Keisuke Ina

The endocrine pancreas of spontaneously diabetic db db mice: microangiopathy as revealed by transmission electron microscopy

Abnormalities in ultrastructures of islet capillaries were detected in db/db mice, with the visual inspection and morphometry of electron micrographs. The observed changes are: (1) capillary scarcity; (2) increase in the mean and diversity of capillary size; (3) pericapillary edema and fibrosis; (4) hypertrophy of the pericyte and abundance therein of actin-like microfilaments; and (5) luminal irregularity. Changes (2), (3) and (4) are conceived to indicate hyperperfusion, capillary hypertension and secondary vascular response. In particular, such pericyte changes were found to be shared by other organs whose capillaries are susceptible to diabetic complications.

Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy

 The developmental mechanism of tubulointerstitial fibrosis in diabetic nephropathy (DN) has not been elucidated. Tubulointerstitial fibrosis, as well as glomerulosclerosis, occurs in DN. Myofibroblasts which overproduce extracellular matrix are present in the renal interstitium in diabetics, although they are almost never seen in normal kidneys. The myofibroblasts appear to originate from interstitial fibroblasts. In addition, transforming growth factor-β1 (TGF-β1), which can evoke myofibroblast transformation, is detected in interstitial cells in the diabetic kidney, but not in the normal kidney. Taken together, these findings led us to speculate that TGF-β1 induces the transformation of interstitial fibroblasts into myofibroblasts, followed by tubulointerstitial fibrosis. Based on this speculation, we discuss the developmental mechanism of tubulointerstitial fibrosis in this review.

Vascular cell adhesion molecule-l expression in the renal interstitium of diabetic KKAy mice

To investigate the mechanism of interstitial inflammation in diabetic nephropathy, we used spontaneously diabetic KKAy mice. Twelve KKAy mice were divided into two groups; six mice were fed standard mouse chow ad libitum and six mice were placed on a diet (i.e. they received the same amount of chow as six control C57BL mice). Diabetic KKAy mice developed hypercholesterolemia and albuminuria. Animals were killed at 16 weeks of age and renal tissues were immunostained for vascular cell adhesion molecule-1 (VCAM-1). In diabetic KKAy mice, the renal interstitium was infiltrated by monocytes, lymphocytes, plasma cells, and other cells. The walls of venules near the infiltrating cells were more intensely stained for VCAM-1 when compared with other sites. In contrast, the VCAM-1 staining of arterioles and peritubular capillaries was not significantly increased. There was weak VCAM-1 staining of the infiltrating cells, including lymphocytes, monocytes, and other cells. Electron microscopy demonstrated immunolabeling for VCAM-1 on the cell surface and in the cytoplasm of both infiltrating cells and vascular endothelial cells. In KKAy mice placed on a diet, there was less staining for VCAM-1 and cellular infiltration was also decreased. Thus, increased expression of VCAM-1 by the endothelial cells of venules and VCAM-1 expression by infiltrating cells were demonstrated in the interstitium of kidneys from diabetic mice. These results suggest that increased expression of VCAM-1 by endothelial cells and infiltrating cells contributes to interstitial inflammation in diabetic nephropathy.

Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Diabetogenic effects of FK506 on renal subcapsular islet isografts in rat

Previously we demonstrated prevention of immune rejection in rat islet allografts by continuous subcutaneous (s.c.) administration of FK506 and also showed that FK506 might have diabetogenic effects (Ryu and Yasunami (1991) Transplantation, 52, 599-605). The purpose of the present study was to characterize further diabetogenic effects of FK506 on renal subcapsular islet isografts in rat. Continuous s.c. administration of FK506 (3 mg/kg/day) for 35 days produced glucose intolerance in the recipients as demonstrated by intravenous (i.v.) glucose tolerance test at the end (35 days) and after discontinuation (90 days) of FK506 administration. Morphologically, beta cells in the grafts of FK506-treated group were degranulated at 35 and 120 days after transplantation. Electron microscopically, degranulation, marked swelling of rough endoplasmic reticulum, Golgi apparatus and mitochondria were detected in beta cells of the grafts treated with FK506 at 35 days, and at 120 days there was moderate structural recovery in the organella. These findings clearly demonstrate that FK506 has diabetogenic effects on renal subcapsular islet isografts in rat and also suggests potential reversibility of damages by FK506 in beta cells of the grafts.

A Clinical Approach to Brown Adipose Tissue in the Para-Aortic Area of the Human Thorax

Background Human thoracic brown adipose tissue (BAT), composed of several subdivisions, is a well-known target organ of many clinical studies; however, the functional contribution of each part of human thoracic BAT remains unknown. The present study analyzed the significance of each part of human thoracic BAT in the association between regional distribution, cellularity, and factors involved in the functional regulation of thoracic BAT. Methods We analyzed 1550 healthy adults who underwent medical check-ups by positron-emission tomography and computed tomography (PET–CT) imaging, 8 cadavers, and 78 autopsy cases in an observational study. We first characterized the difference between the mediastinum and the supraclavicular areas using counts of BAT detection and conditions based on PET–CT outcomes. The measurable important area was then subjected to systematic anatomical and immunohistochemical analyses using anti-uncoupling protein 1 (UCP1) antibody to characterize the cellularity in association with age and sex. Results In PET–CT scanning, the main site of thoracic BAT was the mediastinum rather than the supraclavicular area (P < 0.05). Systemic macroanatomy revealed that the thumb-sized BAT in the posterior mediastinal descending para-aortic area (paBAT) had feeding vessels from the posterior intercostal arteries and veins and sympathetic/parasympathetic innervation from trunks of the sympathetic and vagus nerves, respectively. Immunohistochemical analysis indicated that the paBAT exhibited immunoreactivity for tyrosine hydroxylase and vesicular acetylcholine transporter located in the pericellular nervous fibers and intracellular UCP1. The brown adipose cells of paBAT showed age-dependent decreases in UCP1 expression (P < 0.05), accompanied by a significant increase in vacuole formation, indicating fat accumulation (P < 0.05), from 10 to 37 years of age (P < 0.01). Conclusions paBAT may be one of the essential sites for clinical application in BAT study because of its visible anatomy with feeding vessels and sympathetic/parasympathetic innervation functionally affected by outer condition and senescence.

