Age-related morphologic development of human adrenal zona reticularis (ZR) has not been well examined. Therefore, in this study, 44 human young adrenal autopsy specimens retrieved from large archival files (n=252) were examined for immunohistochemical and morphometric analyses. Results demonstrated that ZR became discernible around 4 years of age, and both thickness and ratio per total cortex of ZR increased in an age-dependent fashion thereafter, although there was no significant increment in total thickness of developing adrenal cortex. We further evaluated immunoreactivity of both KI67 and BCL2 in order to clarify the equilibrium between cell proliferation and apoptosis in the homeostasis of developing human adrenals. Results demonstrated that proliferative adrenocortical cells were predominantly detected in the zona glomerulosa and partly in outer zona fasciculata (ZF) before 4 years of age and in ZR after 4 years of age, but the number of these cells markedly decreased around 20 years of age. The number of BCL2-positive cells increased in ZR and decreased in ZF during development. Adrenal androgen synthesizing type 5 17β-hydroxysteroid dehydrogenase (HSD17B5 or AKR1C3 as listed in the Hugo Database) was almost confined to ZR of human adrenals throughout development. HSD17B5 immunoreactivity in ZR became discernible and increased from around 9 years of age. Results of our present study support the theory of age-dependent adrenocortical cell migration and also indicated that ZR development is not only associated with adrenarche, but may play important roles in an initiation of puberty.

Adrenomedullin 2/intermedin (AM2/IMD) is a new member of calcitonin/calcitonin gene-related peptide family. AM is expressed in various tumors including adrenocortical tumors and modulates tumor growth. The AM2/IMD expression has not been studied, however, in adrenal tumors. The expression of AM2/IMD and AM was therefore studied in human adrenal tumors and attached non-neoplastic adrenal tissues by immunocytochemistry (ICC). Immunoreactive (IR)–AM2/IMD was measured by RIA. Furthermore, the expression of AM2/IMD and its receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity-modifying proteins (RAMPs) 1, 2, and 3 mRNA in these tissues was studied by reverse transcription PCR (RT-PCR). ICC showed that AM2/IMD and AM immunoreactivities were localized in adrenocortical tumors and pheochromocytomas. AM2/IMD and AM immunoreactivities were detected in medulla of attached non-neoplastic tissues, while the degree of immunoreactivity for AM2/IMD and AM in cortices of attached adrenals was relatively weak or undetectable. RIA detected IR-AM2/IMD in adrenal tumors (0.414±0.12 to 0.786±0.27 pmol/g wet weight, mean±s.e.m.) and attached adrenal tissues (0.397±0.052 pmol/g wet weight). Reverse-phase high-performance liquid chromatography showed one broad peak eluted in the similar position to synthetic AM2/IMD with several minor peaks. RT-PCR showed expression of AM2/IMD, CRLR, and RAMP1, RAMP2, and RAMP3 mRNA in tissues of adrenal tumors and attached adrenal glands. In conclusion, AM2/IMD is expressed in human adrenal tumors and attached non-neoplastic adrenal tissues and may play (patho-)physiological roles in normal and neoplastic adrenals as an autocrine/paracrine regulator.

Fasudil, a Rho-kinase inhibitor, may improve insulin signaling. However, its long-term effect on metabolic abnormalities and its preventive effect on diabetic nephropathy are still unknown. We assessed these effects of fasudil in insulin-resistant diabetic rats, comparing them with those of an angiotensin II receptor blocker, olmesartan. Male Otsuka Long–Evans Tokushima fatty (OLETF) and Long–Evans Tokushima Otsuka, non-diabetic control, rats at 15 weeks of age were used. OLETF rats were randomized to receive a low or a high dose of fasudil or olmesartan for 25 weeks. To examine the therapeutic effects after the development of diabetes, OLETF rats at 30 weeks of age were given fasudil for 10 weeks. Administration of high-dose fasudil completely suppressed the development of diabetes, obesity, and dyslipidemia and increased serum adiponectin levels in OLETF rats. High-dose olmesartan also decreased hemoglobin A1c and increased serum adiponectin. There was a significant correlation between hemoglobin A1c and serum adiponectin or free fatty acid levels. The treatment with high-dose fasudil ameliorated proteinuria, glomerulosclerosis, renal interstitial fibrosis, and macrophage infiltration in OLETF rats. Olmesartan, even at the low dose, suppressed renal complications. The treatment with fasudil after the development of diabetes improved the metabolic abnormalities in OLETF rats, but could not suppress the progression of nephropathy. We conclude that the long-term treatment with fasudil prevents the development of diabetes, at least in part, by improving adipocyte differentiation in insulin-resistant diabetic rats. Early use of fasudil may prevent diabetic nephropathy.

Glucose-dependent insulinotropic polypeptide (GIP), a gut hormone secreted from intestinal K-cells, potentiates insulin secretion. Both K-cells and pancreatic β-cells are glucose-responsive and equipped with a similar glucose-sensing apparatus that includes glucokinase and an ATP-sensitive K⁺ (K_ATP) channel comprising KIR6.2 and sulfonylurea receptor 1. In absorptive epithelial cells and enteroendocrine cells, sodium glucose co-transporter 1 (SGLT1) is also known to play an important role in glucose absorption and glucose-induced incretin secretion. However, the glucose-sensing mechanism in K-cells is not fully understood. In this study, we examined the involvement of SGLT1 (SLC5A1) and the K_ATP channels in glucose sensing in GIP secretion in both normal and streptozotocin-induced diabetic mice. Glimepiride, a sulfonylurea, did not induce GIP secretion and pretreatment with diazoxide, a K_ATP channel activator, did not affect glucose-induced GIP secretion in the normal state. In mice lacking K_ATP channels (Kir6.2 ^−/− mice), glucose-induced GIP secretion was enhanced compared with control (Kir6.2 ^{+ / +} ) mice, but was completely blocked by the SGLT1 inhibitor phlorizin. In Kir6.2 ^−/− mice, intestinal glucose absorption through SGLT1 was enhanced compared with that in Kir6.2 ^{+ / +} mice. On the other hand, glucose-induced GIP secretion was enhanced in the diabetic state in Kir6.2 ^{+ / +} mice. This GIP secretion was partially blocked by phlorizin, but was completely blocked by pretreatment with diazoxide in addition to phlorizin administration. These results demonstrate that glucose-induced GIP secretion depends primarily on SGLT1 in the normal state, whereas the K_ATP channel as well as SGLT1 is involved in GIP secretion in the diabetic state in vivo.