Glucocorticoid-induced Insulin Resistance: THE IMPORTANCE OF POSTBINDING EVENTS IN THE REGULATION OF INSULIN BINDING, ACTION, AND DEGRADATION IN FRESHLY ISOLATED AND PRIMARY CULTURES OF RAT HEPATOCYTES

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Abstract

We have recently proposed that “down regulation” of the insulin receptor may be one of the many biological responses of a cell to insulin. In an attempt to further explore this hypothesis we have studied insulin action, binding, and degradation in freshly isolated hepatocytes from rats rendered insulin resistant by the administration of dexamethasone, 1.0 mg/kg every other day, for 1 and 4 wk, and in dexamethasone-treated (0.1 μM for 24 h) primary cultures of hepatocytes from normal rats.

Authors

Jose F. Caro, John M. Amatruda

Regulation of Cathepsin D Metabolism in Rabbit Heart: EVIDENCE FOR A ROLE FOR PRECURSOR PROCESSING IN THE CONTROL OF ENZYME ACTIVITY

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Abstract

Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [35S]methionine. 35S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [35S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing 35S incorporation into cathepsin D with 35S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.

Authors

Allen M. Samarel, Edward A. Ogunro, Alan G. Ferguson, Patricia Allenby, Michael Lesch

Peripheral Serum Thyroxine, Triiodothyronine and Reverse Triiodothyronine Kinetics in the Low Thyroxine State of Acute Nonthyroidal Illnesses: A NONCOMPARTMENTAL ANALYSIS

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Abstract

The low thyroxine (T4) state of acute critical nonthyroidal illnesses is characterized by marked decreases in serum total T4 and triiodothyronine (T3) with elevated reverse T3 (rT3) values. To better define the mechanisms responsible for these alterations, serum kinetic disappearance studies of labeled T4, T3, or rT3 were determined in 16 patients with the low T4 state and compared with 27 euthyroid controls and a single subject with near absence of thyroxine-binding globulin. Marked increases in the serum free fractions of T4 (0.070±0.007%, normal [nl] 0.0315±0.0014, P < 0.001), T3 (0.696±0.065%, nl 0.310±0.034, P < 0.001), and rT3 (0.404±0.051%, nl 0.133±0.007, P < 0.001) by equilibrium dialysis were observed indicating impaired serum binding. Noncompartmental analysis of the kinetic data revealed an increased metabolic clearance rate (MCR) of T4 (1.69±0.22 liter/d per m2, nl 0.73±0.05, P < 0.001) and fractional catabolic rate (FCR) (32.8±2.6%, nl 12.0±0.8, P < 0.001), analogous to the euthyroid subject with low thyroxine-binding globulin. However, the reduced rate of T4 exit from the serum (Kii) (15.2±4.6 d−1, nl 28.4±3.9, P < 0.001) indicated an impairment of extravascular T4 binding that exceeded the serum binding defect. This defect did not apparently reduce the availability of T4 to sites of disposal as reflected by the increased fractional disposal rate of T4 (0.101±0.018 d−1, nl 0.021±0.003, P < 0.001). The decreased serum T3 binding was associated with the expected increases in MCR (18.80±2.22 liter/d per m2, nl 13.74±1.30, P < 0.05) and total volume of distribution (26.55±4.80 liter/m2, nl 13.10±2.54, P < 0.01). However, the unaltered Kii suggested an extravascular binding impairment comparable to that found in serum. The decreased T3 production rate (6.34±0.53 μg/d per m2, nl 23.47±2.12, P < 0.005) appeared to result from reduced peripheral T4 to T3 conversion because of decreased 5′-deiodination rather than from a decreased T4 availability. This view was supported by the normality of the rT3 production rate. The normal Kii values for rT3 indicated a comparable defect in serum and extravascular rT3 binding. The reduced MCR (25.05±6.03 liter/d per m2, nl 59.96±8.56, P < 0.005) and FCR (191.0±41.19%, nl 628.0±199.0, P < 0.02) for rT3 are compatible with an impairment of the rT3 deiodination rate.