Distribution of histaminergic neuronal cluster in the rat and mouse hypothalamus

Histidine decarboxylase (HDC) catalyzes the biosynthesis of histamine from L-histidine and is expressed throughout the mammalian nervous system by histaminergic neurons. Histaminergic neurons arise in the posterior mesencephalon during the early embryonic period and gradually develop into two histaminergic substreams around the lateral area of the posterior hypothalamus and the more anterior peri-cerebral aqueduct area before finally forming an adult-like pattern comprising five neuronal clusters, E1, E2, E3, E4, and E5, at the postnatal stage. This distribution of histaminergic neuronal clusters in the rat hypothalamus appears to be a consequence of neuronal development and reflects the functional differentiation within each neuronal cluster. However, the close linkage between the locations of histaminergic neuronal clusters and their physiological functions has yet to be fully elucidated because of the sparse information regarding the location and orientation of each histaminergic neuronal clusters in the hypothalamus of rats and mice. To clarify the distribution of the five-histaminergic neuronal clusters more clearly, we performed an immunohistochemical study using the anti-HDC antibody on serial sections of the rat hypothalamus according to the brain maps of rat and mouse. Our results confirmed that the HDC-immunoreactive (HDCi) neuronal clusters in the hypothalamus of rats and mice are observed in the ventrolateral part of the most posterior hypothalamus (E1), ventrolateral part of the posterior hypothalamus (E2), ventromedial part from the medial to the posterior hypothalamus (E3), periventricular part from the anterior to the medial hypothalamus (E4), and diffusely extended part of the more dorsal and almost entire hypothalamus (E5). The stereological estimation of the total number of HDCi neurons of each clusters revealed the larger amount of the rat than the mouse. The characterization of histaminergic neuronal clusters in the hypothalamus of rats and mice may provide useful information for further investigations.

Transplantation of normal islets into the portal vein of Otsuka Long Evans Tokushima Fatty rats prevents diabetic progression.

To investigate the long-term effects of normal pancreatic islet transplantation on progression of obese type 2 diabetes mellitus (DM), 1500 normal islets (per rat) from Wistar King A rats at 8 weeks of age were transplanted into the liver through the portal vein of Otsuka Long Evans Tokushima Fatty (OLETF) rats, an animal model of obese type 2 DM, at 12 weeks of age. Body weight in the transplanted OLETF (IT) rats 8 and 28 weeks after islet transplantation did not differ from that in the corresponding sham-operated (SO) rats, but was greater than that in lean littermates (LETO rats; P < 0.05 for each group). In the early phase, 8 weeks after transplantation, rats in both IT and SO groups were normoglycemic, but hyperinsulinemic (P < 0.05 for each compared with LETO rats), probably resulting from increased body weight. In the late phase, 28 weeks after transplantation, hyperglycemia in the IT group was greatly attenuated compared with the SO group (P < 0.05), but hyperinsulinemia remained in both the IT and the SO groups compared with that in the LETO group (P < 0.05 for each). Immunohistochemical studies demonstrated that hypertrophic and fibrotic changes in pancreatic islets, together with mesangial proliferation of the glomerular matrix, an indicator for diabetic nephropathy, were attenuated predominantly in the IT group at the late phase after transplantation compared with those in the corresponding phase of the SO group. Islet transplantation into the liver of OLETF rats thus prevented further progression of obese type 2 DM. A possible mechanism is that islet transplantation may prevent development of hyperglycemia by improving abnormal hepatic glucose metabolism and consequently insulin resistance, which may lead to blockade of a vicious cycle between advancing damage to pancreatic islet cells and increased demand for insulin secretion, thus sparing original pancreatic cells from exhaustion induced by increased demand for insulin secretion.

Contraction of tubulointerstitial fibrosis tissue in diabetic nephropathy, as demonstrated in an in vitro fibrosis model

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-β1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-β1 plus Y27632, a Rho kinase inhibitor. TGF-β1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-β1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-β1 group than in the control group. Collectively, these results indicate that TGF-β1-induced FPCL contraction is attributable to actin–myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.

Ultrastructural and immunohistochemical similarities between glomerular arteriolar and mesangial lesions in experimental diabetes

It has thus far been speculated that plasma proteins have leaked and accumulated in diabetic expanded mesangium. We have previously shown that glomerular arteriolar hyalinosis has frequently occurred and has consisted of basement membrane-like extracellular matrix in alloxan diabetic rats. In the present study, the two diabetic glomerular lesions, arteriolar hyaline nodules and increased mesangial matrix, were comparatively evaluated by means of electron microscopy and immunohistochemistry. It was revealed that both of these lesions contained, in common, finely particulate extracellular matrix mingled with cytoplasmic fragments, and exhibited intense immunoreactions for IgG and IgM. It is indicated that both types of diabetic glomerular lesions are essentially of the same nature.