Jacques Furnelle

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.

Spectrum of fatty acids synthesized in situ and metabolic heterogeneity of free fatty acids and glycerides within isolated rat adipocytes.

Abstract 1. 1. Lipid metabolism was explored in isolated rat adipose cells incubated in the presence of 10 mM glucose and a low concentration (0.05–0.2 mM) of a labelled pre-cursor ([1- 14 C]-cetate or [1- 14 C]palmitate). Six lipid classes were partitioned by thin-layer chromatography and their fatty acids were analyzed by radio gas-liquid chromatography. 2. 2. Since the specific activity of free fatty acids was always lower intracellularly than in the medium, it follows that the intracellular pool of free fatty acids was heterogeneous. Free fatty acids labelled in vitro with acetate left the cells first, while the relatively large amount of free fatty acids derived from bulk triglycerides was esterified more quickly. 3. 3. The specific activity of diglycerides was very high after incubation with both precursors when compared with their concentration. Therefore, it is probable that a portion of recently synthesized diglycerides was very susceptible to lipolytic breakdown. The 1,3-diglycerides were often as well labelled as the 1,2-diglycerides. Theophylline and dibutyryl cyclic AMP increased the total concentration of diglycerides by 50%. They made the fatty acid composition of diglycerides more comparable to the triglycerides ( i.e. richer in unsaturated fatty acids) and reduced their labelling sharply. 4. 4. Myristate, palmitate and palmitoleate labelled with acetate were preferentially esterified into triglycerides and stearate was concentrated into phospholipids. 5. 5. The in vitro differences between mass and radioactivity distribution of fatty acids may be due to the heterogeneity of the lipogenetic process (2-carbon elongation and monodesaturation) and/or to the previous in vivo supply to adipose cells of lipids of hepatic origin rich in unlabelled stearate and oleate. Lipolytic agents increased the relative porportion of myristate labelled with acetate.

Guanosine 5'-O-(2-thiodiphosphate) as a competitive inhibitor of GTP in hormone or cholera toxin-stimulated pancreatic adenylate cyclase

The hormone-sensitive adenylate cyclase of many eucaryotic cells is controlled by the presence of guanyl nucleotides (reviewed [ 1,2]). The catalytic subunit is highly active in the presence of a guanyl nucleotide triphosphate but is inactive or poorly active in the presence of GDP [3-51. Hormones act in altering the configuration of the system to facilitate the access of the activating nucleotide triphosphate 14-71. The switch-off mechanism of adenylate cyclase activity consists of the hydrolysis of the guanyl nucleotide triphosphate by the GTPase present in the system and can be i~ibited by a cholera toxin pretreatment ]&-IO]. According to this mechanism, GDP should be an antagonist of GTP action on adenylate cyclase. However GDP itself cannot be tested since it is rapidly phosphorylated by the ATP-regenerating system present in the adenylate cyclase assay medium. We decided to use GDP& a GDP analog which is only slowly phosphorylated by the regenerating system Ill]. The system tested was the adenylate cyclase present in semi-pulled rat pancreatic plasma membranes. This system consists of at least 4 functionary distinct components: the catalytic unit; hormone receptors; and two classes of guanyl nucleotide sites, the first one N1 allowing the transduction of the hor. mane-generated signal and the second one Nz con-

In vitro effects of ethanol and ethanol metabolism in the rat pancreas.

1. Ethanol provoked no effect on basal and carbamylcholine-stimulated secretion of amylase from rat pancreatic fragments incubated for 1 h. 2. Ethanol enhanced in vitro synthesis of fatty acids from [1-14C]acetate and from 3H2O by 110 and 166%, respectively. The spectrum of fatty acids labelled with [1-14C]acetate in the presence of ethanol pointed to a stimulation of the malonic acid pathway, whereas the elongation of polyenoic fatty acids was unaltered. The in vitro metabolism of [1-14C]ethanol indicates that ethanol itself contributed carbon atoms to lipogenesis dose-dependently. 3. The conversion of [U-14C]glucose into sn-glycero 3-phosphate and the esterification of fatty acids into phosphatidic acids and triacylglycerols was stimulated whereas net lipolysis was unaffected. The oxidation of [U-14C]-glucose and of [1-14C]acetate to 14CO2 and the beta-oxidation of [1-14C]palmitate was reduced by 24--26%. 4. Maximal effects were produced by a 100--200 mM ethanol concentration and the concentration of ethanol evoking a similar half-maximal alteration of most processes was 20--30 mM. A 10--20 min lag period was required for the full development of these effects. 5. It is concluded that ethanol at low concentration alters the redox state of pancreatic fragments therefore favoring de novo lipogenesis and triacylglycerol formation and depressing glucose uptake and fatty acid oxidation.

In vitro lipid metabolism in the rat pancreas. II. Effects of secretagogues on fatty acid metabolism, net lipolysis and ATP levels.

1. The concentration of carbamylcholine, bombesin, pancreozymin, pentagastrin and secretin evoking a similar 4--5-fold maximal increase in amylase secretion from rat pancreatic fragments were 3.10(-6), 10(-7), 10(-8), 3.10(-6), and 3.10(-6) M, respectively. The maximal concentration of vasoactive intestinal peptide tested (3.10(-6) M) increased amylase secretion by 250%. The six secretagogues could be separated into two groups according to their effects on lipid metabolism and ATP levels. 2. When used at their optimal concentrations, carbamylcholine, bombesin, pancreozymin, and pentagastrin lowered pancreatic ATP levels by 18-26% and increased net release of free fatty acids by 68-105%. 3. The effects of 3.10(-6) M carbamylcholine and 10(-8) M pancreozymin on the metabolism of 3H2O, D-[U-14C]glucose and [1-14C]acetate were similar; the incorporation of radioactivity in the fatty acid moiety of glycerolipids decreased by 20--50% whereas the incorporation of 3H from 3H2O and of 14C from [U-14C]glucose increased by 20--35% in the glycerol moiety. In addition, the oxidation of [U-14C]glucose, [1-14C]acetate and [1-14C]palmitate to 14CO2 increased by 15--32% while the esterification of [1-14C]palmitate, [1-14C]-linoleate, and [1-14C]arachidonate was inhibited by 14--23%. The spectrum of fatty acids labeled with [1-14C]acetate indicated an inhibition of the malonic acid pathway whereas the elongation of polyenoic fatty acids was unaltered.

EFFECTS OF LYSINE DEFICIENCY ON THE EPIDIDYMAL ADIPOSE TISSUE OF YOUNG RATS

The small amount of very active enzymic and structural proteins, present in the epididymal adipose tissue of male rats, is under nutritional and hormonal control, both in uivo and in vitro (Christophe & Wodon, 1964). I t has been shown that young rats receiving ad libitum a diet deficient in lysine suffer from a loss of liver, pancreatic and muscle proteins (Vandermeers & Christophe, 1964; Vandermeers-Piret & Christophe, 1964; Sidransky & Baba, 1960), and it seemed worthwhile to see whether the kwashiorkor-like disease found in these malnourished animals had also some effect on the protein metabolism of their epididymal adipose tissue. The differences in the results between adipose tissue metabolism in lysine-deficient rats and control rats were moderate, while the differences in the results between adipose tissue metabolism in rats kept on the lysine-poor diet and rats recovering from their deficiency were striking. It will also be shown in this paper that an acute state of amino acid imbalance artificially created in uitro can already result in a degradation of protein metabolism in adipose tissue.

7-day time study of lipid metabolism in normal and obese-hyperglycemic Bar Harbor mice. Qualitative and quantitative aspects

Summary o 1. Lipid metabolism was followed for 7 days in lean and obese-hyperglycemic 11-week old Bar Harbor mice by measuring the incorporation of a single dose (90 μC) of [1-14C] acetate in the liver and extrahepatic tissues. 2. In the liver of control animals, microsomes were more active than supernatant with respect to lipogenesis at time 10 min. They were also more efficient in desaturating labelled 18 : 0 than 16 : 0. A portion of unlabelled fatty acid was elongated with [1-14C] acetate. The radioactivity of liver glycerides decreased rapidly because of the secretion of lipoproteins rich in labelled 18 : 1. The turnover at the 2-position in triglycerides remaining in the liver appeared to be slower than at positions 1 and 3. 3. In the adipose tissue, the renewal of 1–2 diglycerides was very rapid. The labelling of 1–3 diglycerides in this tissue suggests the existence of a monoglyceride pathway operative in vivo. Triglycerides were better labelled in muscle than in adipose tissue. 4. In all tissues, the secondary alterations in the structure of « precursortriglycerides and phospholipids during the 7-day period indicate that a sizeable proportion was submitted to either complete or partial lipolysis. Transfers of 14C by elongation and/or desaturation occurred in the newly released fatty acids before acyl rearrangements, as a result the divergences between 14C distribution and lipid structures observed at time 10 min were slowly reduced. 5. In obese-hyperglycemic mice twice as much of the radioactivity offered was incorporated into total body lipids. The liver was the chief lipogenetic site, with microsomes as well as supernatant overloaded with radioactive triglycerides. The microsomal 9,10 dehydrogenase was very active, and the 2-position of triglycerides was dominated by labelled 18 : 1 throughout the experiment. 6. Both, the renewal of adipose tissue 1–2 diglycerides and the radioactivity of muscle triglycerides were higher than in lean littermates.