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J. Grinblat and A. Klein

ABSTRACT

A study was carried out to determine the effect on lymphocytic cortisol metabolism (LCM) of plasma from 62 patients with diffuse thyrotoxic goitre (DTG), 14 patients with toxic nodular goitre (TNG) and ten hypothyroid patients. Plasma of 33 healthy donors served as controls. A known concentration of human lymphocytes was incubated with cortisol in media containing 50% phosphate-buffered saline (PBS) and 50% of one of the following additions: (1) PBS, (2) homologous plasma (HP), (3) heterologous plasma, (4) plasma from DTG patients, (5) plasma from TNG patients, (6) plasma from hypothyroid patients, (7) PBS and HP to which l-thyroxine (T4) and tri-iodothyronine (T3) had been added separately and as a mixture up to a concentration ten times the normal, (8) boiled HP and (9) boiled DTG plasma. Plasma from hypothyroid patients gave an LCM-enhancing effect (LCMEE) similar to that of HP. The plasma of DTG and TNG patients had a markedly lesser effect on LCM than did HP. The T4 and T3 had no additional effect when added to PBS or HP. Boiling of HP and DTG plasma resulted in a similar decrease in LCMEE. The findings of this study raise the possibility of the existence of a factor inhibiting LCMEE in the plasma of thyrotoxic patients.

J. Endocr. 1984 101, 149–153

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V Ott, M Fasshauer, A Dalski, HH Klein and J Klein

Ciliary neurotrophic factor (CNTF) plays an important role in regulating neuronal growth. Recently, central anorexigenic effects of this cytokine have been characterized. However, peripheral effects on tissues that actively contribute to the regulation of energy homeostasis have not been described. Here, we report direct potent and selective effects of CNTF on growth factor and metabolic signalling intermediates in mouse brown adipocytes. CNTF stimulates STAT3, MAP kinase, Akt, and p70 S6 kinase. We find that, next to mediating Akt and p70 S6 kinase activation, both phosphatidylinositol 3-kinase and protein kinase C are separately acting, main intermediates for inducing mitogen-activated protein (MAP) kinase activation. On a functional level, CNTF enhances beta3-adrenergic induction of uncoupling protein-1. Thus, these results demonstrate direct effects of CNTF on adipose tissue signalling and metabolism and suggest a novel role for this cytokine in the peripheral regulation of energy homeostasis.

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A. Klein, A. W.-L. Chan and A. Malkin

ABSTRACT

Mononuclear cell preparations are capable of metabolizing cortisol to three metabolites which lack the immunosuppressive effect of their precursor. In the present study we noted a linear correlation, up to a point, between glucose concentration and the rate of human mononuclear cell cortisol metabolism in vitro. The mechanism by which glucose exerts its effect was investigated further. We observed that: (1) the effect of glucose on mononuclear cell cortisol metabolism was not influenced by insulin; (2) NADPH and NADH enhanced cortisol metabolism by disrupted cells, irrespective of whether the homogenates were dialysed or not; (3) lactate and ATP inhibited mononuclear cell cortisol metabolism and (4) almost all the glucose used was converted to lactate. It is concluded that mononuclear cell cortisol metabolism can depend on both nucleotides.

J. Endocr. (1986) 109, 181–185

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I Klein, O Esik, V Homolya, F Szeri and A Varadi

