Insulin effect upon lipogenesis of perfused mouse liver

Lipogenesis of mouse livers was estimated by the incorporation of tritium, from triated water, into triglyceride fatty acids, thus eliminating the problem of varying specific activity of metabolic precursors which is met when using ¹⁴C- or ³H-labelled substrates. Using this procedure, a rapid (within 2 h) stimulatory effect of insulin upon lipogenesis of perfused livers obtained from anti-insulin serum-treated normal or obese-hyperglycemic (ob/ob) micr has been observed. Anti-insulin serum treatment did not alter hepatic glycogen or cyclic AMP content. The smallest effective stimulatory concentration of the hormone was 50 micro unit per ml. Insulin increased lipogenesis in the presence of either glucose or acetate but not in the absence of substrate. It did not relieve the inhibitory effect of added oleate upon the lipogenic process. The clear-cut stimulator effect of insulin upon lipogenesis observed in livers from anti-insulin serum-treated normal or obese-hyperglycemic mice was no longer present when livers from untreated animals were studied. Under the latter conditions, basal lipogenesis was higher than that seen in livers of animals treated with anti-insulin serum prior to the experiment, being highest in livers obtained from hyperinsulinemic obese-hyperglycemic mice. This suggested that the presence of endogenous insulin immediately prior to the experimental sufficed to stimulate hepatic lipogenesis, the degree of this stimulation being apparently related to that of insulinemia. Although the precise site of aciton of the rapid stimulatory influence of insulin upon liver lipogenesis is not determined, it appears to be situated at or beyond the level of acetyl-CoA carboxylase, fatty acid handling and cyclic AMP being apparently not implicated in such hormonal regulations.     

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [¹²⁵I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [¹²⁵I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [¹²⁵I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization.     

Glycerolipid biosynthesis in rat adipose tissue 12. Properties of Mg2+-dependent and -independent phosphatidate phosphohydrolase

The properties of Mg²⁺-dependent and Mg²⁺-independent phosphatidate phosphohydrolase activities were investigated in different subcellular fractions in rat adipose tissue. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. The Mg²⁺-dependent phosphatidate phosphohydrolase was inhibited in the presence of N-methyl- or N-ethylmaleimide, whereas the Mg²⁺-independent activity was unaffected by these agents. The Mg²⁺-dependent phosphatidate phosphohydrolase was more sensitive to proteolysis and to high temperature (55 °C) compared to the Mg²⁺-independent enzyme. The Mg²⁺-dependent phosphatidate phosphohydrolase activity was reduced significantly during aging without any appreciable effects on the Mg²⁺-independent phosphatidate phosphohydrolase activity. These studies demonstrate that, in addition to Mg²⁺-dependency, these two forms of phosphatidate phosphohydrolases differ in several respects irrespective of their location in the adipose cell.     

The effects of insulin and glucose on the induction and intracellular translocation of delta-aminolevulinic acid synthase

The administration of insulin and glucose to young Sprague-Dawley rats (125-150 g) resulted in changes in the intracellular distribution and in the turnover rates of delta-aminolevulinic acid synthase (ALAS) activity in the mitochondria and the cytosol. When starved, allylisopropylacetamide (AIA)-induced rats were injected with either insulin or glucose, the percentage of the total ALAS activity found in the cytosol increased from 27% in control animals to 33-40% in insulin-treated and 50% in glucose-treated rats. Similar increases of the ALAS activity in the cytosol were observed after insulin treatment of noninduced, starved animals. Glucose administration also repressed 25-40% of the AIA induction of ALAS as previously reported; however, this effect apparently was not a result of elevated insulin levels, since there was no observed repression of AIA induction after insulin administration. The effects of insulin and glucose on the turnover rates of ALAS activity in the mitochondria and in the cytosol were investigated by observing changes in the half-lives of ALAS activity in the two intracellular compartments. Administration of both insulin and glucose resulted in an increased half-life of ALAS activity in the cytosol from 20.8 to over 100 min, while the mitochondrial half-life was not significantly changed. When insulin was given to either fed, AIA-induced or to starved, noninduced rats, the half-life of the cytosolic ALAS increased from about 14 to 40 min. In contrast to the starved, induced animals, the mitochondrial ALAS half-life in starved, noninduced animals decreased 50%. These results suggest that insulin and glucose treatment may inhibit the translocation of ALAS from the cytosol into the mitochondrial matrix.     

Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells

Enzymes of glutamate metabolism were studied in the astrocytes isolated from rats injected with a large dose of ammonium acetate and compared with those isolated from controls. The activities of glutamate dehydrogenase (GDH) and glutaminase decreased while those of glutamine synthetase (GS) and aspartate aminotransferase (AAT) increased both in convulsive and comatose states. The activity of alanine aminotransferase (A1AT) increased only in convulsive state. The results suggested that glutamate required for the formation of glutamine in astrocytes might have its origin in nerve endings and the depletion of citric acid cycle intermediates might occur in nerve endings at least in acute ammonia toxicity.     

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.     

Relationship between the subunit structure of insulin receptor and its competence to bind insulin and undergo phosphorylation

Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [γ-³²P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N^?B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with ³²P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) α-dimer, β-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, α₂β₂structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one α and One β Chain.     

Comparative suppressive effects of anti-allotype antibodies on spleen cells of immature and adult rabbits

The suppressive effects of anti-allotype antibody on splenic lymphocytes of adult (>3 months of age) and newborn (1-week-old) rabbits were compared in vitro. An approximately 10-fold lower concentration of anti-b4 sufficed to modulate completely membrane-bound b4 immunoglobulin (Ig) when newborn cells were pulse-treated for 2 or 24 hr than when adult cells were tested. In contrast to splenic lymphocytes from adults, which regenerated most of the original proportion of b4⁺ cells in culture following treatment with up to 300 μg of anti-b4 for 24 hr (4), immature lymphocytes were susceptible to irreversible modulation of membrane-bound Ig even when lower concentrations of anti-b4 were used. However, under conditions permitting reversible modulation of membrane b4, lymphocytes of newborns regenerated the membrane product more rapidly than did adult cells. Both mature and immature splenocytes were shown to be capable of some level of b4 synthesis even in the continuing presence of anti-b4. No evidence for susceptibility to complement-mediated killing of newborn spleen cells by anti-allotypic antibodies was obtained. The observations reported here support the concept that the greater sensitivity of B lymphocytes from the newborn to succumb to irreversible suppressive effects by anti-allotype antibody plays a significant role in the restriction for induction of allotype suppression to the perinatal period.     

Location of rep and inc sequences in the F secondary replicon

Miniplasmids derived by deletion of DNA from the F plasmid secondary replicon have been tested for the ability to replicate and to express incompatibility with the IncFI plasmid, ColV3-K30. The results demonstrate that the minimal rep region of the secondary replicon lies within a 1.9-kb sequence (33.7F-35.6F kb), and that an inc region, presumably involved in replication control, is present in a 0.45-kb portion of the rep region (33.7F-34.15F kb). In addition, the secondary replicon was found not to require DNA polymerase I activity.     

The effects of sulphur supplementation on cellulose digestion in vitro and on nutrient digestion,nitrogen metabolism and rumen characteristics of lambs fed on good quality fescue and tropical star grass hays

Experiments were conducted in vitro and in vivo to determine the effects of sulphur (S) supplementation of a good quality fescue hay containing 0.27% total S and a tropical star grass hay containing 0.20% total S. Addition of S was on an isosulphurous basis of either sodium sulphate or D,L-methionine. Cellulose digestion in vitro was improved (P < 0.001) by the addition of 1% urea. Supplementation of forage with 0.05, 0.10 or 0.15% S from either sodium sulphate or methionine also stimulated cellulose digestion in vitro. There were no differences between S sources. The addition of 0.4 or 0.8% nitrate-nitrogen (nitrate-N) (potassium nitrate) depressed (P < 0.05) cellulose digestion in vitro of both hays. No effect of animal adaptation to nitrate was evident. Addition of S partially counteracted the depression in cellulose digestion due to nitrate. Trials were conducted in vivo in which 12 crossbred wether lambs (fescue experiment) or 12 crossbred intact male lambs (star grass experiment) were randomly assigned to one of three treatments: control (forage with no addition of S); forage plus 0.15% S as sodium sulphate; and forage plus 0.15% S as D,L-methionine. Lambs were housed in metabolism crates and each experiment was replicated twice. Dry matter intakes were highest for methionine-supplemented fescue and for S-supplemented star grass, regardless of S source. Dry matter digestibility tended to increase with S addition (fescue experiment) and was significantly higher for S-supplemented star grass. There was a significant increase (P < 0.05) in neutral detergent fibre (NDF) and acid detergent fibre (ADF) digestibility due to supplemental S, regardless of S source. Nitrogen retention, ammonia-N and ruminal volatile fatty acids were unaffected by S supplementation.     

