Secreted Frizzled-related protein 4 (sFRP4) chemo-sensitizes cancer stem cells derived from human breast, prostate, and ovary tumor cell lines.

This study investigated molecular signals essential to sustain cancer stem cells (CSCs) and assessed their activity in the presence of secreted frizzled-related protein 4 (sFRP4) alone or in combination with chemotherapeutic drugs. SFRP4 is a known Wnt antagonist, and is also pro-apoptotic and anti-angiogenic. Additionally, sFRP4 has been demonstrated to confer chemo-sensitization and improve chemotherapeutic efficacy. CSCs were isolated from breast, prostate, and ovary tumor cell lines, and characterized using tumor-specific markers such as CD44+/CD24-/CD133+. The post-transcription data from CSCs that have undergone combinatorial treatment with sFRP4 and chemotherapeutic drugs suggest downregulation of stemness genes and upregulation of pro-apoptotic markers. The post-translational modification of CSCs demonstrated a chemo-sensitization effect of sFRP4 when used in combination with tumor-specific drugs. SFRP4 in combination with doxorubicin/cisplatin reduced the proliferative capacity of the CSC population in vitro. Wnt/ß-catenin signaling is important for proliferation and self-renewal of CSCs in association with human tumorigenesis. The silencing of this signaling pathway by the application of sFRP4 suggests potential for improved in vivo chemo-responses.

The impact of cholecalciferol supplementation on the systemic inflammatory profile: a systematic review and meta-analysis of high-quality randomized controlled trials.

Causal links between vitamin D status [25(OH)D] and systemic inflammation were examined through a systematic review of randomized controlled trials (RCTs). Selected RCTs were ⩾12 weeks, conducted in adults free of acute inflammatory disease, and of high-quality (Jadad score ⩾3). Of 14 studies that met our criteria, 9 studies (15 study arms) permitted extraction of data. There was no effect on the weighted mean difference (WMD) of IL-6 (WMD (95% confidence interval)=0.1, (-0.166, 0.366) pg/ml, P=0.462) or C-reactive protein (CRP) (WMD=-0.324, (-1.007, 0.359) mg/l, P=0.352). Subgroup analyses of trials achieving ⩾80 nmol/l indicated a trend for lower CRP (WMD=-0.834, (-1.726, 0.058) mg/l, P=0.067), however heterogeneity was significant (I2=66.7%, P=0.017). Studies employing a low dose (<1000 IU/d) showed increased CRP (WMD=0.615, (0.132, 1.098), P=0.013). In contrast, ⩾1000 IU/d had a favourable effect on CRP (WMD=-0.939, (-1.805, -0.073), P=0.034) but heterogeneity was significant (I2=61.3%, P=0.017). Meta-regression indicated that older age predicted a significant decrease in IL-6 (ß=-0.02, (-0.034, -0.006) pg/ml, P=0.013) and CRP (ß=-0.06, (-0.103, -0.017), P=0.01), whereas a greater percentage of females (ß=0.027, (0.011, 0.044), P=0.004) and longer study duration independently predicted a higher WMD for CRP (ß=0.049, (0.018, 0.079), P=0.005). Available high-quality RCTs did not support a beneficial effect of cholecalciferol on systemic IL-6 and CRP. Future studies should consider the confounding effects of age, gender and study duration, while possibly targeting an achieved 25(OH)D ⩾80 nmol/l.

Forearm to fingertip skin temperature gradients in the thermoneutral zone were significantly related to resting metabolic rate: potential implications for nutrition research.

