Pyruvate carboxylase (PC) activity is enhanced in the islets of obese rats, but it is reduced in the islets of type 2 diabetic rats, suggesting the importance of PC in β-cell adaptation to insulin resistance as well as the possibility that PC reduction might lead to hyperglycemia. However, the causality is currently unknown. We used obese Agouti mice (AyL) as a model to show enhanced β-cell adaptation, and type 2 diabetic db/db mice as a model to show severe β-cell failure. After comparison of the two models, a less severe type 2 diabetic Agouti-K (AyK) mouse model was used to show the changes in islet PC activity during the development of type 2 diabetes mellitus (T2DM). AyK mice were separated into two groups: mildly (AyK-M, blood glucose <250 mg/dl) and severely (AyK-S, blood glucose >250 mg/dl) hyperglycemic. Islet PC activity, but not protein level, was increased 1.7-fold in AyK-M mice; in AyK-S mice, islet PC activity and protein level were reduced. All other changes including insulin secretion and islet morphology in AyK-M mice were similar to those observed in AyL mice, but they were worse in AyK-S mice where these parameters closely matched those in db/db mice. In 2-day treated islets, PC activity was inhibited by high glucose but not by palmitate. Our findings suggest that islet PC might play a role in the development of T2DM where reduction of PC activity might be a consequence of mild hyperglycemia and a cause for severe hyperglycemia.
T Vanttinen, J Liu, J Liu, C Hyden-Granskog, M Parviainen, I Penttila and R Voutilainen
Inhibins are gonadal glycoproteins with endocrine effects on pituitary FSH secretion and para/autocrine effects on ovarian and testicular function. The purpose of this study was to investigate the endocrine and para/autocrine regulation of inhibin A and inhibin B secretion in human ovarian granulosa-luteal cells. The cells were obtained from women undergoing in vitro fertilization, and the primary cultures were treated with FSH, LH, human chorionic gonadotropin (hCG), activin A, 8-bromo cyclic AMP (8-BrcAMP), staurosporine (a protein kinase C inhibitor) and an antagonist of IGF action (type-1 IGF receptor antibody alpha IR3). The secretion of inhibins was measured by ELISA assays capable of reliably distinguishing between inhibin A and B. FSH, LH, hCG and 8-BrcAMP increased inhibin A secretion on average up to 180% (P<0.01), 192% (P<0.05), 210% (P<0.01) and 243% (P<0.01) respectively of the control level, while their stimulatory effect on inhibin B secretion was less pronounced (up to 167%, P<0.01; 139%, P<0.05; 127%, P>0.05; 133%, P>0.05 of the controls respectively). alpha IR3 decreased inhibin A and B secretion down to 70% (P<0.01) and 50% (P<0.01) respectively of the control. Staurosporine decreased inhibin B secretion down to 49% (P<0.01) of the control; its effect on inhibin A secretion was not significant. Activin A increased inhibin B secretion up to fourfold of the control (P<0.05) while its effect on inhibin A secretion was insignificant. We conclude that gonadotropins via the protein kinase A signal transduction pathway are the main positive regulators of inhibin A and B secretion in human granulosa-luteal cells. The protein kinase C signal transduction pathway seems to be important especially for inhibin B secretion. Locally produced IGFs are probably important inducers of the production of both forms of inhibin in human ovaries while activins seem to upregulate inhibin B secretion.
K H Liu, J C Huang and J D Godkin
Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5·3–6·3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin.
Journal of Endocrinology (1995) 146, 527–534
J Liu, P Heikkilä, A I Kahri and R Voutilainen
The steroidogenic acute regulatory protein (StAR) has recently been shown to be a factor necessary for cholesterol transport into adrenal and gonadal mitochondria, which is the regulated, rate-limiting step in steroidogenesis. We show here that StAR mRNA is highly expressed in normal adult adrenals (n=9), adrenocortical adenomas (n=16), adrenal hyperplasias (n=6), adrenocortical carcinomas (n=6) and adrenals adjacent to tumor tissues (n=9). There was a good correlation between the expression of StAR and the cholesterol side-chain cleavage enzyme/20,22-desmolase (P450 scc) mRNAs both in normal (r=0·93; P<0·01) and in tumor (r=0·97; P<0·001) tissues. No StAR mRNA was detected in Northern blots of liver, kidney, breast, parathyroid or phaeochromocytoma RNAs.
In cultured adrenocortical cells, adrenocorticotropin (ACTH), (Bu)2cAMP, and cholera toxin increased StAR and P450 scc mRNA accumulation 6- to 18-fold, dose-and time-dependently. StAR (and P450 scc) mRNA increased relatively slowly in response to ACTH treatment, with the maximal increment at 24 h, while the mRNA of the early response gene c-fos peaked within 2 h. The protein kinase inhibitor H-7 inhibited basal and ACTH-induced StAR mRNA expression. Our results show that StAR mRNA is expressed at high levels in normal human adrenals and adrenocortical neoplasms. It is up-regulated in parallel with P450 scc by ACTH in adult adrenocortical cells, which suggests that ACTH is at least one of the key regulators of adrenal StAR expression.
Journal of Endocrinology (1996) 150, 43–50
J Liu, R Voutilainen, A I Kahri and P Heikkilä
Abundant c-myc gene expression in neoplasms has been often linked to poor prognosis. As c-myc mRNA is expressed and hormonally regulated in human adrenals, we examined the c-myc gene expression in adrenal tumors by RNA analysis and immunohistochemistry to find out the possible role of c-myc in adrenal neoplasms. The abundant expression of the c-myc gene in normal adrenals was localized to the zona fasciculata and zona reticularis, with much lower expression in the zona glomerulosa and adrenal medulla. In hormonally active adrenocortical carcinomas (n=6) and in virilizing adenomas (n=4), c-myc mRNA levels were approximately 10% of those in normal adrenals (n=11). In contrast, adrenal adenomas from patients with Cushing's (n=4) and Conn's (n=9) syndrome, non-functional adenomas (n=2), adrenocortical hyperplasias (bilateral, n=5; nodular, n=4), and non-functional adrenocortical carcinomas (n=3) expressed c-myc mRNA to the same extent as normal adrenals. The c-myc mRNA abundance in benign adrenal pheochromocytomas (n=19) was similar to that in normal adrenal medulla. However, in malignant adrenal pheochromocytomas (n=6), the average c-myc mRNA levels were approximately threefold that in benign adrenal pheochromocytomas. There was a good correlation between c-myc mRNA expression and immunohistochemical reactivity in both normal and pathological adrenal tissues. Southern blot analysis revealed no amplification or rearrangement of the c-myc gene in any of the adrenal tumors.
In conclusion, c-myc expression localized to zona fasciculata and reticularis in normal adrenals. Virilizing adenomas and hormonally active adrenocortical carcinomas expressed c-myc mRNA clearly less than the other adrenal neoplasms and normal adrenal tissue. On the other hand, malignant pheochromocytomas contained more c-myc mRNA than benign ones. Further studies are required to clarify the mechanisms and significance for the distinct expression pattern of the c-myc gene in different adrenal neoplasms.
Journal of Endocrinology (1997) 152, 175–181
J. Liu, R. M. Haigh and C. T. Jones
Glucocorticoids are known to regulate the contractility of vascular smooth muscle by increasing its response to noradrenaline. The molecular mechanisms for achieving this remain unclear. Recent results in our laboratory have demonstrated that glucocorticoids affect both α1-adrenoceptor number and coupling to G proteins. Whether this leads to an increase in second-messenger production has to be established. The present experiments, therefore, report the effects of dexamethasone on inositol polyphosphate production in vascular smooth muscle cells in culture. Noradrenaline induced the release of inositol polyphosphates from prelabelled [3H]inositol phosphoinositides in the membrane in a dose-dependent manner. The concentration of noradrenaline which caused half-maximal response was 1·26 μmol/l. Prazosin inhibited noradrenaline-induced inositol monophosphate formation to 10·26 ± 3·67% (mean ± s.e.m.; P < 0·01, n = 5) of control value whereas yohimbine reduced it to only 61·74 ± 11·82% (P < 0·05, n = 5), suggesting an action primarily through α1-adrenergic receptors. Dexamethasone (100 nmol/l, 48 h) enhanced noradrenaline-induced inositol monophosphate, bisphosphate and trisphosphate formation up to twofold (P < 0·001, n = 5). The enhancement of the response occurred despite the fact that dexamethasone reduced [3H]inositol prelabelling of membrane phosphoinositides by 49·5 ± 9·9% (P < 0·05, n = 3). The present results suggest that the potential action of glucocorticoids on vascular smooth muscle contractility is, at least in part, through controlling α1-adrenoceptor-mediated second-messenger production.
Journal of Endocrinology (1992) 133, 405–411
Zhengu Liu, Violeta Stanojevic, Luke J Brindamour and Joel F Habener
Type 2 diabetes, often associated with obesity, results from a deficiency of insulin production and action manifested in increased blood levels of glucose and lipids that further promote insulin resistance and impair insulin secretion. Glucolipotoxicity caused by elevated plasma glucose and lipid levels is a major cause of impaired glucose-stimulated insulin secretion from pancreatic β-cells, due to increased oxidative stress, and insulin resistance. Glucagon-like peptide-1 (GLP1), an insulinotropic glucoincretin hormone, is known to promote β-cell survival via its actions on its G-protein-coupled receptor on β-cells. Here, we report that a nonapeptide, GLP1(28–36)amide, derived from the C-terminal domain of the insulinotropic GLP1, exerts cytoprotective actions on INS-1 β-cells and on dispersed human islet cells in vitro in conditions of glucolipotoxicity and increased oxidative stress independently of the GLP1 receptor. The nonapeptide appears to enter preferably stressed, glucolipotoxic cells compared with normal unstressed cells. It targets mitochondria and improves impaired mitochondrial membrane potential, increases cellular ATP levels, inhibits cytochrome c release, caspase activation, and apoptosis, and enhances the viability and survival of INS-1 β-cells. We propose that GLP1(28–36)amide might be useful in alleviating β-cell stress and might improve β-cell functions and survival.
J Liu, AI Kahri, P Heikkila and R Voutilainen
Adrenomedullin (ADM) is a polypeptide originally discovered in a human pheochromocytoma and is also present in normal adrenal medulla. It has been proposed that ADM could be involved in the regulation of adrenal steroidogenesis via paracrine mechanisms. Our aim was to find out if ADM gene is expressed in adrenocortical tumors and how ADM gene expression is regulated in adrenal cells. ADM mRNA was detectable by Northern blotting in most normal and hyperplastic adrenals, adenomas and carcinomas. The average concentration of ADM mRNA in the hormonally active adrenocortical adenomas was about 80% and 7% of that in normal adrenal glands and separated adrenal medulla respectively. In adrenocortical carcinomas, the ADM mRNA concentration was very variable, but on average it was about six times greater than that in normal adrenal glands. In pheochromocytomas, ADM mRNA expression was about ten times greater than that in normal adrenals and three times greater than in separated adrenal medulla. In primary cultures of normal adrenal cells, a protein kinase C inhibitor, staurosporine, reduced ADM mRNA accumulation in a dose- and time-dependent fashion (P < 0.01), whereas it simultaneously increased the expression of human cholesterol side-chain cleavage enzyme (P450 scc) gene (a key gene in steroidogenesis). In cultured Cushing's adenoma cells, adrenocorticotropin, dibutyryl cAMP ((Bu)2cAMP) and staurosporine inhibited the accumulation of ADM mRNA by 40, 50 and 70% respectively (P < 0.05), whereas the protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), increased it by 50% (P < 0.05). In primary cultures of pheochromocytoma cells, treatment with (Bu)2cAMP for 1 and 3 days increased ADM mRNA accumulation two- to threefold (P < 0.05). Our results show that ADM mRNA is present not only in adrenal medulla and pheochromocytomas, but also in adrenocortical neoplasms. Both protein kinase A- and C-dependent mechanisms regulate ADM mRNA expression in adrenocortical and pheochromocytoma cells supporting the suggested role for ADM as an autocrine or paracrine (or both) regulator of adrenal function.
T Vanttinen, J Liu, T Kuulasmaa, P Kivinen and R Voutilainen
Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.
J Liu, R Voutilainen, A I Kahri and P Heikkilä
We have analyzed the expression of the c-myc proto-oncogene in human adrenal glands in vivo and in primary cell cultures by Northern blot analysis. c-myc mRNA was consistently expressed in all human adrenals studied. Expression in adult adrenals was found to be approximately 50% of that in fetal adrenals, but much higher than that in adult liver and kidney. Adrenocorticotropin (ACTH) treatment increased c-myc mRNA accumulation dose- and time-dependently up to more than 5-fold (on average), with the maximal effect at 2 h. (Bu)2cAMP and 12-O-tetradecanoyl phorbol 13-acetate (TPA) also induced c-myc gene expression. There was no synergistic effect between the ACTH, (Bu)2cAMP and TPA treatments. The basal level of c-myc expression was reduced by the protein kinase inhibitors H-7 (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride), staurosporine and HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). H-7 totally abolished ACTH-, TPA- and (Bu)2cAMP-induced c-myc expression, while staurosporine inhibited the stimulatory effects of ACTH and TPA, and HA1004 weakly inhibited the effects of ACTH and (Bu)2cAMP. Incubation with cycloheximide or 10% fetal calf serum increased c-myc mRNA levels 3- and 4-fold respectively. Our data show that the c-myc gene is expressed abundantly in normal human adrenals, and that this expression can be regulated by multiple factors in the primary cultures.
Journal of Endocrinology (1996) 148, 523–529