The metabolism of prostaglandins F2α and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue,luteal tissue and conceptuses in vitro

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF_2a and PGE₂ and the degree of metabolism of the prostaglandins measured. Prostaglandins and metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.     

Comparative studies on calotropins DI and DII from the latex of Calotropis gigantea

Autodigestion of two cysteine proteinases, calotropins DI and DII isolated from the latex of Calotropis gigantea, has been studied at pH 7.5 and 37 degrees C in the presence of an activating agent. Calotropin DI is more susceptible to autodigestion than calotropin DII. During autodigestion no interconversion of one calotropin to another has occurred, as verified by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Immunologically, both calotropins are closely related, but they differ from papain and ficin. Both calotropins have blocked N-terminal amino acid residues. Their C-terminal amino acid sequences, determined by treatment with carboxypeptidase Y, are -(Pro, Ala)-Ala-Val-Tyr for calotropin DI and -(Ala, Val)-Ala-Pro-Tyr for calotropin DII. The tryptic peptide maps of their reduced and S-carboxymethylated derivatives suggest that both calotropins share a high proportion of common regions in their amino acid sequences. Calotropins DI and DII are two distinct proteinases, and they do not appear to be produced by autodigestion of a single precursor. Although they are inert to the common synthetic substrates of papain and ficin, their specificities toward oxidized insulin B chain are comparable to those of papain and ficin.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

Sugar transport in fat cells: effects of mechanical agitation, cell-bound insulin, and temperature.

The effects of several factors that affect the sugar transport activity in rat epididymal fat cells were studied. The transport activity was assessed semiquantitatively by measuring the uptake of 3-O-methyl-d-glucose by the oil-flotation method. The transport activity was stimulated by mechanical agitation, such as centrifugation of cells. This effect was transient. When agitated cells were incubated at 37 °C with gentle shaking, their transport activity declined. The decline was often facilitated by the addition of glucose or pyruvate. Presumably some cell preparations were low in the source of metabolic energy that was required for this recovery process. When cells were exposed to a high concentration of insulin, washed, and suspended in fresh buffer, the effect of insulin (plus that of mechanical agitation) declined after a certain lag period. The length of this period was a function of the initial insulin concentration. The incubation temperature had different effects on the basal and plus-insulin activities. The basal activity at 25 °C was higher than that at 37 °C, while the plus-insulin activity was lower at 25 °C than at 37 °C.     

Involvement of oxygen and mitochondrial function in the metabolism of D-xylulose by Saccharomyces cerevisiae

Mitochondrial function associated with oxygen was required for growth of Saccharomyces cerevisiae on D-xylulose. The requirement was shown by (i) the inhibition of growth of a wild-type strain under anaerobic conditions, (ii) the inhibition of aerobic growth after treatment with inhibitors of mitochondrial function, and (iii) the lack of aerobic and anaerobic growth of nuclear and cytoplasmic petites. The mitochondrial function was associated with the channeling of catabolites of D-xylulose to growth processes, since ethanol was formed even when growth was inhibited. Mitochondrial function was implicated as well in determining the extent of growth and the concentration of ethanol in aerobic cultures of the wild-type. In such cultures, the concentration of ethanol decreased and growth increased concomitantly as aeration rate increased. A factor in this relation was considered to be the relatively poor ability of D-xylulose to inhibit the oxidative utilization of ethanol.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.