3-L1 Swiss mouse fibroblasts acquired morphological and biochemical characteristics of adipocytes when maintained in medium containing 10% calf serum and added insulin. Identical cultures maintained in the absence of added insulin did not differentiate into adipocytes. Incubation of confluent cultures for 48 h with 0.25 μm dexamethasone and 0.5 mm 1-methyl-3-isobutylxanthine yielded subsequent adipocyte differentiation when the culture medium contained 10% fetal calf serum. In contrast, differentiation did not occur when similarly treated cultures were maintained in medium containing 10% calf serum. The increase in glutamine synthetase which occurred during adipocyte differentiation was closely associated with an increased rate of triglyceride synthesis from acetate, with increased protein, and with increases in the activities of glycerol-3-P dehydrogenase and glucose-6-P dehydrogenase. Glutamine synthetase activity remained undetectable in insulin-treated confluent 3T3-C2 cells maintained under conditions which yielded high glutamine synthetase activity in 3T3-L1 cells. (3T3-C2 cells did not differentiate into adipocytes.) Glutamine accumulated in the culture medium of 3T3-L1 adipocytes, but it did not accumulate in the medium from identically treated 3T3-C2 cells. A half-maximal increase in glutamine synthetase specific activity occurred at a culture medium insulin concentration of 10 ng/ml. Neither adipocyte differentiation nor the rise in glutamine synthetase activity were substantially altered by maintaining confluent cultures in medium lacking added glutamine. Incubation of confluent 3T3-L1 cultures with 3 mml-methionine sulfone, a reversible inhibitor of glutamine synthetase, increased by two-fold both the activity and the cellular content of glutamine synthetase. Incubation of confluent 3T3-L1 cultures with 4 mml-glutamine and l-methionine-dl-sulfoximine, an irreversible inhibitor of glutamine synthetase activity, decreased glutamine synthetase activity to less than 5% of the activity in control cultures; however, neither cellular content of the enzyme nor synthesis rate of the enzyme were substantially altered. In the presence of added glutamine, neither methionine sulfone nor methionine sulfoximine had a significant effect on phenotypic adipocyte conversion. By contrast, when confluent cultures were incubated with methionine sulfoximine and no added glutamine, glutamine synthetase remained absent and there was no evidence of adipocyte conversion. Our data indicate (1) that added insulin is required for adipocyte differentiation of 3T3-L1 cells maintained in medium containing calf serum, (2) that glutamine synthetase activity increases during adipocyte conversion regardless of the culture conditions employed to achieve differentiation, and (3) that glutamine synthetase activity may be required for adipocyte differentiation when cultures are maintained in medium lacking added glutamine.     

Diet and diabetic state modify glycogen synthase activity and expression in rat hepatocytes

Glycogen synthase (GS), a key regulatory enzyme in glycogen synthesis, is controlled by multisite phosphorylation and allosteric regulation and is activated by insulin. This study investigated changes in GS activity and expression in hepatocytes isolated from rats under altered nutritional and diabetic conditions. Experiments were carried out in healthy rats fed a chow diet, rats on high simple sugar (60% of energy from fructose and sucrose) or high fat (46% of energy from fat) diet, and in rats with streptozotocin induced diabetes. In the presence of insulin, activated GS activity (GS(I) form) was increased by 89% in hepatocytes isolated from healthy rats. The stimulatory effect of insulin on GS activity and expression was blunted by cycloheximide and actinomycin treatment. In rats fed a high simple sugar or high fat diet, insulin stimulation of GS(I) in isolated hepatocytes was impaired and GS expression was significantly lower in rats fed the high fat diet in comparison to controls. GLUT-2 protein expression was significantly lowered by both the high fat and high simple sugar diets. In hepatocytes isolated from diabetic rats, total GS activity (GS(T)) was lower than in hepatocytes from healthy animals. Insulin added to the incubation medium did not stimulate GS activity, demonstrating impaired sensitivity to insulin in diabetic rats. However, insulin administration significantly increased GS expression indicating that a defect in synthase phosphorylation may be responsible for impaired GS activity in the diabetic state. The results presented in this study further confirm that GS activity is affected by both dietary and hormonal factors which can be measured in a rat hepatocyte model.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

