Secretion of apolipoprotein B in serum-free cultures of human hepatoma cell line, HepG2

AbstractWe have developed a defined medium which can maintain efficient growth of HepG2 cells sustaining the synthesis of a variety of plasma proteins including apolipoprotein B. This defined system was used to investigate long-term effects of insulin, estrogen, triiodothyronine, cholesterol, and oleate on the growth pattern of HepG2 cells and secretion rate of apolipoprotein B. Oleate and triiodothyronine caused significant increases in secretion of apolipoprotein B. The stimulatory effect of triiodothyronine was only observed after long (6 days) exposure of cells to the hormone. In contrast, insulin caused up to a 4-fold decrease in the secretion rate of apolipoprotein B during the early growth periods. This inhibitory effect appeared to be partially abolished after 6 days. Our data suggest that some important questions on regulation of apolipoprotein B expression can be addressed by the long-term culture of HepG2 cells in defined medium

CALIPER database of paediatric reference intervals: key milestones and future directions

Accurately established reference intervals are essential to interpret laboratory test results and assess patient health. Poorly established reference intervals can lead to misdiagnosis, subjecting patients to anxiety, unnecessary testing, and/or infection risk. The clinical importance of reference intervals is well recognised. However, establishing robust reference intervals is a complex process, especially for the paediatric population. Therefore, available reference intervals are often incomplete, cover a limited paediatric age interval, and/or do not consider gender differences. CALIPER, a collaborative study among Canadian paediatric centres, is addressing these critical gaps by determining age- and sex-specific paediatric reference intervals for over 80 biomarkers using samples collected from over 8,500 children and adolescents. These reference intervals established on the Abbott ARCHITECT have been transferred to other major analytical platforms, broadening the utility of the CALIPER database. The effect of diurnal variation, post-prandial effects, biological variation, and storage temperature on analyte concentration has also been assessed. Knowledge translation initiatives, including peer-reviewed publications, an online database, and a smartphone application, allow physicians and laboratory technicians worldwide to easily access the CALIPER database. This project has made great progress in addressing critical knowledge gaps in paediatric reference intervals, ultimately benefiting paediatric healthcare across Canada and globally

Fructose, insulin resistance, and metabolic dyslipidemia

Obesity and type 2 diabetes are occurring at epidemic rates in the United States and many parts of the world. The "obesity epidemic" appears to have emerged largely from changes in our diet and reduced physical activity. An important but not well-appreciated dietary change has been the substantial increase in the amount of dietary fructose consumption from high intake of sucrose and high fructose corn syrup, a common sweetener used in the food industry. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, perturbs glucose metabolism and glucose uptake pathways, and leads to a significantly enhanced rate of de novo lipogenesis and triglyceride (TG) synthesis, driven by the high flux of glycerol and acyl portions of TG molecules from fructose catabolism. These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose-induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Thus, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives. The present review will discuss the trends in fructose consumption, the metabolic consequences of increased fructose intake, and the molecular mechanisms leading to fructose-induced lipogenesis, insulin resistance and metabolic dyslipidemia

Oral supplementation of β-carotene benefits the hepatic structure and metabolism in mice (Mus musculus) exposed to a chronic ethanol consumption

Ethanol cannot be excreted and must be metabolized in the liver. Alcoholic liver disease is a blanket term for conditions related specifically to the liver and alcohol use. The study aimed to evaluate the metabolic, biochemical and histological effects of the oral supplementation with β-carotene on the liver of C57BL/6 mice exposed to ethanol consumption. Thirty male C57BL/6 mice (Mus musculus) were divided into six experimental groups: Control (C), Low-dose alcohol (LA), Moderate-dose alcohol (MA), β-carotene (B), Low-dose alcohol+β-carotene (LA+B) and Moderate-dose alcohol+β-carotene (MA+B) group. One-way ANOVA was used. The greatest intake of calories was noted in the LA (65.4 ± 12.5 kJ) and MA (68.6 ± 18.6 kJ) groups. The LA+B and MA+B groups shown an improvement in the HOMA-IR index (8.7 ± 2.4 and 6.7 ± 3.5, respectively), increased ADH levels (16.2 ± 1.6 and 18.9 ± 0.5 pmol/minmL-1, respectively) and decreased insulin levels (14.0 ± 3.3 and 10.6 ± 5.7 μUmL-1, respectively). It was also observed that oral supplementation with β-carotene improved the hepatic parenchyma in the LA+B group, showing normal-sized hepatocytes, whereas in the MA+B group it relieved the structural damage, revealing fewer lipid droplets than the MA group

