Durability of protective effect of dulaglutide on pancreatic ß-cells in diabetic mice: GLP-1 receptor expression is not reduced despite long-term dulaglutide exposure.

AIMS: It is well-known that chronic exposure to large amounts of ligand leads to downregulation of its receptor. It is not known, however, whether a GLP-1R agonist downregulates its receptor. For this reason, our study examined whether GLP-1R expression is reduced after long-term exposure to dulaglutide (Dula) in non-diabetic and diabetic mice. METHODS: Seven-week-old male db/db and db/m mice were given either Dula (0.6mg/kg×2/week) or a control vehicle (CTL) for 17 weeks. Various metabolic parameters, such as glucose-stimulated insulin secretion (GSIS), insulin and TG content in islets, were evaluated after the intervention. ß-cell-related gene expression was also analyzed by real-time RT-PCR. RESULTS: In db/m mice, GLP-1R expression in ß-cells did not decrease, not even after long-term administration of Dula, compared with control mice, while GLP-1R expression in 24-week-old db/db mice treated with Dula was augmented, rather than downregulated, compared with 24-week-old CTL db/db mice. This was probably due to improved glycaemic control. In db/db mice treated with Dula, food intake and blood glucose levels were significantly decreased up to 24 weeks of age compared with CTL db/db mice, and their expression levels of various ß-cell-related genes, insulin content and GSIS were also enhanced. In contrast, oxidative and endoplasmic reticulum stress, inflammation, fibrosis and apoptosis were suppressed with Dula treatment. CONCLUSION: Dula exerts beneficial effects on glycaemic control and has long-lasting protective effects on pancreatic ß-cells. GLP-1R expression levels were not reduced at all in non-diabetic as well as diabetic mice despite long-term dulaglutide exposure.

Gene expression profile in rat pancreatic islet and RINm5F cells.

To clarify tissue-specificity of pancreatic beta cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40,000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40,710 3'-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3'- and 5'-ESTs revealed 10,406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon-intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.

Overt congestive heart failure with mitral and aortic regurgitation due to antiphospholipid syndrome in a patient with systemic lupus erythematosus.

A 51-year-old woman with overt congestive heart failure with pleural and pericardial effusion was treated with furosemide and nifedipine, leading to improvement in her condition and a decrease in effusions. An echocardiography demonstrated mitral and aortic regurgitation with mitral valve prolapse, which caused the congestive heart failure. Since leukocytopenia and lymphocytopenia with arthralgia could be observed, serological investigations were performed. She was diagnosed as having systemic lupus erythematosus (SLE) with antiphospholipid syndrome, and started on a treatment of prednisolone and aspirin. Based on the treatment, the pleural and pericardial effusion went into complete remission, indicating that the serositis related to SLE had overlapped the heart failure. Since there was no evidence of any other diseases that could be responsible for the valvular lesions, we concluded that they were due to antiphospholipid syndrome. The administration of prednisolone had no significant effect on valvular morphology or function as demonstrated by echocardiography. When patients with valvular disease are seen, a valvulopathy related to antiphospholipid syndrome should be considered as part of the differential diagnosis.

Role of rat adrenal antioxidant defense systems in the aldosterone turn-off phenomenon.

The mechanism(s) of the "aldosterone turn-off phenomenon", hypoaldosteronemia following chronic ACTH administration, remains unclear. Our previous observation that antioxidants prevented turn-off prompted us to evaluate the chronic effect of ACTH on the enzymatic antioxidant system as well as P450aldo activity and expression of CYP11B2 in adrenal zona glomerulosa. Male Wistar rats were administered ACTH-Z for 5 days with or without antioxidants, vitamin E or DMSO. Adrenal capsules were prepared for P450aldo activity measurement and mRNA content determination by competitive RT-PCR, and immunoreactivity of Mn-SOD in whole adrenals was evaluated. ACTH decreased the P450aldo activity and mRNA level of CYP11B2 in adrenal capsules, while co-administration of vitamin E or DMSO partially blocked this inhibition. ACTH increased Mn-SOD mRNA and immunoreactivity but decreased GPx mRNA. These results suggest that prolonged ACTH treatment increases oxidative stress in the zona glomerulosa and an imbalance in the ratio of Mn-SOD to GPx, possibly via corticosterone overproduction in the zona fasciculata, resulting in the downregulation of CYP11B2. Vitamin E and DMSO might thus protect CYP11B2 expression through their antioxidant actions.

Recurrence of giant adrenocortical carcinoma in the contralateral adrenal gland 6 years after surgery: report of a case.