Medullary thyroid carcinoma (MTC) occurs usually in sporadic form, but about a quarter of the cases are hereditary and appear as part of one of the multiple endocrine neoplasia type 2 (MEN2) syndromes. Mutations in the RET protooncogene are known to be the cause of the MEN2A and familial medullary thyroid carcinoma (FMTC) syndromes in the majority of the families. Direct DNA testing allows prophylactic thyroidectomy to be offered to individuals carrying a mutation in the above codons, and in mutation-negative cases it reduces the yearly screening-related burden on family members at risk of the disease. By DNA sequencing and PCR-restriction fragment length polymorphisms, 65 MTC probands were examined for mutations in residues 609, 611, 618, 620 of exon 10, and in residues 634, 768, 804 of exons 11, 13, and 14 respectively of the RET protooncogene. In our study, mutations in the above codons were detected in all of the 14 clinically MEN2A and FMTC families. One of these mutations, TGC609 TCC has not been reported previously. Of the 14 probands with the mutation, 25 relatives also had the identified mutation and 18 relatives proved to be non-carriers. Among the 51 probands with clinically sporadic MTC, none was found to carry a mutation in the above positions even if indirect signs of MTC, pheochromocytoma or hyperparathyroidism could be detected in some families. The frequency of the TGC634AGC mutation is unexpectedly high in our samples, which can probably be attributed to a founder effect. We conclude that screening for mutations in these codons is effective in families fulfilling the strict clinical criteria of MEN2A or FMTC.

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A. Klein, B. Bruser, J. B. Robinson, P. H. Pinkerton and A. Malkin

ABSTRACT

We have observed previously that the rate of cortisol catabolism by lymphocytes (CCL) was indicative of the vulnerability of these cells to cortisol. We attempted to ascertain whether cortisol-sensitive lymphocytes (e.g. thymocytes) metabolize cortisol at a different rate from cortisol-resistant cells and whether lymphocytes in which cortisol catabolism is inhibited become cortisol sensitive. The work was facilitated by the observation that an ethanol extract plasma from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) had the capacity to inhibit CCL.

The capacity of thymocytes to metabolize cortisol was found to be 11 times lower than that of peripheral lymphocytes. Inhibition of CCL with an ethanol extract of plasma from AIDS/ARC patients made the cells vulnerable to cortisol, causing them to die at a rate seven times greater than that of control samples. It is suggested that these findings may have important implications with regard to the nature of lymphocyte depletion in AIDS/ARC patients or in people at risk of developing the syndrome.

J. Endocr. (1987) 112, 259–264

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S. Matthaei, H. Benecke, H. H. Klein, A. Hamann, G. Kreymann and H. Greten

ABSTRACT

To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (30MG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 30MG (12±4 vs 13±3 fmol/5 × 104 cells, means ± s.e.m.); in contrast, in FR-24 rats insulin-stimulated 30MG was markedly decreased by 43% when compared with that in FR-6 rats (158±14 vs 90±8 fmol/5 × 104 cells; P < 0·01). Insulin binding to adipocytes from FR-6 rats was 2·40±0·38% compared with 2·28±0·47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48·6±7·2 vs 29·8±5·5 pmol/mg membrane protein; P < 0·01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23·9±4·2 (FR-6) vs 14·4±3·1 (FR-24) pmol/mg membrane protein; P < 0·01). These results were confirmed by immunoblotting.

In conclusion, (1) maximal insulin-stimulated 30MG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 30MG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 30MG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats.

Journal of Endocrinology (1990) 126, 99–107

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R. A. Nowak, J. S. Klein, D. M. Pulido and J. M. Bahr

ABSTRACT

The present study was undertaken to determine (1) whether the rabbit feto-placental unit maintains corpora lutea systematically and/or locally and (2) the interrelationships between conceptus number, luteal weight, luteal progesterone concentrations and serum progesterone levels. Thirty-three does were divided into the following treatment groups: (I) bilaterally pregnant, two ovaries; (II) unilaterally pregnant, two ovaries; (III) bilaterally pregnant, one ovary; (IV) unilaterally pregnant, one ovary, contralateral and (V) unilaterally pregnant, one ovary, ipsilateral. Blood samples were obtained from all rabbits on days 6, 9, 12, 15, 18 and 21 post coitum. Does were killed on day 21, and the percentage of viable fetuses, fetal weights, and luteal weights recorded. Blood samples and corpora lutea were analysed for progesterone.

