Efficacy of Dietary Lipid Control in Healing High-Fat and High-Cholesterol Diet-Induced Fibrotic Steatohepatitis in Rats

Nonalcoholic steatohepatitis is related to lifestyle, particularly to dietary habits. We developed diet-induced fibrotic steatohepatitis model stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats showing steatosis, hepatic inflammation, and severe fibrosis induced by high-fat and -cholesterol (HFC) diet feeding. We aimed to clarify the efficacy of dietary intervention on the disease before and after the appearance of fibrosis. Male SHRSP5/Dmcr rats were divided into 9 groups; of these, 6 groups were fed control or HFC diet for several weeks and the remaining 3 groups represented the dietary intervention groups, which were fed the control diet after HFC diet feeding for 2 (before the appearance of fibrosis) or 8 (after the appearance of fibrosis) weeks. Dietary intervention before the appearance of fibrosis significantly improved the steatosis and reset the increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum total cholesterol (TC) levels. However, dietary intervention after the appearance of fibrosis was unable to reset the levels of hepatic TC, serum ALT, and fibrogenesis-related markers and had only a minor influence on hepatic fibrosis, although it reset the increased expression of transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). It was noted that dietary intervention improved the increased AST levels; however, aggregated CD68-positive cells were still observed around the fibrosis area, which may be related to the findings of inflammatory cytokine mRNAs. Taken together, dietary intervention for fibrotic steatohepatitis improved steatosis, although it could not completely improve fibrosis.     

Role of IGFBP7 in Diabetic Nephropathy: TGF-β1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-β1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-β1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-β1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-β1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = −0.376, p = 0.01), urinary β₂-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-β1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-β1-induced tubular injury in DN.     

Human Leukocyte Antigen and Systemic Sclerosis in Japanese: The Sign of the Four Independent Protective Alleles,DRB1*13:02, DRB1*14:06, DQB1*03:01, and DPB1*02:01

ObjectiveSeveral studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic sclerosis (SSc) have been reported. Anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are found in SSc patients. Here, we sought to identify HLA alleles associated with SSc in Japanese, and explored their associations with SSc phenotypes including the presence of autoantibodies.MethodsAssociations of HLA-DRB1, DQB1, and DPB1 were analyzed in 463 Japanese SSc patients and 413 controls.ResultsWe found that DRB1*13:02 (P = 0.0011, Pc = 0.0319, odds ratio [OR] 0.46, 95% confidence interval [CI] 0.29–0.73), DRB1*14:06 (P = 6.60X10^-5, Pc = 0.0020, OR 0.05, 95%CI 0.01–0.41), DQB1*03:01 (P = 0.0009, Pc = 0.0150, OR 0.56, 95%CI 0.40–0.79), and DPB1*02:01 (P = 5.16X10^-6, Pc = 8.77X10^-5, OR 0.52, 95%CI 0.39–0.69) were protectively associated with SSc. In addition, these four alleles seemed to be independently associated with the protection against the susceptibility of SSc. On the other hand, we could not find predisposing alleles for overall SSc. With respect to SSc subsets, a tendency for these four alleles to be protectively associated was observed. However, there was a significant association between DRB1*01:01, DRB1*10:01, DQB1*05:01, and DPB1*04:02 and the susceptibility to SSc with ACA. On the other hand, the presence of DRB1*15:02, DQB1*06:01, DPB1*03:01, and DPB1*09:01 was associated with SSc with ATA.ConclusionThus, the present study has identified protective associations of the four HLA class II alleles with overall Japanese SSc and predisposing associations of HLA class II alleles with Japanese SSc subsets.     

Dapagliflozin,a Sodium-Glucose Co-Transporter 2 Inhibitor,Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.     

Floating culture promotes the maintenance of hematopoietic stem cells

In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine.