UV法测定聚甲基丙烯酸酯纳米粒中胰岛素的包封率

建立一种简便易行的测定聚甲基丙烯酸酯胰岛素纳米粒中游离胰岛素含量方法.用Nanosep OD100C33超滤膜分离纳米粒和游离药物,在276 nm处测定药物的吸光度,建立胰岛素含量测定方法,并对线性、回收率、精密度等指标进行考察,最后测定各种胰岛素和载体比例混合的纳米粒的包封率.结果发现,该超滤膜能较好地分离纳米粒和游离的药物,在0.11～1.10 u/mL范围内,药物在276 nm的吸光度和浓度存在良好的线性关系(r=0.999 8),线性方程为A=0.868 8C-0.001 6,高、中、低3种浓度的回收率和精密度良好.该方法操作简单、结果可靠,可用于胰岛素纳米粒中药物包封率的测定.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

Cell-Free DNA Fragments as Biomarkers of Islet β-Cell Death in Obesity and Type 2 Diabetes

Type 2 diabetes (T2D) typically occurs in the setting of obesity and insulin resistance, where hyperglycemia is associated with decreased pancreatic β-cell mass and function. Loss of β-cell mass has variably been attributed to β-cell dedifferentiation and/or death. In recent years, it has been proposed that circulating epigenetically modified DNA fragments arising from β cells might be able to report on the potential occurrence of β-cell death in diabetes. Here, we review published literature of DNA-based β-cell death biomarkers that have been evaluated in human cohorts of islet transplantation, type 1 diabetes, and obesity and type 2 diabetes. In addition, we provide new data on the applicability of one of these biomarkers (cell free unmethylated INS DNA) in adult cohorts across a spectrum from obesity to T2D, in which no significant differences were observed, and compare these findings to those previously published in youth cohorts where differences were observed. Our analysis of the literature and our own data suggest that β-cell death may occur in subsets of individuals with obesity and T2D, however a more sensitive method or refined study designs are needed to provide better alignment of sampling with disease progression events.     

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer’s Disease Signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.     

Recent Update on the Molecular Mechanisms of Gonadal Steroids Action in Adipose Tissue

The gonadal steroids, including androgens, estrogens and progestogens, are involved in the control of body fat distribution in humans. Nevertheless, not only the size and localization of the fat depots depend on the sex steroids levels, but they can also highly affect the functioning of adipose tissue. Namely, the gonadocorticoids can directly influence insulin signaling, lipid metabolism, fatty acid uptake and adipokine production. They may also alter energy balance and glucose homeostasis in adipocytes in an indirect way, e.g., by changing the expression level of aquaglyceroporins. This work presents the recent advances in understanding the molecular mechanism of how the gonadal steroids influence the functioning of adipose tissue leading to a set of detrimental metabolic consequences. Special attention is given here to highlighting the sexual dimorphism of adipocyte functioning in terms of health and disease. Particularly, we discuss the molecular background of metabolic disturbances occurring in consequence of hormonal imbalance which is characteristic of some common endocrinopathies such as the polycystic ovary syndrome. From this perspective, we highlight the potential drug targets and the active substances which can be used in personalized sex-specific management of metabolic diseases, in accord with the patient’s hormonal status.     

蛋白质化学修饰的研究进展

蛋白质分子具有极其复杂的结构层次，用化学修饰的方法研究蛋白质分子的结构与功能的关系一直是生物化学和分子生物学领域的热点。人们研究出许多小分子化学修饰剂并进行了多种类型的化学修饰。综述了蛋白质化学修饰领域的研究现状与水平，同时强调蛋白质的化学修饰是生化药物研究开发的重要手段之一。     

Preparation of pH‐sensitive poly(vinyl alcohol‐g‐methacrylic acid) and poly(vinyl alcohol‐g‐acrylic acid) hydrogels by gamma ray irradiation and their insulin release behavior

A series of pH‐responsive hydrogels were studied as potential drug carriers for the protection of insulin from the acidic environment of the stomach before releasing in the small intestine. Hydrogels based on poly(vinyl alcohol) networks grafted with acrylic acid or methacrylic acid were prepared by a two‐step process. Poly(vinyl alcohol) hydrogels were prepared by gamma ray irradiation (50 kGy) and then followed by grafting either acrylic acid or methacrylic acid onto these poly(vinyl alcohol) hydrogels with subsequent irradiation (5–20 kGy). These graft hydrogels showed pH‐sensitive swelling behavior and were used as carriers for the controlled release of insulin. The in vitro release of insulin was observed for the insulin‐loaded hydrogels in a simulated intestinal fluid (pH 6.8) but not in a simulated gastric fluid (pH 1.2). The release behavior of insulin in vivo in a rat model confirmed the effectiveness of the oral delivery of insulin to control the level of glucose. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 636–643, 2004     

Interpolymer complex microparticles based on polymethacrylic acid‐chitosan for oral insulin delivery

In the present study, microparticles composed of polymethacrylic acid‐chitosan (PMAA‐CS) were prepared by a novel interionic gelation method. Free‐radical polymerization of methacrylic acid was carried out in the presence of CS, using a water‐soluble initiator, and application of these microparticles toward oral insulin delivery was evaluated. Microparticles obtained were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) studies. From SEM studies, it was observed that microparticles had an aggregated morphology with size ～20 μm, while FTIR confirmed the presence of ionic interaction between PMAA and CS chains. Protein loading was done by diffusion filling method, and from in vitro release study, it was observed that insulin‐loaded microparticles displayed a pH depended release profile at alkaline/acidic pH. Microparticles exhibited sustained release of insulin for 3–4 h at neutral pH, and enzyme linked immunosorbent assay (ELISA) proved that encapsulated protein maintained 100% biological activity at neutral pH. Preliminary study suggests that these microparticles can serve as good candidate for oral protein delivery. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 506–512, 2006