Gene expression with corresponding pathways analysis in Gaucher disease.

Gaucher disease (GD) caused by mutation in the GBA gene has a wide spectrum of phenotypes. Besides the storage disorder, secondary alteration of various pathways occurs with modification of the expression of many genes. In our work we analysed the expression profile of genes in adult patients with type 1 GD. METHODS: This study was an observational, cross-sectional analysis of a group of twenty patients with type 1 GD and ten healthy volunteers as a control group. First, on the group of ten persons, microarray gene analysis was performed. Afterwards, significantly regulated genes were selected, and the microarray results were confirmed by real-time PCR on the whole study group. RESULTS: Based on the microarray results in the pathway analysis, we focused on genes related to chemokines, inflammatory processes, endocytosis, autophagy, and apoptosis. Patients with GD demonstrated up-regulation of genes related to NFkB pathway (NFkB, NKkBR SQSTM1), inflammation (IL-1b), endocytosis and autophagy (BCN1, SMAD), genes coding proteins involved in apoptosis (CASP, NFkB, BCL2) as well as genes related to proteasome degradation (PSMD2, PSMB9) and SNARE complex (SNAP, STX). Simultaneously, we showed down-regulation of genes coding proteins of chemokines and their receptors (GNB4, CCL5). The qRT-PCR results confirmed changes in expression of selected genes. Parallel microarray results showed inhibition of genes related to neurones development and survival (NTRK1) and stimulation of gene expression related to neurodegeneration and apoptosis (BCN1, IL1B). CONCLUSIONS: The work revealed different pathway activation, especially inflammatory processes followed by autophagy and apoptosis. Our results also pay attention to new pathways leading to disorders of the functioning of the nervous tissue in patients with type 1 GD, which may lead to the development of polyneuropathy and chronic pain. These are clinical symptoms that severely decrease the quality of life in GD patients.

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block.

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.

DNA methylation microarrays identify epigenetically regulated lipid related genes in obese patients with hypercholesterolemia.

BACKGROUND: Epigenetics can contribute to lipid disorders in obesity. The DNA methylation pattern can be the cause or consequence of high blood lipids. The aim of the study was to investigate the DNA methylation profile in peripheral leukocytes associated with elevated LDL-cholesterol level in overweight and obese individuals. METHODS: To identify the differentially methylated genes, genome-wide DNA methylation microarray analysis was performed in leukocytes of obese individuals with high LDL-cholesterol (LDL-CH, ≥ 3.4 mmol/L) versus control obese individuals with LDL-CH, < 3.4 mmol/L. Biochemical tests such as serum glucose, total cholesterol, HDL cholesterol, triglycerides, insulin, leptin, adiponectin, FGF19, FGF21, GIP and total plasma fatty acids content have been determined. Oral glucose and lipid tolerance tests were also performed. Human DNA Methylation Microarray (from Agilent Technologies) containing 27,627 probes for CpG islands was used for screening of DNA methylation status in 10 selected samples. Unpaired t-test and Mann-Whitney U-test were used for biochemical and anthropometric parameters statistics. For microarrays analysis, fold of change was calculated comparing hypercholesterolemic vs control group. The q-value threshold was calculated using moderated Student's t-test followed by Benjamini-Hochberg multiple test correction FDR. RESULTS: In this preliminary study we identified 190 lipid related CpG loci differentially methylated in hypercholesterolemic versus control individuals. Analysis of DNA methylation profiles revealed several loci engaged in plasma lipoprotein formation and metabolism, cholesterol efflux and reverse transport, triglycerides degradation and fatty acids transport and ß-oxidation. Hypermethylation of CpG loci located in promoters of genes regulating cholesterol metabolism: PCSK9, LRP1, ABCG1, ANGPTL4, SREBF1 and NR1H2 in hypercholesterolemic patients has been found. Novel epigenetically regulated CpG sites include ABCG4, ANGPTL4, AP2A2, AP2M1, AP2S1, CLTC, FGF19, FGF1R, HDLBP, LIPA, LMF1, LRP5, LSR, NR1H2 and ZDHHC8 genes. CONCLUSIONS: Our results indicate that obese individuals with hypercholesterolemia present specific DNA methylation profile in genes related to lipids transport and metabolism. Detailed knowledge of epigenetic regulation of genes, important for lipid disorders in obesity, underlies the possibility to influence target genes by changing diet and lifestyle, as DNA methylation is reversible and depends on environmental factors. These findings give rise for further studies on factors that targets methylation of revealed genes.