Age- and sex-specific reference intervals for superoxide dismutase enzyme and several minerals in a healthy adult cohort

Introduction The aim of this study was to establish RIs for clinically important markers including superoxide dismutase (SOD), serum copper, zinc, calcium, magnesium, and phosphate in a cohort of healthy Iranian adults. Materials A subsample from MASHAD cohort study was used to assess serum SOD, copper, zinc, calcium, magnesium and phosphate. Serum SOD was measured according to its inhibitory potential of pyrogallol oxidation. Micro- and macro-minerals were measured using flame atomic absorption spectrometry and a BT3000 autoanalyzer, respectively. Sex- and age-specific RIs were then calculated based on CLSI Ep28-A3 guidelines. Results Reference value distributions for studied parameters did not demonstrate any age-specific differences that were statistically significant. In addition, sex partitioning was not required for all parameters, apart from serum magnesium, which showed a wider range in females (0.81–1.26 mg/dl) compared with males (0.82–1.23 mg/dl). Conclusion The RIs established in this study can be expected to improve mineral assessment and clinical decision-making in the Iranian adult population

A Canadian Study of Cisplatin Metabolomics and Nephrotoxicity (ACCENT): A Clinical Research Protocol

Background: Cisplatin, a chemotherapy used to treat solid tumors, causes acute kidney injury (AKI), a known risk factor for chronic kidney disease and mortality. AKI diagnosis relies on biomarkers which are only measurable after kidney damage has occurred and functional impairment is apparent; this prevents timely AKI diagnosis and treatment. Metabolomics seeks to identify metabolite patterns involved in cell tissue metabolism related to disease or patient factors. The A Canadian study of Cisplatin mEtabolomics and NephroToxicity (ACCENT) team was established to harness the power of metabolomics to identify novel biomarkers that predict risk and discriminate for presence of cisplatin nephrotoxicity, so that early intervention strategies to mitigate onset and severity of AKI can be implemented. Objective: Describe the design and methods of the ACCENT study which aims to identify and validate metabolomic profiles in urine and serum associated with risk for cisplatin-mediated nephrotoxicity in children and adults. Design: Observational prospective cohort study. Setting: Six Canadian oncology centers (3 pediatric, 1 adult and 2 both). Patients: Three hundred adults and 300 children planned to receive cisplatin therapy. Measurements: During two cisplatin infusion cycles, serum and urine will be measured for creatinine and electrolytes to ascertain AKI. Many patient and disease variables will be collected prospectively at baseline and throughout therapy. Metabolomic analyses of serum and urine will be done using mass spectrometry. An untargeted metabolomics approach will be used to analyze serum and urine samples before and after cisplatin infusions to identify candidate biomarkers of cisplatin AKI. Candidate metabolites will be validated using an independent cohort. Methods: Patients will be recruited before their first cycle of cisplatin. Blood and urine will be collected at specified time points before and after cisplatin during the first infusion and an infusion later during cancer treatment. The primary outcome is AKI, defined using a traditional serum creatinine-based definition and an electrolyte abnormality-based definition. Chart review 3 months after cisplatin therapy end will be conducted to document kidney health and survival. Limitations: It may not be possible to adjust for all measured and unmeasured confounders when evaluating prediction of AKI using metabolite profiles. Collection of data across multiple sites will be a challenge. Conclusions: ACCENT is the largest study of children and adults treated with cisplatin and aims to reimagine the current model for AKI diagnoses using metabolomics. The identification of biomarkers predicting and detecting AKI in children and adults treated with cisplatin can greatly inform future clinical investigations and practices

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field