Impaired compensatory beta‐cell function and growth in response to high‐fat diet in LDL receptor knockout mice

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr−/− mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr−/− mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr−/− mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr−/− mice showed no significant changes in beta-cell mass, but lower islet–duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr−/− mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.     

RXR/VDR激动剂对糖尿病ApoE-/-小鼠胸主动脉硬化及NF-κB表达的调控

 目的：探讨视黄醇X受体（RXR）激动剂贝沙罗汀（Bex）和维生素D受体（VDR）激动剂骨化三醇（Cal）对链脲佐菌素（STZ）诱导的载脂蛋白E基因敲除(ApoE-/-)小鼠胸主动脉硬化及NF-κB表达的影响。方法: 动物分6组：C57组、ApoE-/-组、STZ-ApoE-/-组、STZ-ApoE-/-+Bex（10 mg·kg-1·d-1）组、STZ-ApoE-/-+Cal（10 μg/kg）组和STZ-ApoE-/-+Bex+Cal组。STZ腹腔注射复制糖尿病动物模型,Western blotting法及免疫组化法测定小鼠胸主动脉中NF-κB的表达,HE染色观察小鼠胸主动脉斑块的面积。结果：与C57组相比,ApoE-/-组的血糖水平无明显差别,血清总胆固醇（TC）及低密度脂蛋白（LDL）水平升高（P<005）;STZ-ApoE-/-组的血糖及血清TC和LDL水平较ApoE-/-组明显增高（P<005）,Bex和Cal对小鼠的血糖及血清TC、LDL无影响。与C57组相比,ApoE-/-和STZ-ApoE-/-小鼠胸主动脉的NF-κB蛋白表达显著增加;与STZ-ApoE-/-组相比,Bex和Cal均显著降低NF-κB的水平（P<0.05）,联合用药降低更明显（P<001）。与STZ-ApoE-/-组相比,Bex和Cal可缩小STZ-ApoE-/-小鼠的胸主动脉斑块面积（均P<005）;与Bex组相比,联合组斑块面积缩小的程度有显著差异（P<005）。结论：Bex和Cal可降低STZ-ApoE-/-小鼠胸主动脉粥样硬化斑块面积及NF-κB的表达,联合有协同作用,由此推论Bex和Cal的抗动脉粥样硬化机制可能与其对NF-κB的调节有关。     

Reducing plasma free fatty acids by acipimox improves glucose tolerance in high‐fat fed mice

To study whether free fatty acids (FFAs) contribute to glucose intolerance in high‐fat fed mice, the derivative of nicotinic acid, acipimox, which inhibits lipolysis, was administered intraperitoneally (50 mg kg^?1) to C57BL/6J mice which had been on a high‐fat diet for 3 months. Four hours after administration of acipimox, plasma FFA levels were reduced to 0.46 ± 0.06 mmol L^?1 compared with 0.88 ± 0.10 mmol L^?1 in controls (P < 0.001). At this point, the glucose elimination rate after an intravenous glucose load (1 g kg^?1) was markedly improved. Thus, the elimination constant (K_G) for the glucose disposal between 1 and 50 min after the glucose challenge was increased from 0.54 ± 0.01% min^?1 in controls to 0.66 ± 0.01% min^?1 by acipimox (P < 0.001). In contrast, the acute insulin response to glucose (1–5 min) was not significantly different between the groups, although the area under the insulin for the entire 50‐min period after glucose administration was significantly reduced by acipimox from 32.1 ± 2.9 to 23.9 ± 1.2 nmol L^?1 × 50 min (P=0.036). This, however, was mainly because of lower insulin levels at 20 and 50 min because of the lowered glucose levels. In contrast, administration of acipimox to mice fed a normal diet did not affect plasma levels of FFA or the glucose elimination or insulin levels after the glucose load. It is concluded that reducing FFA levels by acipimox in glucose intolerant high‐fat fed mice improves glucose tolerance mainly by improving insulin sensitivity making the ambient islet function adequate, suggesting that increased FFA levels are of pathophysiological importance in this model of glucose intolerance.     