Omega-3 fatty acid supplementation influences the whole blood transcriptome in women with obesity, associated with pro-resolving lipid mediator production.

The n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce low-grade inflammation associated with obesity. The relationship between therapeutic response to n-3 PUFAs and modification of the transcriptome in obesity or metabolic syndrome remains to be explored. Blood samples were obtained from women with obesity before and after three-months supplementation with a moderate dose of n-3 PUFAs (1.8g EPA+DHA per day) or from controls. n-3 PUFAs (GC) and plasma concentrations of lipoxins, resolvins, protectin X (GC-MS/MS) and inflammatory markers (ELISA) were measured. Whole blood transcriptome was assayed using microarray. Women supplemented with n-3 PUFAs for 3months had significantly higher levels of EPA and DHA in plasma phosphatidylcholine. n-3 PUFA supplementation, in contrast to placebo, significantly decreased the concentrations of several inflammatory markers (SELE, MCP-1, sVCAM-1, sPECAM-1, and hsCRP), fasting triglycerides and insulin and increased the concentrations of pro-resolving DHA derivatives in plasma. The microarray data demonstrated effects of n-3 PUFAs on PPAR-α, NRF2 and NF-κB target genes. N-3 PUFAs increased DHA-derived pro-resolving mediators in women with obesity. Elevated resolvins and up-regulation of the resolvin receptor occurred in parallel with activation of PPAR-α target genes related to lipid metabolism and of NRF2 up-regulated antioxidant enzymes.

Influence of dietary fatty acids on differentiation of human stromal vascular fraction preadipocytes.

Mediators such as cytokines, eicosanoids, nitric oxide and growth factors may regulate adipogenesis as well as inflammation. It is well documented that production of some form of eicosanoids activates lipid synthesis during adipogenesis but also contributes to the formation of factors maintaining low-level systemic inflammation. Developing nutrients for reduction of adipogenesis and inflammation can enhance preventive efficacy of daily diet. This study examined the effects of free fatty acid influence on changes in lipid biosynthesis and corresponding gene expression during differentiation of human subcutaneous adipose tissue stromal vascular fraction (SVF) cells. Proadipogenic conditions promoted SVF cell differentiation and lipid droplet (LD) formation up to 15 days. This correlated with gene expression of adipocyte differentiation markers as well as inflammatory cytokines and their receptors. Addition of free fatty acids to differentiation medium increased their incorporation during the first period of differentiation (48 h). Presence of eicosanoid acid (EPA) during the initial period of differentiation by elevation of Perilipin 3 protein (TIP47), may be responsible for smaller LD formation. Presence of arachidonic acid (AA) tends to deposit lipids in large form of LDs. Prolongation of differentiation up to 15 days decreased AA or EPA in cellular lipids. PUFA through up-regulation of both phospholipase 2 and enzymes related to eicosanoid production influenced type and quantity of eicosanoids which regulated the extent of SVF cell differentiation. Formation of small LDs and reduction of pro-inflammatory mediators in adipose tissue are the consequence of eicosanoid production with anti-inflammatory potential from EPA.

[DNA methylation in obesity]. / Metylacja DNA a otylosc prosta.

The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes), have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA) synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

Aortic valve and arterial calcification in patients with familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (heFH) is an autosomal dominant lipid metabolism disorder. Its prevalence is 1:250-1:300 people in the population. Patients with heFH have an up to 13-fold increased risk of premature coronary artery disease (CAD). If left untreated, men and women with heFH typically develop early CAD before the ages of 55 and 60, respectively. There is evidence that coronary artery calcification (CAC) and aortic valve calcification (AoVC) are more prevalent in FH patients than in the general population. It is documented that CAC and AoVC are predictors of increased risk of cardiovascular morbidity and mortality in heFH patients, like in the general population. However, the etiology and pathogenesis of vascular calcification in FH patients is not well understood. Risk factors for vascular calcification include age, increased levels of atherogenic lipoproteins, Lp(a), increased blood pressure, and inflammation. There are convincing data from clinical studies and animal atherosclerotic mouse models using low-density lipoprotein receptor (LDL-R) knockout mice that the vascular calcification processes in FH are associated with LDL-R mutations, probably partly due to a higher total cholesterol burden of FH subjects. Data from animal models as well as clinical studies indicate that the Wnt/beta-catenin pathway components and LDL receptor-related proteins 5 and 6 (LRP-5/6) might be involved in calcification processes in FH patients. The purpose of the review is to describe the prevalence of coronary and aortic calcification and its risk factors in FH patients. The review covers data about the role of the Wnt/beta-catenin pathway and factors modulating calcification processes.