BACKGROUND: Resting metabolic rate (RMR) should be measured in the thermoneutral zone (TNZ). Forearm to fingertip skin temperature gradients (FFG) could serve as an objective measure of this pre-condition. SUBJECTS/METHODS: Eighty-six adult Australians were studied at 25 °C in a temperature-controlled chamber. Measurements of overnight fasted RMR, respiratory quotient (RQ) and FFG were complemented by clinical biochemistry. McAuley's Index of insulin sensitivity (McA_ISI) and presence of metabolic syndrome was determined. Physical activity was estimated from the short version of the International Physical Activity Questionnaire. Fat mass (FM) and fat-free mass (FFM) were obtained from dual-energy x-ray absorptiometry. Twenty-nine participants were assessed for changes in RMR (ΔRMR), RQ (ΔRQ) and FFG (ΔFFG) following a 6-month free-living period. Multiple linear regression analyses of RMR and RQ on FFG, and of ΔRMR and ΔRQ on ΔFFG were conducted after controlling for 12 known determinants of energy metabolism. RESULTS: There were wide between-subject variations in unadjusted FFG ranging from -4.25 to +7.8 °C. The final parsimonious model for cross-sectional observations of RMR included age, FM, FFM, McA_ISI and FFG (ß=63 kJ/d (95% confidence interval (CI): 14.2, 112.1, P=0.012)). However, FFG was unrelated to RQ.In the longitudinal cohort, adjusted ΔRMR significantly associated only with ΔFFG (ß=100 kJ/d (95% CI: 10.3, 189.1; P=0.030)), and adjusted ΔRQ associated with ΔFFG (-0.003 (95% CI: -0.005, 0.0002, P=0.038)), age and McA_ISI. CONCLUSIONS: Sizeable between-subject variations in FFG at 25 °C were associated with RMR and RQ. Monitoring FFG may serve as an objective assessment of the TNZ during RMR measurements.

The bioenergetics of inflammation: insights into obesity and type 2 diabetes.

Diabetes mellitus is one of the most common chronic metabolic disorders worldwide, and its incidence in Asian countries is alarmingly high. Type 2 diabetes (T2DM) is closely associated with obesity, and the staggering rise in obesity is one of the primary factors related to the increased frequency of T2DM. Low-grade chronic inflammation is also accepted as an integral metabolic adaption in obesity and T2DM, and is believed to be a major player in the onset of insulin resistance. However, the exact mechanism(s) that cause a persistent chronic low-grade infiltration of leukocytes into insulin-target tissues such as adipose, skeletal muscle and liver are not entirely known. Recent developments in the understanding of leukocyte metabolism have revealed that the inflammatory polarization of immune cells, and consequently their immunological function, are strongly connected to their metabolic profile. Therefore, it is hypothesized that dysfunctional immune cell metabolism is a central cellular mechanism that prevents the resolution of inflammation in chronic metabolic conditions such as that observed in obesity and T2DM. The purpose of this review is to explore the metabolic demands of different immune cell types, and identify the molecular switches that control immune cell metabolism and ultimately function. Understanding of these concepts may allow the development of interventions that can correct immune function and may possibly decrease chronic low-grade inflammation in humans suffering from obesity and T2DM. We also review the latest clinical techniques used to measure metabolic flux in primary leukocytes isolated from obese and T2DM patients.

Nutrient regulation of ß-cell function: what do islet cell/animal studies tell us?

Diabetes mellitus is widely recognised as one of the most serious metabolic diseases worldwide, and its incidence in Asian countries is growing at an alarming rate. Type 2 diabetes (T2DM) is closely associated with age, sedentary lifestyle and poor diet. In T2DM, ß-cell dysfunction will occur before hyperglycaemia develops. Excessive levels of glucose, lipid and various inflammatory factors interact at the level of the pancreatic islet to promote ß-cell dysfunction. Pancreatic ß-cell lines have been widely utilised since the early 1980s and have contributed a large volume of important information regarding molecular, metabolic and genetic mechanisms that regulate insulin secretion. The purpose of this review is to describe the origin and characteristics of the most commonly used ß-cell lines and their contribution to discovery of fundamental regulatory processes that control insulin production and release. Pancreatic islets obtained from rodents as well as other animals have additionally provided information on the architecture and three-dimensional design of this endocrine tissue that allows precise regulation of hormone release. Understanding the nature of failure of physiologic and metabolic processes leading to insufficient insulin release and subsequent diabetes has allowed development of novel anti-diabetic therapeutics, now in common use, worldwide.