Evaluation of mild hyperhomocysteinemia during the development of atherosclerosis in apolipoprotein E-deficient and normal mice

We focused on the effect of mild hyperhomocysteinemia (HHcy) on the development of atherosclerosis, using apolipoprotein E-deficient (apoE^−/−) and normal mice. Mice received diets enriched in methionine with low or high levels of folate, B₁₂ and B₆ (diets B and C, respectively), and diet only with low levels of folate, B₁₂ and B₆ (diets D), to induce mild HHcy. Normal mice fed on diets B, C and D presented mild HHcy, but they did not develop atherosclerotic lesions after 24 weeks of diet. In addition, increased endoplasmic reticulum stress was present in normal mice fed on diet B, compared to others groups. ApoE^−/− mice fed on diet B for 20 weeks presented the greatest atherosclerotic lesion area at the aortic sinus than other groups. These results suggest that the methionine may have a toxic effect on endothelium, and the B-vitamins addition on diet may have a protective effect in the long term, despite the increase on homocysteine levels. Mild HHcy accelerated the development of atherosclerosis in apoE^−/− mice, and supplementation with B-vitamins is important for prevention of vascular disease, principally in the long term.     

Japanese herbal medicines shosaikoto,inchinkoto, and juzentaihoto inhibit high‐fat diet‐induced nonalcoholic steatohepatitis in db/db mice

Few studies have investigated the effects of Japanese herbal medicines on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). To the best of our knowledge, only one study has examined whether high‐fat (HF) diet‐fed db/db mice are appropriate animal models of NASH. We investigated the effects of four types of Japanese herbal medicines (shosaikoto (TJ‐9), inchinkoto (TJ‐135), juzentaihoto (TJ‐48), and keishibukuryogan (TJ‐25)) on hepatic lesions of HF diet‐fed db/db mice. Db/db mice were divided into six groups: control diet (control); HF diet (HF); and HF diet supplemented with TJ‐9, TJ‐135, TJ‐48, or TJ‐25 (TJ‐9, TJ‐135, TJ‐48, and TJ‐25, respectively). Mice were killed after 6 weeks of treatment, and biochemical and pathological analyses were performed. Mice in the HF group consistently developed histopathological features consistent with definite NASH, and marked necroinflammation occurred. Serum alanine aminotransferase levels in the TJ‐9, TJ‐135, and TJ‐48 groups were significantly improved compared with those in the HF group. With regard to liver histology, TJ‐9 and TJ‐48 significantly improved lobular inflammation, and TJ‐135 significantly improved ballooning degeneration. We have shown that HF diet‐fed db/db mice are animal models that correctly recapitulate the histopathology of human NASH and that TJ‐9, TJ‐135, and TJ‐48 inhibit necroinflammatory activity in this model.     

Inhibitory effect of co‐administration of atorvastatin and endothelin‐1 receptor antagonist on the progression of atherosclerosis in rabbit

Atorvastatin, a hydroxymethylglutaryl‐CoA reductase inhibitor, and endothelin‐1 (ET‐1) receptor antagonist have been separately indicated to ameliorate disease progression in atherosclerosis. However, no study has evaluated the effect of their combination on atherosclerosis. The objective of the current study was to evaluate the direct in vivo effects of a combination regimen of atorvastatin and ET‐1 receptor antagonist on male New Zealand white rabbit models of atherosclerosis (injury‐induced). Thirty‐two atherosclerotic rabbits were divided into four experimental groups: (a) injury group – fed high‐fat diet; (b) ET‐1 receptor antagonist preventive group – fed high‐fat diet, but with intragastric administration of the ET‐1 receptor antagonist, darusentan; (c) combined preventive group – fed high‐fat diet, but with intragastric administration of both darusentan and atorvastatin; and (d) treatment group – fed high‐fat diet for the first 8 weeks, followed by normal diet and intragastric administration of both darusentan and atorvastatin up to 16 weeks. A further eight non‐atherosclerotic rabbits were fed normal diet and classified as the control group. At the end of 8 and 16 weeks, compared with the injury group, the combined preventive group had significant reduction in both the concentration of serum lipids and inflammatory factors and atherosclerosis formation, indicative of a multifaceted anti‐atherosclerotic impact. The relative area of atherosclerotic lesions in the injury group (30.84%) was significantly higher than the control group (4.62%; p < 0.05). The combined preventive group showed a significantly robust effect on lowering serum lipid, inflammatory cytokines, and maintained homeostatic balance of free radicals, and important downstream effectors like ET‐1 and matrixmetalloproteinase‐9. Our data show that atorvastatin and ET‐1 receptor antagonist co‐administration may decrease lipid levels, stabilize plaques and relieve vascular inflammation. By reducing the plaque burden, this regimen may minimize the risk of atherosclerotic plaque rupture or arterial occlusion.     