Analysis of tafazzin and deoxyribonuclease 1 like 1 transcripts and X chromosome sequencing in the evaluation of the effect of mosaicism in the TAZ gene on phenotypes in a family affected by Barth syndrome.

Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the TAZ gene in two affected patients with congenital cardiomyopathy. Furthermore, women in this family had no mutations in their blood cells, whereas they only had mutations in the oral epithelial cells. The objective of the project was to evaluate the effect of intertissue mosaicisms on the Barth syndrome phenotypes, searching for another disease-related loci on chromosome X and finally to assess the consequences of the mutation. We conducted the advanced genetic study including cytogenetic research (constitutional karyotyping in blood and fibroblasts), NGS sequencing (with custom chromosome X sequencing together with the evaluation of loss of heterozygosity (LOH) and aberrations (CNV) in the whole genome) in four different tissues and sequencing of tafazzin and deoxyribonuclease 1 like 1 transcripts. The presence of deletions within the 5'untranslated region of the TAZ gene and/or the noncoding regions of the DNASE1L1 gene were detected in several tissues. Whereas, there is no intertissue mosaicism regarding point mutation in TAZ gene in all investigated tissues in female carriers. Only the male patient presented biochemical markers and neurological symptoms of Barth syndrome. All the female carriers are healthy and have normal tafazzin and deoxyribonuclease 1 like 1 transcripts in 2 analyzed tissues. The conclusion of this study is that we cannot rule out or confirm mosaicism in the noncoding regions of TAZ or DNASE1L1 genes, but this is not clinically relevant in female carriers because they are healthy. Finally, it has been proven that mutation (c.83T>A, p.Val28Glu) is responsible for disease in males in this family.

A Period 2 genetic variant interacts with plasma SFA to modify plasma lipid concentrations in adults with metabolic syndrome.

Genetic variants of Period 2 (PER2), a circadian clock gene, have been linked to metabolic syndrome (MetS). However, it is still unknown whether these genetic variants interact with the various types of plasma fatty acids. This study investigated whether common single nucleotide polymorphisms (SNPs) in the PER2 locus (rs934945 and rs2304672) interact with various classes of plasma fatty acids to modulate plasma lipid metabolism in 381 participants with MetS in the European LIPGENE study. Interestingly, the rs2304672 SNP interacted with plasma total SFA concentrations to affect fasting plasma TG, TG-rich lipoprotein (TRL-TG), total cholesterol, apoC-II, apoB, and apoB-48 concentrations (P-interaction < 0.001-0.046). Carriers of the minor allele (GC+GG) with the highest SFA concentration (>median) had a higher plasma TG concentration (P = 0.001) and higher TRL-TG (P < 0.001) than the CC genotype. In addition, participants carrying the minor G allele for rs2304672 SNP and with a higher SFA concentration (>median) had higher plasma concentrations of apo C-II (P < 0.001), apo C-III (P = 0.009), and apoB-48 (P = 0.028) compared with the homozygotes for the major allele (CC). In summary, the rs2304672 polymorphism in the PER2 gene locus may influence lipid metabolism by interacting with the plasma total SFA concentration in participants with MetS. The understanding of these gene-nutrient interactions could help to provide a better knowledge of the pathogenesis in MetS.

Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance.