Clearing-factor lipase in adipose tissue. Studies with puromycin and actinomycin

1. When adipose tissue from starved rats is incubated in a medium containing glucose, insulin, heparin and actinomycin (5mug./ml.) the total clearing-factor lipase activity of the system increases at least tenfold over a period of 9hr. In the absence of actinomycin, enzyme activity also increases, but to a lesser extent and for only about 3hr. Some enzyme activity appears in the incubation medium in both the presence and the absence of actinomycin. 2. When the glucose and insulin of the incubation medium are replaced by pyruvate and heparin is omitted, an increase in the total clearing-factor lipase activity in the presence of actinomycin still occurs, but only after a lag of several hours. When only heparin is omitted from the medium, the rise in enzyme activity begins immediately, but there is a shoulder in the time-course curve after a few hours. In the absence of heparin, little enzyme activity appears in the incubation medium. 3. The increases in enzyme activity in the presence of actinomycin are prevented if puromycin (0.5mg./ml.) is present in the incubation medium. 4. Catecholamines and corticotrophin inhibit the increase in enzyme activity caused by actinomycin. 5. The clearing-factor lipase activity of adipose tissue from fed animals declines with a half-life of between 1 and 1.5hr. when the tissue is incubated in the presence of puromycin. The clearing-factor lipase activity of adipose tissue from starved animals is stable under similar circumstances, as is the raised activity found after such tissue has been incubated in the presence of actinomycin. 6. Clearing-factor lipase extracted from adipose tissue of fed animals is less stable in solution than that extracted from the tissue of starved animals after this has been incubated in the presence of actinomycin.     

Deposition of docosahexaenoic acid (DHA) is limited in forebrain of young obese fa/fa Zucker rats fed a diet high in α-linolenic acid but devoid of DHA

Docosahexaenoic acid (DHA) is required for neurotransmitter synthesis and learning. Conversion of α-linolenic acid (ALA) to DHA is considered adequate to support brain function in youth, but it is unknown if brain DHA can be maintained in insulin resistant states. This study investigated brain fatty acid and desaturase activities in young insulin resistant Zucker rats on diets with and without DHA. Male fa/fa and lean rats were fed diets enriched with flaxseed (FXO, ALA: 35.5% fatty acids), menhaden (MO, DHA: 9.2%) or safflower oil (SO, linoleic acid: 54.1%) for 9 weeks, n=8 per diet per genotype. Compared to lean, the 15 week old fa/fa rats were obese (56% heavier) and insulin-resistant (>18-fold in homeostasis model assessment of insulin resistance). The forebrain of fa/fa rats had higher palmitoleic (16:1n-7) and dihomo-γ-linolenic (20:3n-6) acids, and higher Δ9, Δ6 but lower Δ5 (all P≤.006) desaturase indices than lean. The Δ9 and Δ6 desaturase indices positively, while the Δ5 negatively (all P≤.01) correlated with insulin resistance. The Δ9 desaturase index positively correlated with adiposity index. The percentage of forebrain DHA of fa/fa rats was lower (P=.011) than lean rats when fed FXO diet while there was no difference (P>.05) between fa/fa and lean rats fed MO or SO diet. Thus, the alterations in the fatty acid and desaturase indices in the brain were consistent inhibited forebrain synthesis of DHA in the fa/fa rats. ALA may not have potential to effectively serve as a precursor for synthesizing DHA for youth forebrain during insulin resistance since Δ5 desaturase activity is limited.     

Use of hepatocytes in primary culture for biochemical studies on liver functions

Summary Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary culture. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its -adrenergic action. The -action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland.These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.     