Vitamin D status and insulin sensitivity are novel predictors of resting metabolic rate: a cross-sectional analysis in Australian adults.

PURPOSE: Resting metabolic rate (RMR) accounts for two-thirds of the total energy expenditure in sedentary individuals. After accounting for traditional factors, there still remains a considerable unexplained variance in RMR. There is a pandemic of obesity and metabolic syndrome (MetS) which coexists with a high prevalence of vitamin D insufficiency. The aim of this study was to evaluate the potential effects of vitamin D status, insulin sensitivity (IS) and the metabolic syndrome (MetS) on RMR in Australian adults. METHODS: RMR, respiratory quotient (RQ), McAuley's insulin sensitivity index, fat mass (FM), fat-free mass (FFM) and vitamin D status were assessed in Australian adults. The presence of MetS was evaluated by current standard criteria. Predictors of RMR were examined through multiple linear regression based on stepwise and backward regression approaches with attention to multi-collinearity. All analyses were conducted on SPSS version 21. RESULTS: One hundred and twenty-seven participants (45 men, 82 women), aged 53.4 ± 11.7 years and BMI 31.9 ± 5.2 kg/m(2), were included. Forty-one subjects were insufficient in vitamin D status (<50 nmol/L), and 75 participants had the MetS. A parsimonious regression model explained 85.8 % of RMR and was given by: RMR (kJ/d) = 1931 + 83.5 × FFM (kg) + 29.5 × FM (kg) + 5.65 × 25(OH)D (nmol/L) - 17.6 × age (years) - 57.51 × IS. CONCLUSION: Vitamin D status and IS are novel independent predictors of RMR in adults. Future studies could validate a causal role for these factors in human energy metabolism.

Epigenetic regulation of the secreted frizzled-related protein family in human glioblastoma multiforme.

Glioblastoma multiforme (GBM) are intracranial tumors of the central nervous system and the most lethal among solid tumors. Current therapy is palliative and is limited to surgical resection followed by radiation therapy and temozolomide treatment. Aberrant WNT pathway activation mediates not only cancer cell proliferation but also promotes radiation and chemotherapeutic resistance. WNT antagonists such as the secreted frizzled-related protein (sFRP) family have an ability to sensitize glioma cells to chemotherapeutics, decrease proliferation rate and induce apoptosis. During tumor development, sFRP genes (1-5) are frequently hypermethylated, causing transcriptional silencing. We investigated a possible involvement of methylation-mediated silencing of the sFRP gene family in human GBM using four human glioblastoma cell lines (U87, U138, A172 and LN18). To induce demethylation of the DNA, we inhibited DNA methyltransferases through treatment with 5-azacytidine. Genomic DNA, RNA and total protein were isolated from GBM cells before and after treatment. We utilized bisulfite modification of genomic DNA to examine the methylation status of the respective sFRP promoter regions. Pharmacological demethylation of the GBM cell lines demonstrated a loss of methylation in sFRP promoter regions, as well as an increase in sFRP gene-specific mRNA abundance. Western blot analysis demonstrated an increased protein expression of sFRP-4 and increased levels of phosphorylated-ß-catenin. These data indicate an important role of methylation-induced gene silencing of the sFRP gene family in human GBM.

Cytotoxicity and cytoprotective effects of citrus flavonoids on insulin-secreting cells BRIN-BD11: beneficial synergic effects.

