Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Novel chemiluminescent immunoassay to measure plasma aldosterone and plasma active renin concentrations for the diagnosis of primary aldosteronism.

Determination of plasma aldosterone concentrations (PAC) and plasma active renin concentrations (ARC) is essential for the diagnosis of primary aldosteronism (PA). In Japan, although PAC and ARC are measured by radioimmunoassay and immunoradiometric assay, respectively, non-radioisotopic methods with better detection sensitivity, measurement accuracy, and technical simplicity are needed. We developed two-site sandwich chemiluminescent enzyme immunoassays (CLEIAs) to measure both PAC and ARC using monoclonal antibodies immobilized onto ferrite particles. The results of both assays are obtained simultaneously from a single plasma sample within 30 min using a fully automated system. The novel CLEIAs were validated using plasma samples from patients with PA (n = 52) and essential hypertension (n = 23). The PAC determined by the CLEIA was significantly correlated with that measured by liquid chromatography/mass spectrometry or conventional radioimmunoassay. The ARC determined by the CLEIA was significantly correlated with that measured by immunoradiometric assay. The limits of detection of the CLEIAs for PAC and ARC were 0.1 ng/dl and 0.04 pg/ml, respectively, which were better than those of conventional methods (PAC: 2.5 ng/dl; ARC: 5 pg/ml). The PAC and PAC/ARC ratio (ARR) were significantly higher, and the ARC significantly lower, in patients with PA than in those with essential hypertension. An ARR cut-off of 1.31 ng/dl per pg/ml showed a sensitivity of 96.2% and specificity of 78.3% for PA screening. The newly developed CLEIAs for measuring PAC and ARC could provide a clinically powerful alternative to conventional methods used for hypertension screening in clinical practice.

Paternal allelic mutation at the Kcnq1 locus reduces pancreatic ß-cell mass by epigenetic modification of Cdkn1c.

Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic ß-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic ß-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Pancreatic ß-cell failure mediated by mTORC1 hyperactivity and autophagic impairment.

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in ß-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in ß-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with ß-cell-specific deletion of Tsc2 (ßTsc2(-/-)) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on ß-cell failure. mTORC1 hyperactivation drove an early increase in ß-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older ßTsc2(-/-) mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in ß-cells of ßTsc2(-/-) mice, but mitophagy was also impaired under these circumstances. We provide evidence of ß-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to ß-cell failure.

Constitutive activation of Rac1 in pancreatic ß cells facilitates F-actin depolymerization but exerts no influence on the increase of pancreatic ß cell mass and facilitation of insulin secretion.

Insulin secretion from pancreatic ß cells has an important role in the onset of type 2 diabetes. Insulin secretion from pancreatic ß cells is regulated by pancreatic ß cell mass and their insulin secretory function. By using pancreatic ß cell-specific Rac1-knockout mice, we recently showed that Rac1 deletion, even with no reduction in pancreatic ß cell mass, inhibits F-actin depolymerization, which causes insulin secretion to decline. However, the effect of Rac1 deficiency on the growth and apoptosis of pancreatic ß cells was not clarified. Further, the effect of constitutive Rac1 activation on the secretion of insulin from pancreatic ß cells has not been studied. Here, we used pancreatic islets isolated from pancreatic ß cell-specific Rac1-knockout mice to evaluate the growth and apoptosis of pancreatic ß cells. We found that Rac1 deficiency does not influence the growth or apoptosis of pancreatic ß cells. Further, when a constitutively activated form of Rac1 (G12V) is expressed, F-actin depolymerization was increased in the pancreatic ß cell lines, which had no effect on pancreatic ß cell growth or glucose-stimulated insulin secretion. These findings indicate that excessive Rac1 expression or activation in pancreatic ß cells facilitates F-actin depolymerization, but has no effect on insulin secretion.

DPP4 inhibitor vildagliptin preserves ß-cell mass through amelioration of endoplasmic reticulum stress in C/EBPB transgenic mice.

The development of type 2 diabetes is accompanied by a progressive decline in ß-cell mass and function. Vildagliptin, a dipeptidyl peptidase 4 inhibitor, is representative of a new class of antidiabetic agents that act through increasing the expression of glucagon-like peptide-1. The protective effect of this agent on ß cells was studied in diabetic mice. Diabetic pancreatic ß cell-specific C/EBPB transgenic (TG) mice exhibit decreased ß-cell mass associated with increased apoptosis, decreased proliferation, and aggravated endoplasmic reticulum (ER) stress. Vildagliptin was orally administered to the TG mice for a period of 24 weeks, and the protective effects of this agent on ß cells were examined, along with the potential molecular mechanism of protection. Vildagliptin ameliorated hyperglycemia in TG mice by increasing the serum concentration of insulin and decreasing the serum concentration of glucagon. This agent also markedly increased ß-cell mass, improved aggravated ER stress, and restored attenuated insulin/IGF1 signaling. A decrease in pancreatic and duodenal homeobox 1 expression was also observed in ß cells isolated from our mouse model, but this was also restored by vildagliptin treatment. The expression of C/EBPB protein, but not mRNA, was unexpectedly downregulated in vildagliptin-treated TG mice and in exenatide-treated MIN6 cells. Activation of the GLP1 pathway induced proteasome-dependent C/EBPB degradation in ß cells as the proteasome inhibitor MG132 restored the downregulation of C/EBPB protein by exenatide. Vildagliptin elicits protective effects on pancreatic ß cells, possibly through C/EBPB degradation, and has potential for preventing the progression of type 2 diabetes.