Authors

Elaine M. Kaptein, William J. Robinson, Deborah A. Grieb, John T. Nicoloff

Leukocyte Elastase Release during Blood Coagulation: A POTENTIAL MECHANISM FOR ACTIVATION OF THE ALTERNATIVE FIBRINOLYTIC PATHWAY

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Abstract

Immunological detection of elastase, an enzyme present within leukocyte granules, has been used as a marker for polymorphonuclear leukocyte activation. Polymorphonuclear leukocytes contained 4.6 μg/107 cells, whereas erythrocytes, mononuclear cells, and platelets contained <1% of this level. In plasma that was separated from blood cells after 1 h at 22°C, the mean level of elastase-related antigen in seven normal donors was 25±6 ng/ml. This level was unaltered by immediate separation of the plasma from the cells, by inclusion of protease inhibitors, or by anticoagulation of the plasma with either EDTA or acidcitrate-dextrose (the level in heparinized plasma was approximately threefold higher). In serum, the level of elastase-related antigen was 288±125 ng/ml, representing an 11.5-fold increase above plasma levels. The antigen detected in serum was immunochemically indistinguishable from the leukocyte enzyme. Release of elastase was observed when isolated polymorphonuclear leukocytes were added to nonanticoagulated platelet-rich or platelet-poor plasma, recalcified plasma, or to serum. Addition of a chelating agent to serum prevented elastase release, but calcium or magnesium did not induce release in the absence of plasma. Coagulation induced by addition of thrombin to plasma also failed to induce release. In whole blood or in anticoagulated plasma reconstituted with polymorphonuclear leukocytes and then recalcified, initial release of elastase occurred concomitantly with or slightly after clotting and reached maximal levels within 20-40 min after clot formation. The data indicates that early events in coagulation or other pathways that occur in parallel with coagulation induce leukocyte release. The release of elastase, a major fibrinolytic protease of leukocytes, from the cells provides a mechanism for this enzyme or other granule proteases to participate in physiological events.

Authors

Edward F. Plow

Selective Protection against Conidia by Mononuclear and against Mycelia by Polymorphonuclear Phagocytes in Resistance to Aspergillus: OBSERVATIONS ON THESE TWO LINES OF DEFENSE IN VIVO AND IN VITRO WITH HUMAN AND MOUSE PHAGOCYTES

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Abstract

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages.

Authors

Andreas Schaffner, Herndon Douglas, Abraham Braude

Mechanisms of Epinephrine-induced Glucose Intolerance in Normal Humans: ROLE OF THE SPLANCHNIC BED

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Abstract

To evaluate the role of the splanchnic bed in epinephrine-induced glucose intolerance, we selectively assessed the components of net splanchnic glucose balance, i.e., splanchnic glucose uptake and hepatic glucose production, and peripheral glucose uptake by combining infusion of [3-3H]glucose with hepatic vein catheterization. Normal humans received a 90-min infusion of either glucose alone (6.5 mg/kg−1 per min−1) or epinephrine plus glucose at two dose levels: (a) in amounts that simulated the hyperglycemia seen with glucose alone (3.0 mg/kg−1 per min−1); and (b) in amounts identical to the control study. During infusion of glucose alone, blood glucose rose twofold, insulin levels and net posthepatic insulin release increased three- to fourfold, and net splanchnic glucose output switched from a net output (1.65±0.12 mg/kg−1 per min−1) to a net uptake (1.56±0.18). This was due to a 90-95% fall (P < 0.001) in hepatic glucose production and a 100% rise (P < 0.001) in splanchnic glucose uptake (from 0.86±0.14 to 1.71±0.12 mg/kg−1 per min−1), which in the basal state amounted to 30-35% of total glucose uptake. Peripheral glucose uptake rose by 170-185% (P < 0.001). When epinephrine was combined with the lower glucose dose, blood glucose, insulin release, and hepatic blood flow were no different from values observed with glucose alone. However, hepatic glucose production fell only 40-45% (P < 0.05 vs. glucose alone) and, most importantly, the rise in splanchnic glucose uptake was totally blocked. As a result, splanchnic glucose clearance fell by 50% (P < 0.05), and net splanchnic glucose uptake did not occur. The rise in peripheral glucose uptake was also reduced by 50-60% (P < 0.001). When epinephrine was added to the same dose of glucose used in the control study, blood glucose rose twofold higher (P < 0.001). The initial rise in splanchnic glucose uptake was totally prevented; however, beyond 30 min, splanchnic glucose uptake increased, reaching levels seen in the control study when severe hyperglycemia occurred. Splanchnic glucose clearance, nevertheless, remained suppressed throughout the entire study (40%-50%, P < 0.01).

Authors

Luigi Saccà, Carlo Vigorito, Marco Cicala, Biagio Ungaro, Robert S. Sherwin

Elastase of U-937 Monocytelike Cells: COMPARISONS WITH ELASTASES DERIVED FROM HUMAN MONOCYTES AND NEUTROPHILS AND MURINE MACROPHAGELIKE CELLS

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Abstract

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized 14C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [3H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at ∼30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.