Substrate specificity of a high-affinity, monovalent cation-dependent amino acid carrier.

When mammary gland explants from mid-pregnant rats were incubated with insulin (5 μg/ml) and [³H]cortisol (5 μg/ml) for one day, the tissue accumulated 1.69 μg cortisol/g wet tissue. During a second incubation with insulin and prolactin (5 μg/ml), only 20% of the steroid was lost per day. Such retention of glucocorticoid had an important biological consequence: the tissue exposed for one day to insulin and cortisol showed a transient stimulation of casein synthesis during a subsequent, five-day incubation with insulin and prolactin. No casein synthesis was detected, if the first culture medium contained only insulin. In conclusion, mammary gland explants from mid-pregnant rats require a glucocorticoid for casein synthesis, but this requirement may be obscured if the explants are initially incubated in medium containing cortisol, since they are capable of accumulating and retaining this steroid. Similar interpretative difficulties may arise in studies on other steroid-tissue relationships.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

The regulation of hepatic stearoyl-coenzyme A desaturase in obese-hyperglycaemic (ob/ob) mice by food intake and the fatty acid composition of the diet.

1. The effects of food intake and the fatty acid composition of the diet on the hepatic stearoyl-CoA desaturase activity of obese-hyperglycaemic (ob/ob) mice were investigated. 2. Obese mice fed on a commercial mouse diet, ad libitum, had 6.5-fold more activity per liver cell than had lean mice. 3. On a diet containing 14% corn oil the activity was 65% less in obese mice and 62% less in lean mice compared with animals fed on the commercial diet. 4. Feeding with 14% saturated fat in the diet doubled the activity in lean mice compared with those on the commercial diet, but had no effect on the activity in obese mice. 5. Obese mice fed on the corn-oil diet contained a higher proportion of linoleic acid in the liver lipids than did lean mice fed on the commercial diet, but the acyl-CoA desaturase activity was 125% higher than in the lean mice. 6. Limiting the food intake of obese mice by pair-feeding with lean mice decreased their acyl-CoA desaturase activity when the animals were fed on the saturated-fat diet, but the activity remained 75% higher than in lean mice, whereas in obese mice pair-fed on the corn-oil diet the activity was the same as in lean mice. 7. During starvation the acyl-CoA desaturase activity in livers from obese mice decreased more slowly and proportionately less than in livers from lean mice. 8. It is concluded that increased substrate supply as a result of hyperphagia and not low concentration of linoleic acid is the main factor causing high acyl-CoA desaturase activity in obese mice.     

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants

Chicken embryo liver explants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 microM in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the delta 9-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5-1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative from cell surface insulin receptor to intracellular sites.     

The effect of dexamethasone on pyruvate kinase activity in primary cultures of hepatocytes

Pyruvate kinase activity in primary cultures of hepatocytes isolated from a normal rat was maintained at a constant level similar to that found in vivo (14.0 +/- 2.8 units per mg of DNA) for over 6 days when both dexamethasone and insulin were included in the medium. Yet the pyruvate kinase activity decreased 50% when the cells were cultured for 2 days and 4 days, respectively, in the presence of either dexamethasone or insulin alone. A brief, 10 min incubation of hepatocytes in the presence of dexamethasone was sufficient to maintain the enzyme activity of cells subsequently cultured for 4 days in the presence of insulin. The optimal dexamethasone concentration was 1 microM. Three other glucocorticoids were able to maintain the pyruvate kinase activity in cells cultured in medium containing insulin. The presence of the protein synthesis inhibitors, actinomycin D or cyclohexamide in cells cultured in the presence of dexamethasone and insulin resulted in a 25% decrease in the pyruvate kinase activity. Therefore, it is suggested that the synergistic effect of glucocorticoids and insulin to maintain pyruvate kinase activity in primary cultures of hepatocytes is dependent upon the ability of these cells to maintain protein synthesis.