We report herein the case of a patient in whom a giant adrenocortical carcinoma was found to have recurred in the contralateral adrenal gland and intrapelvic cavity 6 years after his initial operation. A 52-year-old man had consulted our hospital complaining of right upper abdominal pain and weight loss, and was subsequently diagnosed as having a giant adrenal tumor by computed tomography scans and echography. A laparotomy was performed and the tumor, located in the right retroperitoneal cavity and infiltrating the liver and right kidney, was surgically removed. The lesion, 29 x 19 x 10 cm in size and 4700 g in weight, was histopathologically diagnosed as an adrenocortical carcinoma. Adjuvant chemotherapy with mitotane was given for 3 months and his postoperative course was uneventful until a recurrence in the contralateral adrenal gland and peritoneal cavity was found 6 years later. The second resection was successful, and he is currently alive with no further sign of recurrence 8 years after his first operation. We report this unusual case as it provides much useful information on the biological features of adrenocortical carcinomas and the state of tumor dormancy.

High molecular weight corticotropin measured with immunoradiometric assay in a patient with asymptomatic pituitary corticotropinoma.

A 57-yr-old female with corticotropinoma showing no Cushingoid stigmata is reported. Basal plasma levels of ACTH measured with immunoradiometric assay and beta-endorphin were high, 12.6-15.9 pmol/l and 3.5 pmol/l, respectively. Plasma cortisol level and urinary free cortisol excretion were normal, 303-359 nmol/l and 171-226 nmol/day, respectively. Plasma ACTH markedly increased to 70.5 pmol/l with intravenous administration of 100 microg CRH. Diurnal rhythm of plasma ACTH was seen, but its level in the night was still high. Plasma ACTH suppression with dexamethasone was insufficient. CRH stimulation after dexamethasone suppression increased plasma ACTH level from 4.4 to 13.7 pmol/l. Intravenous administration of 4 microg desmopressin increased plasma ACTH from 15.6 to 19.6 pmol/l. Oral administration of 16 mg lepramide insufficiently decreased plasma ACTH from 7.3 to 5.3 pmol/l. However, plasma cortisol responses in these conditions were normal. Postoperative pathological study revealed subtype 1 corticotropinoma immunohistochemically and electron-microscopically. Postoperative basal plasma ACTH decreased to 3.9 pmol/l, although plasma cortisol did not change. Diurnal rhythm and dexamethasone suppressibility of plasma ACTH became normal. Plasma sample was chromatographed on a Sephadex G-75 column. The elution profile showed two peaks of ACTH, one of which was compatible with 1-39 ACTH and another with higher molecular weight ACTH which was probably secreted from corticotropinoma. Anomaly in processing of proopiomelanocortin was suspected.

Mutants of 11beta-hydroxysteroid dehydrogenase (11-HSD2) with partial activity: improved correlations between genotype and biochemical phenotype in apparent mineralocorticoid excess.

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.

Marked increase in plasma ACTH with tumor reduction after chemotherapy in ectopic ACTH syndrome.

We report on a case of rapid and marked hormone release as a result of rapid tumor reduction due to chemotherapy in a 36-year-old woman with ectopic ACTH syndrome due to small cell lung cancer. Treatment of the cancer with cisplatin and etoposide resulted in an 80% reduction in tumor size on computed tomographic scan within two weeks. Concurrently, plasma ACTH exhibited an unexpected and astonishing increase from 373 pg/ml before treatment to more than 1200 pg/ml. There were no biochemical characteristics observed in tumor lysis syndrome of solid tumors such as azotemia, increased LDH and hyperkalemia. The present case indicates that anticancer chemotherapy instituted in patients with ectopic ACTH syndrome could result in an acute increase of plasma ACTH and exacerbation of hypercortisolism, similar to tumor lysis syndrome, which is a potentially fatal complication following anti-cancer chemotherapy.

Coexpression of mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenase 2 in human gastric mucosa.