BACKGROUND: High fat diet impairs nitric oxide (NO) bioavailability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. The purpose of this study was to investigate the effect of high fat diet on metabolism in mice with decreased (eNOS-/-) and increased (DDAH overexpressed) NO bioavailability. METHODS: eNOS-/- (n = 16), DDAH (n = 24), and WT (n = 19) mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and insulin were monitored. The matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis. Gene expression in adipose tissues was analyzed by microarray and Real Time PCR. Comparisons of the mean values were made using the unpaired Student t test and p < 0.05 were considered statistically significant. RESULTS: eNOS-/- mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose level was observed. Fasting insulin and cholesterol levels remained unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-regulated, whereas in eNOS-/- mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were upregulated. CONCLUSIONS: Our results indicate that increased NO availability attenuates some HFD induced alterations in metabolism and gene expression associated with insulin resistance.

[Humanin and its derivatives as peptides with potential antiapoptotic and confirmed neuroprotective activities]. / Humanina i jej analogi jako peptydy o potencjalnym dzialaniu antyapoptotycznym i potwierdzonych wlasciwosciach neuroprotekcyjnych.

Humanin (HN) is a newly discovered 24-amino acid peptide, which may suppress neuronal cell death. HN cDNA includes the open reading frame (HN-ORF) of 75 bases, located 950 bases downstream of the 5' end of the HN cDNA. It was demonstrated that HN cDNA is 99% identical with mitochondrial DNA (mtDNA) sequence. HN homologues have been identified as expressed sequence tags (ESTs) in rat and nematode. Certain regions homologous to the HN cDNA exist on human chromosomes. HN forms homodimers and multimers and this seems to be essential for the peptide functions. HN acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2). It was demonstrated that HN plays a protective role by an antiapoptotic activity interfering with Bax activation, and suppressing Bax-dependent apoptosis. HN is also shown to suppress the c-Jun N-terminal kinase (JNK) and ASK/JNK-mediated neuronal cell death. Several studies also confirm that HN could be important in prevention of angiopathy-associated Alzheimer's disease dementia, diseases related to mitochondrial dysfunction (MELAS), and other types of beta-amyloid accumulation associated neurodegeneration. A very recent study demonstrated a pluripotent cytoprotective effect and mechanisms of HNs in cells other than from the CNS, such as germ cells, or panreatic b-cells, and potent physiological consequences that result from HN interaction with IGFBP3 and STAT3. The in vivo studies suggest that humanin may protect against cognitive impairment, also due to ischemia/reperfusion injury.

Proteomic biomarkers in Gaucher disease.

AIMS: The research work was conducted to find new biomarkers and potential drug targets in Gaucher disease type 1 (GDt1) by analysing the serum proteins. METHODS: This study was an observational, cross-sectional analysis of a group of 12 adult participants: six Gaucher disease (GD) patients and six healthy control. Fasting venous blood underwent proteomics analysis and molecular tests. Over 400 proteins were analysed, and in case of significantly different concentrations between the study and control group, we checked corresponding genes to confirm changes in their expression and consistency with protein alteration. RESULTS: We found 31 proteins that significantly differed in concentration between GDt1 patients and a control group. These were mostly proteins involved in the regulation of the inflammatory processes and haemostasis. The levels of proteins such as alpha-1-acid glycoprotein 2, S100-A8/A9, adenyl cyclase-associated protein 1, haptoglobin or translationally controlled tumour protein related to inflammation process were significantly higher in GD patients than in control group, whereas the levels of some proteins such as heavy constant mu and gamma 4 or complement C3/C4 complex involved in humoral response like immunoglobulins were significantly decreased in GD patients. Alteration in two proteins concentration was confirmed in RNA analysis. CONCLUSIONS: The work revealed few new targets for further investigation which may be useful in clinical practice for diagnosis, treatment and monitoring GDt1 patients.

Epigenetic Regulation of Processes Related to High Level of Fibroblast Growth Factor 21 in Obese Subjects.

