The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

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 synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Lipogenesis in rabbit isolated fat-cells

1. Fat-cells isolated from rabbit perirenal adipose tissue were incubated with the following U-(14)C-labelled substrates: 5mm-glucose (+insulin), 5mm-pyruvate, 5mm-lactate, 5mm-glucose+5mm-acetate (+insulin), and the relative rates of incorporation of these substrates into glyceride fatty acids determined. In general total rates of fatty acid synthesis were similar whatever substrate was supplied to the cells. 2. Rabbit fat-cells incorporated [U-(14)C]acetate into fatty acids and CO(2) as well in the absence of glucose as in the presence of this substrate. 3. The disposition of the utilization of glucose-derived carbon through various metabolic pathways was determined. 4. Extramitochondrial and mitochondrial activities were determined for 11 enzymes. The cells contained a very low activity of pyruvate carboxylase, undetectable NADP-malate dehydrogenase activity and a high mitochondrial phosphoenolpyruvate carboxylase activity. 5. Various rabbit fat-cell metabolic parameters based on the measurement of (14)C incorporation and enzyme activity were compared with the same parameters previously measured in rat and guinea-pig fat-cells. In general guinea pig occupied a position between rat and rabbit with respect to these parameters. 6. The profiles of substrate incorporation into fatty acids and of relative enzyme activities in rabbit fat-cells indicated that the operation of a ;citrate-cleavage' pathway may not be obligatory for the supply of lipogenic acetyl units.     

Rapid effects of insulin and glucose on the hepatic incorporation of gluconeogenic substrates into glyceride glycerol and glycogen

1. The hepatic utilization of gluconeogenic substrates was investigated shortly after portal infusion of either insulin or glucose in fasted rats. 2. After 20 min of insulin infusion blood glucose concentration decreased. However, neither glucose generation from precursors such as alanine or pyruvate nor their incorporation into fatty acids was modified. Under these conditions, insulin rapidly increased the incorporation of gluconeogenic substrates into the hepatic glyceride glycerol fraction. Insulin treatment led to a decrease in substrate incorporation into liver glycogen. 3. After 20 min of portal glucose infusion both plasma insulin and glucose concentrations increased and the incorporation of pyruvate into hepatic glyceride glycerol and into glycogen was also stimulated. 4. A close relationship was observed between blood glucose concentrations and the level of incorporation of gluconeogenic substrates into liver glycogen. 5. In conclusion, during fasting insulin stimulates the incorporation of gluconeogenic substrates into the glycerol moiety of hepatic glycerides, which may be the preferential mechanism through which fatty acid esterification is accomplished during refeeding. This effect of insulin is rapid and detected even before other classical modifications induced by the hormone such as gluconeogenesis inhibition or lipogenesis activation. Furthermore, the effect is not related to insulin-induced hypoglycemia since glucose infusion mimics insulin action on glyceride glycerol synthesis.     

Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue: Effects of insulin, adrenaline and alloxan-diabetes

1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents

1. 0.5mm-Palmitate stimulated incorporation of [U-¹⁴C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.     

Regulation of pyruvate dehydrogenase activity in rat epididymal fat-pads and isolated adipocytes by adrenaline.

1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.

1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle)

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.     

The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Roles of coenzyme A derivatives, citrate and l-glycerol 3-phosphate

1. Methods are described for the extraction and assay of acetyl-CoA and of total acid-soluble and total acid-insoluble CoA derivatives in rat epididymal adipose tissue. 2. The concentration ranges of the CoA derivatives in fat pads incubated in vitro under various conditions were: total acid-soluble CoA, 0.20-0.59mm; total acid-insoluble CoA, 0.08-0.23mm; acetyl-CoA, 0.03-0.14mm. 3. An investigation was made of some postulated mechanisms of control of fatty acid and triglyceride synthesis in rat epididymal fat pads incubated in vitro. The concentrations of intermediates of possible regulatory significance were measured at various rates of fatty acid and triglyceride synthesis produced by the addition to the incubation medium (Krebs bicarbonate buffer containing glucose) of insulin, adrenaline, albumin, palmitate or acetate. 4. The whole-tissue concentrations of glucose 6-phosphate, l-glycerol 3-phosphate, citrate, acetyl-CoA, total acid-soluble CoA and total acid-insoluble CoA were assayed after 30 or 60min. incubation. The rates of fatty acid and triglyceride synthesis, calculated from the incorporation of [U-(14)C]glucose into fatty acids and glyceride glycerol respectively, and the rates of glucose uptake, lactate plus pyruvate output and glycerol output were measured over a 60min. incubation. 5. The rate of triglyceride synthesis could not be correlated with the concentrations of either l-glycerol 3-phosphate or long-chain fatty acyl-CoA (measured as total acid-insoluble CoA). Factor(s) other than the whole-tissue concentrations of these recognized precursors appear to be involved in the determination of the rate of triglyceride synthesis. 6. No relationship was found between the rate of fatty acid synthesis and the whole-tissue concentrations of the intermediates, citrate or acetyl-CoA, or with the two proposed effectors of acetyl-CoA carboxylase, citrate (as activator) or long-chain fatty acyl-CoA (as inhibitor). The control of fatty acid synthesis appears to reside in additional or alternative factors.     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes.

Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+-free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo-inositol that were found previously to be associated with this period of development.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

In vitro lipid metabolism in the rat pancreas. III. Effects of carbamylcholine and pancreozymin on the turnover of phosphatidylinositols, 1,2-diacylglycerols and phosphatidylcholines

1. The turnover of phosphatidylinositols and other glycerolipids was examined in rat pancreatic fragments incubated in the presence of carbamylcholine and pancreozymin used at a concentration inducing maximal alpha-amylase hypersecretion. 2. In stimulated tissue, [1-14C]acetate-labeled fatty acids were incorporated into phosphatidylinositols, 1,2-diacylglycerols, and phosphatidic acids in preference to phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, monoacylglycerols, and free fatty acids. Variations in the percent distribution of 14C among fatty acids and in specific activity of individual fatty acids in each lipid class suggested that the secretagogues reduced selection of newly synthesized 1,2-diacylglycerols which occurred in the resting state before their incorporation into phosphatidylinositols. Secretagogues also promoted recycling of endogenous 1,2-diacylglycerols (produced from hydrolysis of unlabeled glycerolipids) for the biosynthesis of phosphatidylinositols. 3. Increased rate of incorporation of [1-14C]palmitate, [1-14C]linoleate, [1-14C]arachidonate and [1(3)(n)-3H]glycerol into phosphatidylinositols was detrimental to phosphatidylcholines. 4. The lipolytic effects of carbamylcholine and pancreozymin as illustrated by the release of 1,2-diacylglycerols and free fatty acids, were markedly inhibited in calcium-free medium enriched with 1 mM EGTA but increased turnover of phosphatidylinositols as determined from incorporation of radioactive precursors was only moderately affected.