Serum progesterone levels were similar for all groups until day 9 post coitum. Levels in groups III, IV and V declined significantly between days 9 and 12 following removal of one ovary at day 9. Fetal viability, fetal weights and luteal progesterone concentrations did not differ among any of the groups. Luteal weights did not differ among groups I, III, IV and V, but luteal weights of animals in group II were lower than those of group I (P<0·05). Ratios of viable fetuses to number of corpora lutea ranged from 1:11–10:5. No differences were observed in serum progesterone, luteal weights or luteal progesterone concentrations among animals with two conceptuses and those with seven or more, but serum progesterone levels in does with only one conceptus were lower than those in does with more (P<0·05). These results indicate that the feto-placental unit maintains corpora lutea systemically and that the high rate of pregnancy failure by day 21 in does with only one conceptus is due to the inability of a single conceptus to maintain normal serum progesterone levels even though the corpus luteum weight is not affected.

J. Endocr. (1986) 109, 107–110

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A Iida-Klein, S Shou Lu, R Kapadia, M Burkhart, A Moreno, D W Dempster and R Lindsay

Parathyroid hormone (PTH) stimulates bone resorption as well as bone formation in vivo and in organ culture. The catabolic actions of PTH have been recognized in patients with hyperparathyroidism, or with acute infusion of the N-terminal 1–34 fragment of human PTH (hPTH1–34). Whereas the anabolic actions of daily injection with PTH have been well studied in both humans and mice, the catabolic actions of PTH on murine bone remain to be defined. To do this we sought to create a model with short-term, sustained hyperparathyroidism using osmotic infusion pumps. We treated 10-week-old female C57BL/J6 mice with continuous infusion of hPTH1–34 (8.1 pmol/0.25 μl per h, equivalent to 40 μg/kg per day) or vehicle for 2 weeks, using Alzet osmotic pumps. Bone mineral density (BMD), serum total calcium, hPTH1–34, mouse intact PTH (mPTH1–84), osteocalcin and mouse tartrate-resistant acid phosphatase (mTRAP) activity, and microarchitectural variables of the distal femur were measured. Separately, we compared the effects of intermittent daily injection of hPTH1–34 (40 μg/kg per day) with continuous infusion of hPTH1–34 on BMD and bone markers. Exogenous hPTH1–34 was detected only in the PTH-infused mice. Both intermittent and continuous treatment with hPTH1–34 markedly suppressed endogenous mPTH1–84, but only the latter induced hypercalcemia. Daily PTH injection significantly increased both serum osteocalcin and mTRAP, while continuous PTH infusion showed a strong trend to stimulate mTRAP, with a slight but non-significant increase in osteocalcin. There were significant differences in BMD at all sites between animals treated with the same daily dose of intermittent and continuous hPTH1–34. Microcomputed tomography (μCT) analysis of the distal femurs revealed that hPTH1–34 infusion significantly decreased trabecular connectivity density (P<0.05). Thus, the murine bone response to continuous PTH infusion was quite different from that seen with daily PTH injection. Short-term infusion of hPTH1–34 appears to be a good model to study the mechanisms underlying the catabolic action of PTH in mice.

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A J Tilbrook, D M de Kretser, F R Dunshea, R Klein, D M Robertson, I J Clarke and S Maddocks