Authors

Robert M. Senior, Edward J. Campbell, Jill A. Landis, Fraya R. Cox, Charles Kuhn, Hillel S. Koren

Induction of Membranous Nephropathy in Rabbits by Administration of an Exogenous Cationic Antigen: DEMONSTRATION OF A PATHOGENIC ROLE FOR ELECTRICAL CHARGE

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Abstract

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk.

Authors

Wayne A. Border, Harry, J. Ward, Elaine S. Kamil, Arthur H. Cohen

Regulation of Extraadrenal Steroid 21-Hydroxylase Activity: INCREASED CONVERSION OF PLASMA PROGESTERONE TO DEOXYCORTICOSTERONE DURING ESTROGEN TREATMENT OF WOMEN PREGNANT WITH A DEAD FETUS

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Abstract

We measured deoxycorticosterone (DOC) and progesterone (P) in plasma of 47 women pregnant with a dead fetus and sequentially throughout gestation in 35 women pregnant with a live fetus. When P levels in plasma were low, the plasma levels of DOC in women pregnant with a dead fetus varied but usually were similar to those in women pregnant with a live fetus. However, when P levels were high, the levels of DOC in some women pregnant with a dead fetus were considerably lower than those in women pregnant with a live fetus. To test whether this finding was due to loss of transfer of DOC from fetus to mother or else loss of extraadrenal steroid 21-hydroxylase activity in the mother after death of the fetus, we conducted several studies. The levels of P and DOC in plasma of one woman remained constant from 30 min after fetal death until delivery occurred 13 h later. Estrogen treatment of four women pregnant with a dead fetus brought about an increase in plasma levels of DOC in three of the women. In one woman the ratio of plasma DOC to P was 0.015, a value similar to that found before fetal death, but was 0.003 after fetal death but before estrogen treatment. In two women pregnant with a dead fetus the transfer constants of conversion of plasma P to DOC were 0.011 and 0.005 before, and 0.024 and 0.013, respectively, during estrogen treatment. In one woman pregnant with a deformed fetus with adrenal agenesis, the metabolic clearance rates of DOC before and during estrogen treatment were similar, whereas the plasma production rates of DOC were 2.75 before and 4.31 mg/24 h during estrogen treatment. We suggest that (a) the DOC in plasma of near-term pregnant women arises in part by extraadrenal 21-hydroxylation of plasma P and (b) estrogen stimulates steroid 21-hydroxylase activity in extraadrenal tissues.

Authors

Paul C. MacDonald, Susan Cutrer, Sue C. MacDonald, M. Linette Casey, C. Richard Parker Jr.

Regulation of Episodic Growth Hormone Secretion by the Central Epinephrine System: STUDIES IN THE CHRONICALLY CANNULATED RAT

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Abstract

Catecholamines are postulated to regulate growth hormone (GH) secretion by their influence on the release of two hypothalamic substances, somatostatin, which inhibits GH release, and GH-releasing factor, as yet unidentified. Extensive pharmacologic studies in man and animals indicate a stimulatory effect of central norepinephrine and dopamine on GH, but the function of epiphephrine (EPI) is uncertain. Furthermore, many of the agents used to study the role of catecholamines in GH regulation are not selective in that they affect adrenergic as well as nor-adrenergic and/or dopaminergic neurotransmission. In the present investigation, central nervous system (CNS) EPI biosynthesis was selectively interrupted with the specific norepinephrine N-methyltransferase inhibitors, SK & F 64139 (Smith, Kline & French Laboratories) and LY 78335, (Eli Lilly & Co. Research Laboratories) and the effects of central EPI depletion on episodic GH secretion in the chronically cannulated rat model were determined. Inhibition of CNS EPI synthesis with SK & F 64139 caused complete suppression of episodic GH secretion and concomitantly reduced the EPI level in the hypothalamus without affecting dopamine or norepinephrine. Administration of LY 78335 produced similar effects on pulsatile GH. Morphine-induced, but not clonidine-induced, GH release also was blocked by SK & F 64139. These results indicate that (a) the central EPI system has a major stimulatory function in episodic GH release, (b) morphine-induced GH release is mediated by the central EPI system, and (c) clonidine stimulates GH release by activation of postsynaptic α-adrenergic receptors. Drugs that affect CNS adrenergic systems have a potential role in the diagnosis and treatment of disorders of GH secretion.

Authors

L. Cass Terry, W. R. Crowley, M. D. Johnson