Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Heart rate variability characteristics of patients with irritable bowel syndrome and associations with symptoms.

BACKGROUND: Disturbed brain-gut interactions are assumed to be of importance for symptom generation in patients with irritable bowel syndrome (IBS). The autonomic nervous system (ANS) is part of the bidirectional brain-gut communication, but previous studies in IBS show diverging results. We aimed to identify subgroups of IBS patients with distinct ANS characteristics differentiating them from healthy controls (HC), and to study associations between ANS status and symptoms. METHODS: Heart rate variability (HRV) was measured in IBS patients and HC (Holter monitoring: supine and standing positions with controlled respiration and ambulatory 24-hour period). Frequency (5 minutes, supine, standing) and time domains (24 hours, day, night) were analyzed. Validated questionnaires were used to measure gastrointestinal and psychological symptoms in patients. Patients and HC were compared on a univariate and multivariate level (principal component analysis [PCA] and orthogonal partial least squares discriminatory analysis (OPLS-DA)). KEY RESULTS: We analyzed 158 IBS patients (Rome III) and 39 HC. Patients differed significantly from HC in HRV parameters during daytime and in standing position. In the PCA, a majority of patients overlapped with HC, but the weighted means differed (P < .01). A subset of patients (n = 30; 19%) with an aberrant global HRV profile was identified through PCA and OPLS-DA; these patients reported more severe symptoms of frequent (P < .05) and loose stools (P = .03), as well as urgency (P = .01). CONCLUSIONS AND INFERENCES: Altered ANS function was demonstrated in patients with IBS, and this might be of particular relevance for symptoms in a subset of the patients.

A combined nutrient and lactulose challenge test allows symptom-based clustering of patients with irritable bowel syndrome.

OBJECTIVES: The aim of the present pilot study was to evaluate the usefulness of a test meal containing lactulose in the non-invasive assessment of visceral sensitivity in irritable bowel syndrome (IBS), and to identify subsets of IBS patients based on gastrointestinal (GI) symptom generation. METHODS: We included 43 patients with IBS (Rome III) and 29 healthy controls. The fasted subjects were served three test meals consisting of a 400-ml liquid breakfast alone or containing lactulose (15 or 25 g) in a double-blind crossover design. Seven GI symptoms, overall digestive comfort, and exhaled H2/CH4 were assessed at baseline and every 15 min during 4 h after meal intake. Anxiety and depression were assessed only at baseline. A mapping of the seven GI symptoms was done using a Principal Component Analysis (4 h mean area under the curve, AUC). Independently, a hierarchical cluster analysis was performed on the same parameters to identify GI symptom-based IBS clusters. RESULTS: All three tests were well tolerated. The 25 g lactulose challenge enabled discrimination of IBS from healthy controls according to the symptom response. This challenge also enabled clustering of IBS subjects in two subgroups based mainly on bloating, distension, and discomfort symptoms (2,457 (2,043-2,872), 2,450 (1,910-2,990), 2,602 (2,126-3,079) vs. 537 (383-691), 619 (458-780), 643 (432-854); 4 h mean AUC; P<0.0001), overall digestive comfort (1807 (1318-2295) vs. 3350 (2942-3758); 4 h mean AUC; P<0.0001), and anxiety at baseline (9.2 (7.0-11.5) vs. 5.5 (4.2-6.9); Hospital Anxiety and Depression scale anxiety mean scores; P=0.003). This clustering was independent of the Rome III subtype and the amount of exhaled H2/CH4. CONCLUSIONS: The lactulose challenge test seems to be a promising tool to assess visceral sensitivity in IBS, and to subgroup IBS patients based on their symptom pattern.

TCF7L2 rs7903146 impairs islet function and morphology in non-diabetic individuals.

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a Wnt-signalling-associated transcription factor. Genetic studies have clearly demonstrated that DNA polymorphisms within TCF7L2 confer the strongest known association with increased risk of type 2 diabetes. However, the impact of the TCF7L2 type-2-diabetes-associated rs7903146 T allele on biological function and morphology of human pancreatic islets is unknown. METHODS: Paraffin sections of pancreases from 187 brain-deceased donors (HbA(1c) <6.5% [48 mmol/mol]) were used to genotype the TCF7L2 variant rs7903146 and evaluate its impact on islet morphology and alpha and beta cell subpopulations following immunostaining for glucagon and C-peptide. Following islet isolation, we investigated the correlation between TCF7L2 genotype and in vitro islet functional variables from our in-house pancreatic database. RESULTS: TCF7L2 rs7903146 (T/T) was associated with reduced basal and glucose-stimulated insulin secretion in isolated human islets, and reduced islet density in whole pancreas. Morphological analysis demonstrated islet size was increased in T/T carriers. Furthermore, rs7903146 was associated with an increased glucagon/C-peptide ratio, especially in bigger islets. CONCLUSION/INTERPRETATION: The TCF7L2 variant rs7903146 risk allele is associated with impaired insulin secretion, reduction of total islet number and quantitative as well as qualitative morphological changes in human islets. Understanding how the TCF7L2 genotype modulates its activity and how TCF7L2 impacts the islet morphology may aid the design of new therapeutic approaches for the treatment of type 2 diabetes.

Minor contribution of SMAD7 and KLF10 variants to genetic susceptibility of type 2 diabetes.

BACKGROUND: Transgenic mice over-expressing SMAD7 in pancreatic beta-cells develop type 2 diabetes (T2D). The expression of SMAD7 is affected by KLF11, which contains gene variants that have previously been shown to be involved in genetic susceptibility to T2D, and by the highly homologous KLF10. This study aims to assess the genetic contribution of SMAD7 and KLF10 gene variants to T2D susceptibility in the French population. METHODS: We screened both genes to identify rare and frequent variants by direct sequencing and then genotyped these variants. Six frequent variants of SMAD7 and six of KLF10 were analyzed in 349 T2D patients and 349 normoglycaemic adult subjects. Variants with statistically significant differences in allele and/or genotype distribution were further analyzed in a population sample of 1.712 T2D patients and 1.072 normoglycaemic subjects. RESULTS: Two variants showed a significant association under a recessive model: The intronic SMAD7 IVS2 -21 had an odds ratio of 0.62 (P=0.007, 95% CI=0.44-0.88; P=0.034 when adjusting for age, sex and BMI by logistic regression), and the KLF10 3'UTR +1002 variant had an Odds Ratio of 0.81 (P=0.009, 95% CI=0.69-0.95; P=0.042 when adjusting for age, sex and BMI). CONCLUSION: Although the observed association of SMAD7 and KLF10 gene variants with T2D is modest, they may weakly contribute to a particular genetic background that increases the susceptibility to development of T2D.

Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.

Individual genotyping of single nucleotide polymorphisms (SNPs) remains expensive, especially for linkage disequilibrium mapping strategies involving high-throughput SNP genotyping. On one hand, current methods may suit scientific and laboratory needs in regard to accuracy, reproducibility/robustness, and large-scale application. On the other hand, a cheaper and less time-consuming alternative to individual genotyping is the use of SNP allelefrequencies determined in DNA pools. We have developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals). The measured correlation (R2) in large DNA pools was 0.980. In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (e.g., type 2 diabetes and obesity) and control groups detected by either individual genotyping or Pyrosequencing of DNA pools. In large pools, the variation between the two methods was 1.5 +/- 0.9%. It may be concluded that the allele frequency determination protocol could reliably detect over 4% differences between populations. The method is economical in regard to amounts of DNA, PCR, and primer extension reagents required. Furthermore, it allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.

PPARalpha agonists inhibit tissue factor expression in human monocytes and macrophages.

BACKGROUND: Monocytic tissue factor (TF) expression may contribute to thrombogenicity associated with plaque rupture and may propagate thrombus formation at the site of vascular lesions. Induction of monocytic TF expression by endotoxin is mediated by the activation of transcription factors such as AP-1 and NF-kappaB. Both these signaling pathways are modulated by peroxisome proliferator-activated receptor-alpha (PPARalpha). Therefore, we have studied the effects of fibrates and other PPARalpha agonists on the expression of TF. METHODS AND RESULTS: We show that PPARalpha protein, like primary human monocytes, is also expressed in the human monocytic THP-1 cell line. Fenofibric acid, WY14643, and GW2331 inhibited TF mRNA upregulation after stimulation of THP-1 cells with lipopolysaccharide or interleukin-1ss. In primary human monocytes and macrophages, the lipopolysaccharide- or interleukin-1ss-mediated induction of TF activity was also inhibited by fenofibric acid, WY14643, or GW2331. CONCLUSIONS: These data indicate that activation of PPARalpha results in the downregulation of the TF gene. Our results suggest a novel role for PPARalpha in the control of atherosclerotic plaque thrombogenicity through its effects on TF expression in monocytes and macrophages.

PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

Role of the peroxisome proliferator-activated receptors (PPAR) in atherosclerosis.

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors which form a subfamily of the nuclear receptor gene family. PPAR activators have effects on both metabolic risk factors and on vascular inflammation related to atherosclerosis. PPAR have profound effects on the metabolism of lipoproteins and fatty acids. PPAR alpha binds hypolipidemic fibrates, whereas PPAR gamma has a high affinity for antidiabetic glitazones. Both PPAR are activated by fatty acids and their derivatives. Activation of PPAR alpha increases the catabolism of fatty acids at several levels. In the liver, it increases uptake of fatty acids and activates their beta-oxidation. The effects that PPAR alpha exerts on triglyceride-rich lipoproteins is due to their stimulation of lipoprotein lipase and repression of apolipoprotein CIII expression, while the effects on high-density lipoproteins depend upon the regulation of apolipoproteins AI and AII. PPAR gamma has profound effects on the differentiation and function of adipose tissue, where it is highly expressed. PPAR are also expressed in atherosclerotic lesions. PPAR are present in vascular endothelial cells, smooth muscle cells, monocytes, and monocyte-derived macrophages. Via negative regulation of nuclear factor-kappa B and activator protein-1 signalling pathways, PPAR alpha inhibits expression of inflammatory genes, such as interleukin-6, cyclooxygenase-2, and endothelin-1. Furthermore, PPAR alpha inhibits expression of monocyte-recruiting proteins such as vascular cell adhesion molecule (VCAM)-1 and induces apoptosis in monocyte-derived macrophages. PPAR gamma activation in macrophages and foam cells inhibits the expression of activated genes such as inducible nitric oxide synthase, matrix metalloproteinase-9 and scavenger receptor A. PPAR gamma may also affect the recruitment of monocytes in atherosclerotic lesions as it is involved in the expression of VCAM-1 and intracellular adhesion molecule-1 in vascular endothelial cells. The involvement of PPAR in atherosclerosis, a disease with a chronic inflammatory character, suggests that they may play a role in other inflammatory-related diseases as well.

Intracellular activation of rat hepatic lipase requires transport to the Golgi compartment and is associated with a decrease in sedimentation velocity.

Hepatic lipase (HL) is an N-glycoprotein that acquires triglyceridase activity somewhere during maturation and secretion. To determine where and how HL becomes activated, the effect of drugs that interfere with maturation and intracellular transport of HL protein was studied using freshly isolated rat hepatocytes. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), castanospermine, monensin, and colchicin all inhibited secretion of HL without affecting its specific enzyme activity. The specific enzyme activity of intracellular HL was decreased by 25-50% upon incubation with CCCP or castanospermine, and increased 2-fold with monensin and colchicin. Glucose trimming of HL protein was not affected by CCCP, as indicated by digestion of immunoprecipitates with jack bean alpha-mannosidase. Pulse labeling experiments with [(35)S]methionine indicated that conversion of the 53-kDa precursor to the 58-kDa form, nor the development of endoglycosidase H-resistance, were essential for acquisition of enzyme activity. In sucrose gradients, HL protein from secretion media sedimented as a homogeneous band of about 5.8 S, whereas HL protein from the cell lysates migrated as a broad band extending from 5.8 S to more than 8 S. With both sources, HL activity was exclusively associated with the 5.8 S HL protein form. We conclude that glucose trimming of HL protein in the endoplasmic reticulum is not sufficient for activation; full activation occurs during or after transport from the endoplasmic reticulum to the Golgi and is associated with a decrease in sedimentation velocity.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Maturation and secretion of rat hepatic lipase is inhibited by alpha1B-adrenergic stimulation through changes in Ca2+ homoeostasis: thapsigargin and EGTA both mimic the effect of adrenaline.