Abstract

The aims of this study were to determine the plasma concentrations of follistatin in rams and to assess if the testis contributes to circulating follistatin and if there is uptake or production of follistatin by the head in rams. Catheters were inserted in the carotid artery, jugular vein and spermatic vein of intact rams during the non-breeding season (experiment 1; n=5) and breeding season (experiment 2; n=4). In experiment 1, blood samples were collected from 5 rams every 10 min for 4 h, commencing 20–60 min after surgery. After 2 h of sampling 1 μg gonadotrophin-releasing hormone (GnRH) was injected intravenously. In experiment 2, blood samples were collected from 4 of the rams used in experiment 1 by venipuncture 30 and 15 min before surgery and every 15 min throughout surgery. Commencing 1 h after surgery, matched samples were taken from each of the vessels every 10 min for 4 h (1–4 h after surgery), then every hour for 20 h (4–24 h after surgery) and then every 10 min for 4 h (24–28 h after surgery). In both experiments, follistatin secretion was non-pulsatile and there were no significant differences between the concentrations of follistatin in any of the vessels. There was a significant (P<0·05) increase in the concentrations of follistatin in each of the vessels throughout the 4 h of 10-min sampling in both experiments. In experiment 2 plasma concentrations of follistatin in the jugular vein were significantly (P<0·05) lower before surgery than at other stages of the experiment. During the non-breeding season (experiment 1) the concentrations of follistatin in all vessels were about 2-fold higher (P<0·001) than during the breeding season (experiment 2). Concentrations of follistatin were measured in the testicular tissue of the ram, bull, monkey and rat and were found to be 13·6, 2·1, 2·5, 0·8 ng/g testis respectively. In experiment 3, blood samples were collected every 15 min for 4 h from castrated rams (n=6) in the absence of treatment with testosterone propionate (TP) and after 7 days of treatment with a physiological dose of TP during the breeding and non-breeding seasons. There was no effect of stage of breeding season or TP on the plasma concentrations of follistatin and these concentrations in the castrated rams were similar to the concentrations in the intact rams in experiment 2. In experiment 4, the function of Leydig cells was stimulated by administration of human chorionic gonadotrophin but this had no effect on plasma concentrations of follistatin.

These experiments show that the concentrations of follistatin in the plasma of rams are measurable, that the testis is not the major contributor to circulating follistatin and that there is no significant uptake or production of follistatin by the head in rams. It appears that the contribution of the testis to circulating follistatin may vary with the stage of the breeding season, being greater during the non-breeding season than the breeding season. The gonadotrophins and testosterone do not appear to have a direct effect on the secretion of follistatin in rams. The increase in concentrations of circulating follistatin during surgery and more frequent blood sampling suggest a stress-related effect on the production of follistatin.

Journal of Endocrinology (1996) 149, 55–63

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S Matthaei, B Trost, A Hamann, C Kausch, H Benecke, H Greten, W Höppner and H H Klein

Abstract

To examine the effect of thyroid hormone status on insulin action in isolated rat adipocytes, age- and weight-matched Sprague–Dawley rats were rendered hypothyroid (h) by i.p. injection of 2 mCi [131I]/kg. Another group of rats was made hyperthyroid (H) by i.p. injection of 500 μg l-thyroxine/kg/day for 7 days. The T4 levels in experimental groups were: controls, 33·5±0·95; h, 12·3±1·59: H, 133·2±8·8 μg/l. Adipocytes were isolated and 3-O-methylglucose transport (GT), insulin binding (IB) and insulin receptor kinase activity (IRKA) were determined. Subcellular membrane fractions (low-density microsomes, plasma membranes) were prepared and GLUT1 and GLUT4 glucose transporter immunodetected.

Hyperthyroidism caused no significant effect on either IB or IRKA but increased insulin-stimulated GT by 43·6%. This increase of GT was associated with an increase of primarily GLUT4 glucose transporters. Hypothyroidism was associated with both increased insulin receptor affinity and enhanced IRKA. Despite a marked reduction of primarily GLUT4 glucose transporters, basal and insulinstimulated GT was not reduced when compared with control.

These results suggest that (1) in hyperthyroidism, increased insulin-stimulated glucose transport is associated with an increase of primarily GLUT4 glucose transporters, which may be responsible for the increment of peripheral glucose utilization in hyperthyroidism, and (2) the effect of hypothyroidism on insulin action in adipocytes is characterized by a state of increased insulin sensitivity, as indicated by the increase in insulin receptor affinity and tyrosine kinase activity. Despite the marked reduction of primarily GLUT4 glucose transporters, insulin-stimulated glucose transport is not diminished, which may suggest that functional activity of plasma membrane glucose transporters is enhanced in hypothyroidism.

Journal of Endocrinology (1995) 144, 347–357