Flavonoids, in general, have potent antioxidant activity and they can be used in treating chronic diseases involving oxidative stress, such as diabetes mellitus. The purpose of this study was to evaluate the cytotoxicity and cytoprotective effects of citrus flavonoids on the functionality of BRIN-BD11 cells. The assessment of cytotoxic and cytoprotective flavonoid tested was performed using the MTT reduction assay. The flavonoids did not show cytotoxic effects in any of the tested concentrations (5-20 µM) and also negative insulinotropic effects were not observed. To cytoprotective assay, the IC50 of H2O2 in treatment of 2 h (acute oxidative stress) was measured (350 µM). Moreover, under acute oxidative stress, the isolated flavonoids (10 µM) had no cytoprotective effects. Besides an antioxidant role of the flavonoids was only observed when using in association. Thus future experiments are needed, varying the experimental condition, to better evaluate the possible mechanisms of action of these flavonoids.

Reactive oxygen and nitrogen species generation, antioxidant defenses, and ß-cell function: a critical role for amino acids.

Growing evidence indicates that the regulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels is essential for maintaining normal ß-cell glucose responsiveness. While long-term exposure to high glucose induces oxidative stress in ß cells, conflicting results have been published regarding the impact of ROS on acute glucose exposure and their role in glucose stimulated insulin secretion (GSIS). Although ß cells are considered to be particularly vulnerable to oxidative damage, as they express relatively low levels of some peroxide-metabolizing enzymes such as catalase and glutathione (GSH) peroxidase, other less known GSH-based antioxidant systems are expressed in ß cells at higher levels. Herein, we discuss the key mechanisms of ROS/RNS production and their physiological function in pancreatic ß cells. We also hypothesize that specific interactions between RNS and ROS may be the cause of the vulnerability of pancreatic ß cells to oxidative damage. In addition, using a hypothetical metabolic model based on the data available in the literature, we emphasize the importance of amino acid availability for GSH synthesis and for the maintenance of ß-cell function and viability during periods of metabolic disturbance before the clinical onset of diabetes.

Insights into the critical role of NADPH oxidase(s) in the normal and dysregulated pancreatic beta cell.

It is now widely accepted that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage in diabetes. The source of ROS in the insulin secreting pancreatic beta cells has traditionally been considered to be the mitochondrial electron transport chain. While this source is undoubtedly important, we fully describe in this article recent information and evidence of NADPH oxidase-dependent generation of ROS in pancreatic beta cells and identify the various isoforms that contribute to O(2)(*-) and H(2)O(2) production in various conditions. While glucose-stimulated ROS generation may be important for acute regulation of insulin secretion, at higher levels ROS may disrupt mitochondrial energy metabolism. However, ROS may alter other cellular processes such as signal transduction, ion fluxes and/or cell proliferation/death. The various beta cell isoforms of NADPH oxidase (described in this review) may, via differences in the kinetics and species of ROS generated, positively and negatively regulate insulin secretion and cell survival.

Amino acid metabolism, insulin secretion and diabetes.

In addition to the primary stimulus of glucose, specific amino acids may acutely and chronically regulate insulin secretion from pancreatic beta-cells in vivo and in vitro. Mitochondrial metabolism is crucial for the coupling of glucose, alanine, glutamine and glutamate recognition with exocytosis of insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP (the main coupling messenger in insulin secretion) and other factors that serve as sensors for the control of the exocytotic process. The main factors that mediate the key amplifying pathway over the Ca(2+) signal in nutrient-stimulated insulin secretion are nucleotides (ATP, GTP, cAMP and NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and glutamate. In addition, after chronic exposure, specific amino acids may influence gene expression in the beta-cell, which have an impact on insulin secretion and cellular integrity. Therefore amino acids may play a direct or indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.

Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity.

It is now widely accepted, given the current weight of experimental evidence, that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage caused by glucolipotoxicity in diabetes. The source of ROS in the insulin secreting pancreatic beta-cells and in the cells which are targets for insulin action has been considered to be the mitochondrial electron transport chain. While this source is undoubtably important, we provide additional information and evidence for NADPH oxidase-dependent generation of ROS both in pancreatic beta-cells and in insulin sensitive cells. While mitochondrial ROS generation may be important for regulation of mitochondrial uncoupling protein (UCP) activity and thus disruption of cellular energy metabolism, the NADPH oxidase associated ROS may alter parameters of signal transduction, insulin secretion, insulin action and cell proliferation or cell death. Thus NADPH oxidase may be a useful target for intervention strategies based on reversing the negative impact of glucolipotoxicity in diabetes.

Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line.

AIMS/HYPOTHESIS: Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). METHODS: Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. RESULTS: Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. CONCLUSIONS/INTERPRETATION: NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

The importance of redox shuttles to pancreatic beta-cell energy metabolism and function.

The coupling of cytosolic glycolytic NADH production with the mitochondrial electron transport chain is crucial for pancreatic beta-cell function and energy metabolism. The activity of lactate dehydrogenase in the beta-cell is low, thus glycolysis-derived electrons are transported towards the mitochondrial matrix by a NADH shuttle system, which in turn regenerates cytosolic NAD+. Mitochondrial electron transport then produces ATP, the main coupling factor for insulin secretion. Aralar1, a Ca2+-sensitive member of the malate-aspartate shuttle expressed in beta-cells, has been found to play a significant role in nutrient-stimulated insulin secretion and beta-cell function. Increased capacity of Aralar1 enhances the responsiveness of the cell to glucose. Conversely, inhibition of the malate-aspartate shuttle results in impaired glucose metabolism and insulin secretion. Current research investigates potentiating or attenuating activities of various amino acids on insulin secretion, mitochondrial membrane potential and NADH production in Aralar1-overexpressing beta-cells. This work may provide evidence for a central role of Aralar1 in the regulation of nutrient metabolism in the beta-cells.

Detrimental actions of metabolic syndrome risk factor, homocysteine, on pancreatic beta-cell glucose metabolism and insulin secretion.

Elevated plasma homocysteine has been reported in individuals with diseases of the metabolic syndrome including vascular disease and insulin resistance. As homocysteine exerts detrimental effects on endothelial and neuronal cells, this study investigated effects of acute homocysteine exposure on beta-cell function and insulin secretion using clonal BRIN-BD11 beta-cells. Acute insulin release studies in the presence of various test reagents were performed using monolayers of BRIN-BD11 cells and samples assayed by insulin radioimmunoassay. Cellular glucose metabolism was assessed by nuclear magnetic resonance (NMR) analysis following 60-min exposure of BRIN-BD11 cell monolayers to glucose in either the absence or presence of homocysteine. Homocysteine dose-dependently inhibited insulin release at moderate and stimulatory glucose concentrations. This inhibitory effect was reversible at all but the highest concentration of homocysteine. 13C-glucose NMR demonstrated decreased labelling of glutamate from glucose at positions C2, C3 and C4, indicating that the tricarboxylic acid (TCA) cycle-dependent glucose metabolism was reduced in the presence of homocysteine. Homocysteine also dose-dependently inhibited insulinotropic responses to a range of glucose-dependent secretagogues including nutrients (alanine, arginine, 2-ketoisocaproate), hormones (glucagon-like peptide-1 (7-36)amide, gastric inhibitory polypeptide and cholecystokinin-8), neurotransmitter (carbachol), drug (tolbutamide) as well as a depolarising concentration of KCl or elevated Ca2+. Insulin secretion induced by activation of adenylate cyclase and protein kinase C pathways with forskolin and phorbol 12-myristate 13-acetate were also inhibited by homocysteine. These effects were not associated with any adverse action on cellular insulin content or cell viability, and there was no increase in apoptosis/necrosis following exposure to homocysteine. These data indicate that homocysteine impairs insulin secretion through alterations in beta-cell glucose metabolism and generation of key stimulus-secretion coupling factors. The participation of homocysteine in possible beta-cell demise merits further investigation.

Molecular mechanisms of glutamine action.

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions.