Regulation of proinsulin synthesis in isolated rat islets.

(1) A system is described for studying the short-term effects of agents on proinsulin synthesis in vitro, as measured by the incorporation of [3H]leucine into isolated proinsulin. (2) Of the agents tested, glucose has the most marked, and apparently earliest, effect on proinsulin synthesis. (3) The adenyl cyclase system participates in the regulation of proinsulin synthesis since exogenous cyclic AMP, glucagon, and caffeine are stimulatory. When cyclic AMP is added to the medium in the presence of glucose, it is the most potent agent acting on the adenyl cyclase-phosphodiesterase system. (4) The addition of NADPH to isolated rat islets inhibits proinsulin and Bulk Protein synthesis in vitro.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Summary Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals. This work is supported by National Institutes of Health Grants AM 25647-03 (M. Dawson, Principal Investigator) and GM 28158-01 (C. Tyson, Principal Investigator). The technical assistance of Mr. Jack E. Dabbs, Mr. Charles Hart, and Mr. Randy Douglas is acknowledged.     

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals.     

Insulin-like effects of dithiothreitol on isolated rat adipocytes.

The rate of 3H2O incorporation into lipid in vivo progressively decreased in liver but increased in parametrial adipose tissue during the last 3 days of gestation. These changes seem to be related to those occurring in plasma insulin and progesterone concentrations during the same period. Foetal liver showed a high rate of lipogenesis, which sharply decreased before parturition. foetal lung lipogenesis increased during days 20 and 21 of gestation.     

Lipolytic action of glucagon-like peptides in isolated rat adipocytes.

The lipolytic effect of GLP-1(1-36)-amide, GLP-1(7-36) amide and GLP-2 [proglucagon(126-159)] has been studied in isolated rat adipocytes. Glycerol release and cyclic AMP content were measured after incubation of adipocytes with GLPs and results have been compared with those obtained in the presence of glucagon. GLP-1(7-36)-amide and GLP-1(1-36)-amide at 10(-8), 10(-7) and 10(-6) M concentrations activated glycerol release, the truncated peptide having a more potent effect. On the other hand, GLP-2 had no effect on glycerol release. Also, it has been found that 10(-6) M GLP-1(7-36)-amide increases cyclic AMP content in adipocytes and does not compete with glucagon binding. These results demonstrate that GLP-1(7-36)-amide has a lipolytic effect on isolated rat adipocytes through different receptors than glucagon.     

Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes

Conversion of proinsulin and intermediate forms of proinsulin into insulin were studied with rat liver cell fractions and purified lysosomal proteinases by using the technique of polyacrylamide disc-electrophoresis. Both substrates were degraded very rapidly by homogenates and crude lysosomal fractions to split products not detectable on disc-electropherograms. Neither breakdown nor conversion were detected with the cytosol and the microsomal fraction. With partially purified lysosomal fractions (mol. wt. approx. 25 000) or with highly purified cathepsin L or cathepsin B (B1) proinsulin was converted into products migrating like the intermediate forms and insulin, and the intermediates were converted into products migrating like insulin and deoctapeptide-insulin in disc-electropherograms. The mechanism of conversion seems to be different for both enzymes. The results force us to conclude that lysosomal cathepsins, especially cathepsins L and B might be involved in the process of conversion of proinsulin into insulin and perhaps also of other precursors into biologically active proteins in vivo.     

Glutamine metabolism in isolated incubated adipocytes of the rat.

1. Phosphate-dependent glutaminase activity in the epididymal fat-pad was 15.1 nmol/min per mg of protein. Glutaminase activity demonstrated differences with respect to adipose-tissue sites. Considerable variation was found in different sites of adipose tissue from lean control and Zucker obese animals. 2. Adipocytes incubated in the presence of 2 mM-glutamine utilized glutamine at a rate of 1.8 mumol/h per g dry wt., and glutamate, ammonia, lactate and alanine were produced. Addition of glucose plus insulin increased the rates of glutamine utilization and glutamate, ammonia, lactate and alanine production. Isoprenaline alone or plus glucose further stimulated the rate of glutamine utilization and formation of end products. 3. The rate of incorporation of 14C from glutamine into CO2 was similar to that of glucose, but the rate of incorporation into triacylglycerol was much less. Addition of unlabelled glucose or glucose plus insulin stimulated the rate of incorporation of [14C]glutamine into triacylglycerol, but had no effect on that of 14CO2 formation. Isoprenaline plus glucose increased the rate of incorporation of [14C]glutamine into CO2, but decreased the rate of incorporation into triacylglycerol. 4. In the absence of insulin, the rate of [14C]glutamine incorporation into triacylglycerol was related to the glucose concentration (0-10 mM). However, in the presence of insulin, the rate of incorporation of [14C]glutamine was maximal at 1 mM-glucose.     

Comparisons of insulin and biosynthetic human proinsulin actions in cultured hepatocytes. Kinetics and biologic potencies

The binding and biologic potencies as well as kinetics of action of human biosynthetic proinsulin (HPro) were studied in primary cultures of rat hepatocytes. HPro had 3% (on a molar basis) of the potency of porcine insulin for displacing (125I)-TyrA14-insulin from receptors. Maximally effective concentrations of insulin and HPro caused similar stimulations of 14C-glucose incorporation into glycogen and glycogen synthase activity. However, the dose response curve for HPro stimulation of glycogen synthase was shifted far to the right (EC50 = 4.1 +/- 1.1 nM) of that for insulin (.09 +/- .01). The relative biologic potency of HPro was approximately 3%. Biologically equivalent maximal doses of insulin (8.3 nM) and HPro (53.2 nM) stimulated glycogen synthase activity with similar time courses; half maximal between 15-30 min with maximal effects at 60 min. Deactivation of glycogen synthase upon removal of the hormone was very rapid for both hormones. The relative binding and biologic potencies of HPro compared to insulin in liver (approximately 3%) were similar to values previously seen in adipocytes. This fact, together with the similarity of kinetics of action, suggest that the in vivo hepatoselectivity of HPro is not a property of the target cell itself.     

Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution.

Insulin increases phosphatidylinositol-3-kinase (PI-3-kinase) activity in Chinese hamster ovary cells transfected with human insulin receptor (Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). The subcellular distribution of PI-3-kinase has not been investigated, and it is unclear if insulin has a stimulatory effect on PI-3-kinase in a nonproliferating target tissue, and, if so, whether this effect is subject to counter-regulation. To address these questions, we studied the effect of insulin on PI-3-kinase activity in isolated rat adipocytes. Activity was measured in plasma membranes, intracellular membranes, and cytosol of control and insulin-treated adipocytes, and in anti-Tyr(P) immunoprecipitates prepared from these fractions and from whole cell lysates. Treatment of adipocytes with insulin (200 nM) caused a half-maximal increase in anti-Tyr(P)-immunoprecipitable PI-3-kinase activity in whole cell lysates within 2 min. This effect was concentration-dependent, and it was sensitive to inhibition by norepinephrine. In insulin-stimulated cells, 75% of anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was found in the low density microsomes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in response to insulin nor was it anti-Tyr(P)-immunoprecipitable. These data demonstrate that insulin increases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma membranes.     

Inhibitory effect of insulin on prostacyclin production by isolated rat adipocytes

Physiologic concentrations of insulin completely inhibited the norepinephrine-induced increment in the production of 6-keto-prostaglandin (PG) F_1α, the stable derivative of prostacyclin (PGI₂), by isolated rat adipocytes. The inhibition of PGI₂ production by insulin in isolated rat adipocytes supports the view that the elevated plasma level of 6-keto-PGF_1α in rats with non-ketotic diabetes mellitus and diabetic ketoacidosis is derived at least in part from production of PGI₂ by the adipocyte cell mass.     

Microcalorimetric measurements of heat production in isolated rat brown adipocytes.

Heat production, oxygen consumption, and lipolysis in isolated interscapular brown adipocytes from the rat were investigated. Epinephrine, norepinephrine, and isoproterenol increased heat production in a concentration-dependent manner, showing, about 6-, 4-, and 5-fold higher effects than controls, respectively. The concentration of isoproterenol for threshold heat production and glycerol release were 10(-10) M and 10(-9) M, respectively. The fact that 10(-9) M isoproterenol increased heat production by about 3-fold while glycerol release had no effect at all indicates that calorimetry is more appropriate for investigation of brown adipocytes. At least the method is more sensitive than that of measuring glycerol release.     

Characterization of antilipolytic action of polyamines in isolated rat adipocytes.

The interactions of polyamines with the lipolytic system were studied in isolated rat adipocytes. Spermine, spermidine and putrescine significantly inhibited adenosine deaminase-stimulated lipolysis. An antilipolytic effect of spermine was detectable at a concentration of 0.25 mM (P less than 0.05). At a concentration of 10 mM all three polyamines inhibited the stimulated lipolysis by 50-60% (P less than 0.001). In addition, spermine enhanced the antilipolytic sensitivity of insulin. Spermine (1 mM) decreased the half-maximal inhibitory concentration of insulin from 320 +/- 70 pM to 56 +/- 20 pM (P less than 0.01). The antilipolytic effects and the cyclic-AMP-lowering effects of the polyamines were almost completely prevented in the presence of different phosphodiesterase (PDE) inhibitors (3-isobutyl-1-methylxanthine and RO 20-1724) and, in addition, polyamines had no effect on lipolysis stimulated by dibutyryl cyclic AMP, indicating that polyamines may inhibit lipolysis by activating the PDE enzyme. This latter suggestion was confirmed by demonstrating that spermine (5 mM) significantly enhanced the low-Km PDE enzyme activity (P less than 0.01). Finally, the amounts of polyamines present in isolated adipocytes were measured, and the estimated cytoplasmic concentrations were 0.02 mM (putrescine), 0.86 mM (spermidine), and 1.0 mM (spermine). It is concluded that polyamines may possibly be involved in the physiological regulation of triacylglycerol mobilization in adipocytes.     

Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes.

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.     

Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.     

The metabolism and hormonal responses of human eccrine sweat glands isolated by collagenase digestion.

1. Collagenase digestion of biopsies of human skin yields eccrine sweat glands that can be picked out under binocular light microscopy. The glands are viable as determined by the exclusion of Trypan Blue, the uptake of Methylene Blue, electron microscopy, the rate of lactate dehydrogenase release, ATP content and the rates of glucose oxidation and lactate release. 2. It is proposed that eccrine sweat glands engage in aerobic glycolysis, which accounts for the high content of lactate in sweat (15--60 mM) and the high lactate/pyruvate ratio (100: 1) [Emrich & Zwiebel (1966) Pfluegers Arch. 290, 315--319]. 3. Acetylcholine causes a 4-fold increase in cyclic GMP content, dilatation of the intercellular canaliculi and a reversible, atropine-sensitive, 2-fold increase in the rates of glucose oxidation and lactate release. 4. Isoprenaline causes a 2.5-fold increase in cyclic AMP content. Phenylephrine does not significantly alter cyclic nucleotide metabolism.     

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.