Regulation of T‐lymphocyte motility,adhesion and de‐adhesion by a cell surface mechanism directed by low density lipoprotein receptor‐related protein 1 and endogenous thrombospondin‐1

T lymphocytes are highly motile and constantly reposition themselves between a free‐floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF‐1 influence motility and adhesion through this mechanism. Targeting cell surface‐expressed low‐density lipoprotein receptor‐related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin‐1 (TSP‐1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM‐1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP‐1 and a 130 000 MW TSP‐1 fragment while preventing formation of a de‐adhesion‐coupled 110 000 MW TSP‐1 fragment. The appearance of the 130 000 MW TSP‐1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP‐1 binding site in the LRP1‐associated protein, calreticulin, stimulated adhesion to ICAM‐1 through intact TSP‐1 and CD47. Shear flow enhanced cell surface expression of intact TSP‐1. Hence, chemokines and integrin ligands up‐regulate a dominant motogenic pathway through LRP1 and TSP‐1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP‐1 and CD47. This regulation of T‐cell motility and adhesion makes pro‐adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.     

Elevated complement activities of sera from patients with high density lipoprotein deficiency (Tangier disease): the presence of normal level of clusterin and the possible implication in the atherosclerosis.

Clusterin (apolipoprotein J, SP-40,40), as well as apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II), are apolipoprotein components of high density lipoprotein (HDL), but not of low density lipoprotein. In spite of the deficiencies of apo A-I, apo A-II and HDL in the sera of patients with Tangier disease, clusterin was found in them at normal level. While clusterin was present as the component of HDL with apo A-I in sera of normal donors, it was present as a protein which did not form a complex in sera of Tangier patients. SC5b-9 made from the sera of Tangier patients contained normal amounts of clusterin and was deficient in apo A-I, indicating that clusterin could be incorporated into the SC5b-9 complex without apo A-I. The complement activities of the sera of the patients were higher than those of normal donors. These results may be explained by the deficiencies of apo A-I, apo A-II and HDL in the patients, because they were suggested to be the inhibitors of the reactive haemolysis of complement. The elevated complement activities of the patients might be related to the severe atherosclerotic lesions in Tangier disease.     

Opposing effects of actin signaling and LFA‐1 on establishing the affinity threshold for inducing effector T‐cell responses in mice

Mature CD8⁺ T cells use a narrow antigen affinity threshold to generate tissue‐infiltrating cytotoxic effector T cells and induce autoimmune pathology, but the mechanisms that establish this antigen affinity threshold are poorly understood. Only antigens with affinities above the threshold induce stable contacts with APCs, polarization of a T cell, and asymmetric T‐cell division. Previously published data indicate that LFA‐1 inside‐out signaling might be involved in establishing the antigen affinity threshold. Here, we show that subthreshold antigens weakly activate all major distal TCR signaling pathways. Low‐affinity antigens are more dependent on LFA‐1 than suprathreshold antigens. Moreover, augmenting the inside‐out signaling by hyperactive Rap1 does not increase responses to the subthreshold antigens. Thus, LFA‐1 signaling does not contribute to the affinity‐based antigen discrimination. However, we found that subthreshold antigens do not induce actin rearrangement toward an APC, mediated by Rho‐family GTPases, Cdc42, and Rac. Our data suggest that Rac and Cdc42 contribute to the establishment of the antigen affinity threshold in CD8⁺ T cells by enhancing responses to high‐affinity antigens, or by reducing the responses to low‐affinity antigens.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.     

Deletion of the α2A/α2C‐adrenoceptors accelerates cutaneous wound healing in mice

The α2‐adrenoceptors regulate the sympathetic nervous system, controlling presynaptic catecholamine release. However, the role of the α2‐adrenoceptors in cutaneous wound healing is poorly understood. Mice lacking both the α2A/α2C‐adrenoceptors were used to evaluate the participation of the α2‐adrenoceptor during cutaneous wound healing. A full‐thickness excisional lesion was performed on the dorsal skin of the α2A/α2C‐adrenoceptor knockout and wild‐type mice. Seven or fourteen days later, the animals were euthanized and the lesions were formalin‐fixed and paraffin‐embedded or frozen. Murine skin fibroblasts were also isolated from α2A/α2C‐adrenoceptor knockout and wild‐type mice, and fibroblast activity was evaluated. The in vivo study demonstrated that α2A/α2C‐adrenoceptor depletion accelerated wound contraction and re‐epithelialization. A reduction in the number of neutrophils and macrophages was observed in the α2A/α2C‐adrenoceptor knockout mice compared with wild‐type mice. In addition, α2A/α2C‐adrenoceptor depletion enhanced the levels of nitrite and hydroxyproline, and the protein expression of transforming growth factor‐β and vascular endothelial growth factor. Furthermore, α2A/α2C‐adrenoceptor depletion accelerated blood vessel formation and myofibroblast differentiation. The in vitro study demonstrated that skin fibroblasts isolated from α2A/α2C‐adrenoceptor knockout mice exhibited enhanced cell migration, α‐smooth muscle actin _protein expression and collagen deposition compared with wild‐type skin fibroblasts. In conclusion, α2A/α2C‐adrenoceptor deletion accelerates cutaneous wound healing in mice.     

Hepatocellular carcinoma‐associated single‐nucleotide variants and deletions identified by the use of genome‐wide high‐throughput analysis of hepatitis B virus

This study investigated hepatitis B virus (HBV) single‐nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety‐three HCC patients and 108 non‐HCC patients were enrolled for HBV genome‐wide next‐generation sequencing (NGS) analysis. A systematic literature review and a meta‐analysis were performed to validate NGS‐defined HCC‐associated SNVs and deletions. The experimental results identified 60 NGS‐defined HCC‐associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n = 24) or genotype C (n = 34), except for nt53C, which was present in both genotypes. The pathogenic frequencies of these HCC‐associated SNVs showed a distinct U‐shaped distribution pattern. According to the meta‐analysis and literature review, 167 HBV variants from 109 publications were categorized into four levels (A–D) of supporting evidence that they are associated with HCC. The proportion of NGS‐defined HCC‐associated SNVs among these HBV variants declined significantly from 75% of 12 HCC‐associated variants by meta‐analysis (Level A) to 0% of 10 HCC‐unassociated variants by meta‐analysis (Level D) (P < 0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P = 0.030) and C (P = 0.049). For genotype C, preS deletions involving a specific fragment (nt2977–3013) were significantly associated with HCC (HCC versus non‐HCC, 6/34 versus 0/32, P = 0.025). Meta‐analysis of preS deletions showed significant association with HCC (summary odds ratio 3.0; 95% confidence interval 2.3–3.9). Transfection of Huh7 cells showed that all of the five novel NGS‐defined HCC‐associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including endoplasmic reticulum‐stress and DNA repair systems, as shown by microarray, real‐time polymerase chain reaction and western blot analysis. Their carcinogenic mechanisms are worthy of further research. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.