In rats, the daily changes in hepatic lipase (HL) activity in the liver follow the diurnal rhythm of the catecholamines. To study the underlying mechanism, the effect of adrenaline on maturation and secretion of HL was determined in freshly isolated rat hepatocytes. Adrenaline (10 microM) acutely inhibited the secretion of HL. This effect was abolished by 0.1 microM prazosin, but not by 1 microM propranolol, indicating the involvement of the alpha1-adrenergic pathway. Prazosin was at least 1000-fold more potent than WB4101, a selective alpha1A-antagonist. Adrenaline had no effect on HL secretion in hepatocytes pretreated with chloroethylclonidine, an irreversible alpha1B-selective antagonist. Inhibition of HL was not induced by 10 microM methoxamine, a alpha1A-selective agonist. Thus, adrenaline inhibited HL secretion through activation of the alpha1-adrenoceptors subtype B, which have been shown to signal through Ca2+ as well as cAMP. A similar reduction in HL secretion was induced by the Ca2+-mobilizing hormones angiotensin II (100 nM) and vasopressin (12 nM), the Ca2+ ionophore A23187 (2 microM), and by thapsigargin (1 microM), which inhibits the ER Ca2+-ATPase pump. HL secretion was unaffected by elevating cAMP with 10 microM forskolin or 1 microM 8-Br-cAMP. These results suggest that the alpha1B-adrenergic effects on HL expression are mainly mediated through elevation of intracellular Ca2+. Chelation of extracellular Ca2+ and subsequent lowering of intracellular Ca2+ with EGTA also inhibited HL secretion. In pulse-chase experiments, adrenaline was shown to inhibit the maturation of HL from the 53 kDa, Endo H-sensitive precursor to the Endo H-resistant, catalytically active protein of 58 kDa. In addition, adrenaline induced intracellular degradation of newly synthesized HL. Similar post-translational effects, both qualitatively and quantitatively, were observed with A23187, thapsigargin and EGTA. We conclude that the inhibition of HL maturation and increase in intracellular degradation induced by catecholamines, A23187, thapsigargin and EGTA is evoked by changes in Ca2+ homoeostasis, possibly through lowering ER Ca2+.

Growth hormone restores hepatic lipase mRNA levels but the translation is impaired in hepatocytes of hypothyroid rats.

During hypothyroidism, hepatic lipase (HL) activity is decreased. The low HL may be due to thyroid hormone insufficiency or to the concomitant fall in growth hormone (GH) activity. We studied HL expression in hepatocytes freshly isolated from hypothyroid rats with and without additional GH-substitution. In all animals HL mRNA was detected by RT-PCR in the hepatocytes, but not in the non-parenchymal cells. In hypothyroid cells HL mRNA levels were reduced by 40%, and the in vitro secretion of HL-activity and HL-protein was decreased by about 50%. In cells from GH-substituted hypothyroid rats, HL mRNA level was normalised, but the secretion of HL remained low. The specific enzyme activity of secreted HL was similar under all conditions. The discrepancy between HL mRNA and HL secretion in GH-supplemented rats may be due to (post)translational effects. Therefore we studied the HL synthesis and maturation in hepatocytes from hypothyroid and GH-substituted rats. Pulse-labelling experiments with [(35)S]methionine showed that the incorporation of [(35)S]methionine into HL protein was lower both in hypothyroid cells and in GH-supplemented cells than in control cells. During the subsequent chase, the intracellular processing and transport of newly synthesized HL protein in the hepatocytes from hypothyroid rats, whether or not supplemented with GH, was similar to control cells. We conclude that in livers of hypothyroid, GH-substituted rats translation of HL mRNA is inhibited despite restoration of HL mRNA levels.

Acute effects of adrenaline on hepatic lipase secretion by rat hepatocytes.

Catecholamines are responsible for the daily changes in hepatic lipase (HL) expression associated with feeding and fasting. We have studied the mechanism by which adrenaline decreases HL secretion in suspensions of freshly isolated rat hepatocytes. Adrenaline acutely inhibited HL activity through activation of the alpha1-adrenergic pathway. The cells had significantly less HL activity in the presence of adrenaline versus cycloheximide, where protein de novo synthesis is completely blocked. The specific enzyme activity of secreted HL was not affected. Intracellular HL activity was decreased by adrenaline treatment. Pulse-labeling with [35S]methionine showed that de novo synthesis of the 53-kd endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive HL protein was unaffected by adrenaline. During subsequent chase of the control cells, the 53-kd form was converted to a 58-kd Endo H-resistant HL protein, which was rapidly secreted into the medium. In the presence of adrenaline, formation of the 58-kd protein was markedly reduced, whereas the 53-kd protein disappeared at a rate similar to the rate in controls. This suggests that part of the HL protein was degraded. In contrast to adrenaline, inhibition of HL secretion by colchicine was accompanied by an intracellular accumulation of HL activity and of the 58-kd protein. We conclude that adrenaline inhibits HL secretion posttranslationally by retarding the maturation of the 53-kd HL precursor to an active 58-kd protein, possibly by stimulating degradation of newly synthesized HL protein.

A novel method for measuring protein expression at the cell surface.

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)