The role of mineralocorticoids in human gastrointestinal tract is well established. In the stomach, aldosterone is thought to regulate electrolyte transport associated with gastric acid secretion. In mineralocorticoid target organs, the action of the glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) facilitates aldosterone binding to a nonselective mineralocorticoid receptor (MR) in the face of high levels of circulating glucocorticoids. In the present study, we examined 25 specimens of human stomach for the presence of MR and 11beta-HSD2 using a [3H]aldosterone binding assay, Northern blot analysis, RT-PCR, and immunohistochemistry. Specific [3H]aldosterone binding sites were detected in gastric fundic mucosa, but not in the antrum. In fundic mucosa the Kd was 0.72+/-0.05 nmol/L (mean +/- SE), and Bmax was 6.0+/-1.4 fmol per milligram of protein. Northern blot analysis demonstrated a faint band for MR mRNA at 6.0 kb, although message for 11beta-HSD2 was undetectable. However, RT-PCR demonstrated specific PCR products for both MR and 11beta-HSD2. Immunohistochemistry demonstrated the colocalization of MR and 11beta-HSD2 only in parietal cells. MR-positive cells were further characterized by electron microscopy, confirming the identity of parietal cells. This study shows that parietal cells contain both MR and 11beta-HSD2, suggesting that the human stomach is a novel target organ for mineralocorticoids. Aldosterone may, therefore, regulate biological functions of parietal cells including gastric acid secretion.

Expression of human kidney 11beta-hydroxysteroid dehydrogenase (11-HSD2) in bacteria.

The kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) protects the mineralocorticoid receptor from spurious activation by glucocorticoids. To explore structure-function relationships, human 11-HSD2 cDNA was subcloned into the bacterial expression vector, pET25b. E. coli transformed with wild-type cDNA produced active enzyme that retained biochemical characteristics of the native protein. The addition of 6 histidine residues to the C-terminus of the wild-type enzyme (11-HSD2/His) increased activity 2-fold. Whereas wild-type activity was almost completely sedimented following 100,000g centrifugation, 10-30% of total activity of 11-HSD2/His remained in the supernatant. The 11-HSD2 isozyme normally contains three N-terminal hydrophobic domains. Mutant 11-HSD2/His possessing a single hydrophobic domain retained partial activity, but elimination of all domains inactivated the enzyme. Thus, the N-terminal hydrophobic domains are essential for complete activity of 11-HSD2 but association with an intact cell membrane is not.

DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

In vivo and in vitro effects of AVP and V1a receptor antagonist on Cushing's syndrome due to ACTH-independent bilateral macronodular adrenocortical hyperplasia.

We examined the possibility that AVP and V1a receptors were involved in regulating cortisol production in a 49 year old man with ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH), and investigated the effects of a V1a receptor antagonist. An i.v. injection of a small dose (0.1 or 0.3 U) of AVP, insulin-induced hypoglycaemia, upright posture tests, and oral administration of a V1a receptor antagonist (OPC-21268; 300 mg), and its repeated administration at a dose of 600 mg/day for 8 days were performed. An in vitro study of dispersed cells obtained from resected AIMAH tissue was also conducted. Plasma ACTH, AVP and cortisol levels and 24-h urinary free cortisol excretion were measured in the in vivo studies and cortisol concentrations in incubation media in the in vitro study. Injection of small doses of AVP stimulated cortisol secretion without any elevation of plasma ACTH. Insulin-induced hypoglycaemia caused a rise in plasma AVP followed by an increase in plasma cortisol. Although plasma cortisol levels were not affected by single or repeated administrations of OPC-21268, 24-h urinary free cortisol excretion was significantly decreased by the repeated treatment. In the in vitro study, more cortisol was stimulated by AVP from adrenal cells of the AIMAH tissue than from those of a normal adrenal gland, and this secretion was completely suppressed by OPC-21268. These results suggested that hypersensitivity to AVP may have contributed to overproduction of cortisol in this case of ACTH-independent bilateral macronodular adrenocortical hyperplasia, and may have contributed to its pathogenesis.

11 beta-Hydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess.

Whereas aldosterone is normally a much stronger mineralocorticoid than cortisol in vivo, mineralocorticoid receptors have identical in vitro affinities for these hormones. The in vivo specificity of the receptors is, at least in part, the result of activity of 11-HSD, an enzyme located in most mineralocorticoid target tissues that converts cortisol to cortisone. Cortisone is not a ligand for the receptor, whereas aldosterone is not a substrate of the enzyme. The syndrome of AME is a rare form of juvenile hypertension in which 11-HSD is defective. This deficiency allows mineralocorticoid receptors to be occupied by cortisol, leading to hypertension, because plasma concentrations of cortisol are much higher than those of aldosterone. Licorice, which contains 11-HSD inhibitors, causes a similar syndrome. There are two known isozymes of 11-HSD. The liver or type I isozyme is expressed at high levels in the liver, has a relatively low affinity for steroids (micromolar Km), catalyzes both dehydrogenation and the reverse reductase reaction, and utilizes NADP+ or NADPH as cofactors. The kidney or type 2 isozyme is expressed at high levels in the kidney and placenta, has a high affinity (nanomolar Km) for steroids, catalyzes only dehydrogenation, and utilizes NAD+ as a cofactor. Mutations in the HSD11B2 (HSD11K) gene encoding the kidney isozyme of 11-HSD have been detected in all kindreds with AME studied thus far. This gene represents a candidate locus for the common, "essential" form of hypertension.

11 beta-Hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal tubules and cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine. Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may results in hypokalemia, suppressed plasma renin activity, and hypertension. The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. Because mineralocorticoid receptors themselves have similar affinities for cortisol and aldosterone, it is hypothesized that the deficiency allows these receptors to be occupied by cortisol, which normally circulates at levels far higher than those of aldosterone. We cloned cDNA and genes encoding two isozymes of 11 beta-HSD. The liver (L) or type 1 isozyme has relatively low affinity for steroids, is expressed at high levels in the liver but poorly in the kidney, and is not defective in AME. The kidney (K) or type 2 isozyme has high steroid affinity and is expressed at high levels in the kidney and placenta. Mutations in the gene for the latter isozyme have been detected in all kindreds with AME. Moreover, the in vitro enzymatic activity conferred by each mutation is strongly correlated with the ratio of cortisol to cortisone metabolites in the urine [tetrahydrocortisone (THF) +allo-THF]/THE. This suggests that the biochemical phenotype of AME is largely determined by genotype.

Molecular analysis of 11 beta-hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11 beta-hydroxysteroid dehydrogenase (11-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. The deficiency allows mineralocorticoid receptors to be occupied by cortisol, because these receptors themselves have similar affinities for cortisol and aldosterone. There are two isozymes of 11-HSD, a liver (L) or type 1 isozyme with a relatively low affinity for steroids, and a kidney (K) or type 2 isozyme with high steroid affinity. Mutations in the gene for the kidney isozyme of 11-HSD have been detected in all kindreds with AME. We expressed enzymes carrying all known missense mutations in cultured cells and determined their activity. For each patient with AME, we compared the enzymatic activity predicted by the genotype with the ratio of cortisol to cortisone metabolites in the urine, (THF + aTHF)/THE. These were strongly correlated, suggesting that the biochemical phenotype of AME is largely determined by genotype. The K isozyme of 11-HSD is also expressed in high levels in the placenta, where its function is unclear. AME patients often have low birth weight. By analogy with AME, low placental 11-HSD K activity in humans might be a risk factor for low birth weight and subsequent hypertension. However, we found that there was no significant correlation between 11-HSD activity, mRNA levels, and either fetal or placental weight.

ACTH-induced cortisol secretion is mediated by cAMP and PKC in various adrenocortical adenomas.

We examined the role of PKC in cortisol secretion from adrenocortical adenomas. Isolated cells were prepared from aldosterone producing adenoma (APA, n=5), APA complicated with pre-clinical Cushing's syndrome (APA+PC, n=1), PC (n=2), and cortisol producing adenoma (CPA, n=5). They were stimulated with 100 nM ACTH, 1 microM forskolin (FS), 1 microM tetradecanoyl phorbol 13-acetate (TPA), and 100 nM angiotensin II (AngII). ACTH was most potent to secret cortisol. FS also stimulated cortisol secretion, but did less-potently. TPA and AngII also stimulated cortisol secretion significantly in cells from CPA. Furthermore, ACTH- and TPA-induced PKCalpha and beta translocations from cytosol to membrane were observed in adenoma cells from APA+PC, PC, and CPA. In conclusion, it was suggested that ACTH-induced cortisol secretion may be mediated by both PKC and protein kinase A in adrenocortical adenomas, and that PKC-mediated signal transduction may be involved in ACTH-induced cortisol secretion in CPA.

Familial isolated hypoparathyroidism: case report with serum PTHrP examination.

We report a family, with isolated hypoparathyroidism. The proband was a 24-year-old woman, who presented with paresthesia of both hands. She had a mild degree of extrapyramidal signs, such as rigidity and decrease in arm swinging. Laboratory examinations revealed low PTH levels, mild hypocalcemia and hyperphosphatemia in the proband, her father, a younger brother and a younger sister, whereas her mother had normal serum calcium, phosphorus and PTH levels. These results indicate that four members of the family were affected, suggesting autosomal dominant inheritance. Brain CT revealed calcification of basal ganglia in the proband, her father and a younger sister, but not in her younger brother. Serum PTH-related protein (PTHrP) levels were examined, and found to be slightly high only in the father of the proband.