We hypothesised that epigenetics may play an important role in mediating fibroblast growth factor 21 (FGF21) resistance in obesity. We aimed to evaluate DNA methylation changes and miRNA pattern in obese subjects associated with high serum FGF21 levels. The study included 136 participants with BMI 27-45 kg/m2. Fasting FGF21, glucose, insulin, GIP, lipids, adipokines, miokines and cytokines were measured and compared in high serum FGF21 (n = 68) group to low FGF21 (n = 68) group. Human DNA Methylation Microarrays were analysed in leukocytes from each group (n = 16). Expression of miRNAs was evaluated using quantitative PCR-TLDA. The study identified differentially methylated genes in pathways related to glucose transport, insulin secretion and signalling, lipid transport and cellular metabolism, response to nutrient levels, thermogenesis, browning of adipose tissue and bone mineralisation. Additionally, it detected transcription factor genes regulating FGF21 and fibroblast growth factor receptor and vascular endothelial growth factor receptor pathways regulation. Increased expression of hsa-miR-875-5p and decreased expression of hsa-miR-133a-3p, hsa-miR-185-5p and hsa-miR-200c-3p were found in the group with high serum FGF21. These changes were associated with high FGF21, VEGF and low adiponectin serum levels. Our results point to a significant role of the epigenetic regulation of genes involved in metabolic pathways related to FGF21 action.

MiRNA Expression in Patients with Gaucher Disease Treated with Enzyme Replacement Therapy.

AIMS: The aim of the work was to establish potential biomarkers or drug targets by analysing changes in miRNA concentration among patients with Gaucher disease (GD) compared to in healthy subjects. METHODS: This study was an observational, cross-sectional analysis of 30 adult participants: 10 controls and 20 adults with GD type 1. Patients with GD type 1 were treated with enzyme replacement therapy (ERT) for at least two years. The control group was composed of healthy volunteers, unrelated to the patients, adjusted with age, sex and body mass index (BMI). The miRNA alteration between these groups was examined. After obtaining preliminary results on a group of six GD patients by the high-output method (TaqMan low-density array (TLDA)), potential miRNAs were selected for confirming the results by using the qRT-PCR method. With Diane Tools, we analysed miRNAs of which differential expression is most significant and their potential role in GD pathophysiology. We also determined the essential pathways these miRNAs are involved in. RESULTS: 266 dysregulated miRNAs were found among 753 tested. Seventy-eight miRNAs were downregulated, and 188 were upregulated. Thirty miRNAs were significantly altered; all of them were upregulated. The analysis of pathways regulated by the selected miRNAs showed an effect on bone development, inflammation or regulation of axonal transmission in association with Parkinson's disease. CONCLUSIONS: We revealed few miRNAs, like miR-26-5p, which are highly altered and fit the GD pathophysiological model, might be considered as novel biomarkers of disease progression but need further evaluation.

Specific gene expression in type 1 diabetic patients with and without cardiac autonomic neuropathy.

We hypothesized that some molecular pathways might interact to initiate the process of nervous tissue destruction, promoting cardiac autonomic neuropathy (CAN) in the course of diabetes type 1 (T1D). The study group consisted of 60 T1D patients (58.33% women/41.67% men), on standard therapy. The control group consisted of twenty healthy volunteers recruited in accordance with age, gender and body weight. The presence of CAN was documented by the Ewing test method (ProSciCard apparatus). A microarray data analysis was performed using Gene Spring version 13. The microarray results for selected genes were confirmed by real-time PCR (qRT-PCR), using specific TaqMan Gene Expression Assays. Plasma IL-6 content was measured by an enzyme-linked immunosorbent assay (ELISA). The p < 0.05 value was considered as statistically significant. The microarray analysis, confirmed by qRTPCR, showed significant up-regulation of autophagy, quantity of mitochondria, quality regulatory genes (mTOR, GABARAPL2) apoptosis, ER-stress and inflammation (NFKB1, IL1b, IL1R1, SOD1), in T1D when compared to the control group. A significantly higher IL-6 protein level was observed in T1D patients, in comparison to the control group. We concluded that the observed changes in gene expression and activation of intracellular pathways give a coherent picture of the important role of oxidative stress in inflammation and the activation of apoptosis in the pathomechanism of DM. The significance of the inflammatory process, confirmed by the increased level of the inflammation biomarker IL-6 in the pathomechanisms of CAN was shown even in patients with properly treated T1D.

Epigenetic mechanism in search for the pathomechanism of diabetic neuropathy development in diabetes mellitus type 1 (T1DM).

OBJECTIVE: The aim of this study was to check the hypothesis concerning the crucial role of DNA methylation (one of the epigenetic mechanisms) within selected genes related to the destruction and regeneration of neural cells and its input in the pathogenesis of diabetic neuropathy, using a model of the DNA in peripheral blood cells. METHODS: A cross-sectional, case-control study was conducted, consisting of 24 adult Type 1 Diabetes Melitus (T1DM) patients with autonomic neuropathy (CAN), 25 T1DM patients without neuropathy and 25 matched, healthy adults acting as a control (Ctrl). The Ewing's tests, using the ProSciCard apparatus (Mewicon CATEEM-Tec GmbH), was employed to assess the severity of the patients' symptoms of autonomic neuropathy. For DNA methylation analysis, DNA material of each sample DNA after bisulfite conversion was used for the hybridization of BeadChips (Infinium Methylation EPIC Kit, Illumina), and imaged on the Illumina HiScan. The changes in the expression of selected genes were examined using real-time PCR. Probes were labeled using fluorescein amidite, FAM (Thermo Fisher Scientific). Amplification was performed using the continuous fluorescence detection 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific). The expression ratio of the target mRNA was normalized to the level of 18s RNA and compared with the control. Statistical analysis was performed using Statistica version 13.1. The statistically significant results were recognized, with a value of p < 0.05. RESULTS: Clinical analysis of the investigated groups revealed a significantly higher percentage of personal insulin pump users in the group without neuropathy. The glucose metabolic control, based on the HbA1c level analysis, was also significantly better in T1DM patients without CAN. The Bumphunter method for DNA methylation analysis showed statistically significant regions related to the genes involved in nerve regeneration ninjurin 2 (NINJ2) and functionality (BR serine/threonine kinase 2 BRSK2, claudin 4 CLDN4). When compared with T1DM patients without neuropathy, T1DM patients with neuropathy showed significantly increased methylation in the first NINJ2 axon, and a lower level of DNA methylation in the region of the first intron of BRSK2, as well as the CLDN4 5'UTR regions. The qRT-PCR results confirmed the decreased expression of NINJ2 and CLDN4 genes in patients with T1DM with CAN. CONCLUSIONS: The different DNA methylation profiles, correlating with the expression of genes related to nervous tissue development and regeneration in patients with T1DM with autonomic neuropathy provide evidence for the role of epigenetic mechanisms promoting the development of CAN, a chronic complication of T1DM.

Enhanced GIP Secretion in Obesity Is Associated with Biochemical Alteration and miRNA Contribution to the Development of Liver Steatosis.

Nutrient excess enhances glucose-dependent insulinotropic polypeptide (GIP) secretion, which may in turn contribute to the development of liver steatosis. We hypothesized that elevated GIP levels in obesity may affect markers of liver injury through microRNAs. The study involved 128 subjects (body mass index (BMI) 25-40). Fasting and postprandial GIP, glucose, insulin, and lipids, as well as fasting alanine aminotransferase (ALT), γ-glutamyltransferase (GGT), cytokeratin-18, fibroblast growth factor (FGF)-19, and FGF-21 were determined. TaqMan low density array was used for quantitative analysis of blood microRNAs. Fasting GIP was associated with ALT [ß = 0.16 (confidence interval (CI): 0.01-0.32)], triglycerides [ß = 0.21 (95% CI: 0.06-0.36], and FGF-21 [ß = 0.20 (95%CI: 0.03-0.37)]; and postprandial GIP with GGT [ß = 0.17 (95%CI: 0.03-0.32)]. The odds ratio for elevated fatty liver index (>73%) was 2.42 (95%CI: 1.02-5.72) for high GIP versus low GIP patients. The miRNAs profile related to a high GIP plasma level included upregulated miR-136-5p, miR-320a, miR-483-5p, miR-520d-5p, miR-520b, miR-30e-3p, and miR-571. Analysis of the interactions of these microRNAs with gene expression pathways suggests their potential contribution to the regulation of the activity of genes associated with insulin resistance, fatty acids metabolism, and adipocytokines signaling. Exaggerated fasting and postprandial secretion of GIP in obesity are associated with elevated liver damage markers as well as FGF-21 plasma levels. Differentially expressed microRNAs suggest additional, epigenetic factors contributing to the gut-liver cross-talk.

Angiogenesis in the New Zealand obese mouse model fed with high fat diet.

BACKGROUND: Obesity and its complications lead to vascular injury, atherosclerosis, diabetes and pathological angiogenesis. One of the models to study the obesity and its entanglements is the New Zealand Obese mice model. Aim of this study was to check the effect of high fat diet on changes in biochemical parameters as well as on process of angiogenesis in NZO mice. METHODS: NZO mice were fed with standard (ST) or high fat (HF) diet for seven weeks. Body weight and serum biochemical parameters were monitored. The PECAM1 positive vessel-like structures immunostaining, as well as the gene expression of the matrigel penetrating cells by microarray (confirmed by real-time PCR method) were analyzed. RESULTS: Mice fed with HF diet developed obesity. Number of newly created vessels with lumen was correlated with hyperglycemia and animal weight gain. The number of PECAM1 positive cells in matrigel tended to increase during HF diet. Microarray results revealed changes in gene expression (activation of the oxidative stress and insulin resistance, inhibition of apoptosis and cell differentiation), however without markers of endothelial cell network maturation. CONCLUSION: Observed changes in the NZO mice on HF diet argue for the hyperglycemia related activation of angiogenesis, leading to the formation of pathological, immature network.

Effect of insulin resistance on whole blood mRNA and microRNA expression affecting bone turnover.

OBJECTIVE: To evaluate the effect of insulin resistance in obesity on the expression in whole blood of mRNA and miRNA affecting bone homeostasis as well as to estimate the influence of oral glucose load (OGTT) on serum osteocalcin concentration in obese individuals with and without insulin resistance. DESIGN: Cross-sectional study. METHODS: Carboxylated (cOC), undercarboxylated (ucOC) and total osteocalcin were measured by ELISA in the serum of obese subjects with insulin resistance (n = 41) and obese subjects without insulin resistance (n = 41) (control group) during OGTT. Analysis of gene expression (microarray) and miRNAs (real-time PCR) was performed in venous blood (representating samples) collected before OGTT from obese with insulin resistance and controls. RESULTS: Obese subjects with insulin resistance (higher HOMA-IR and lower oral glucose insulin sensitivity index) presented significantly increased expression of WNT signalling inhibitors (DKK1, DKK2, SOST, SFRP1) and downregulation of the key factor in WNT signalling - ß catenin participating in osteoblasts differentiation. Expression of miRNA involved in osteoblastogenesis was also inhibited (miR-29b, miR-181a, miR-210, miR-324-3p). During OGTT, contrary to the control group, subjects with insulin resistance presented suppression of cOC and total OC decrease after 1 and 2 h of oral glucose load. CONCLUSIONS: Obese subjects with insulin resistance may have defects in osteoblastogenesis that was demonstrated via key signalling molecules mRNA downregulation, and increased expression of WNT antagonists as well as inhibition of expression of miRNA participating in the regulation of osteoblast differentiation. Disturbed osteoblastogenesis in insulin-resistant subjects results in the suppression of blood carboxylated and total osteocalcin decrease during OGTT.

Case report of dysregulation of primary bile acid synthesis in a family with X-linked adrenoleukodystrophy.

RATIONALE: X-linked adrenoleukodystrophy (X-ALD) is a rare disorder caused by mutations in the ABCD1 gene, coding for peroxisomal membrane transporter adrenoleukodystrophy (ALD) protein. The disease is characterized by accumulation of very long chain fatty acids (VLCFAs) in tissues. Adult adrenomyeloneuropathy (AMN) and the cerebral inflammatory form of ALD are the main phenotypes presenting various symptoms. PATIENT CONCERNS: We report a case of 37-year-old patient with diagnosis of X-ALD, confirmed based on elevated VLCFA concentrations and genetic testing of ABCD1 gene. The complete clinical picture in the patient indicates AMN phenotype with cerebral involvement. DIAGNOSES: The reduced synthesis of unconjugated cholic and chenodeoxycholic acids, and the reduction to 28% to 29% of peroxisomal beta-oxidation of behenic acid and normal peroxisomal metabolism of pristanic and palmitic acid were observed in the X-ALD patient. Sanger sequencing of major genes involved in primary bile acid (BA) synthesis failed to identify pathogenic mutations of the investigated set of genes. INTERVENTIONS: Plasma concentrations of BAs, VLCFAs, and beta-oxidation of C22:0, C16:0, and pristanic acid were studied in primary skin fibroblasts of the patient. In addition, we performed sequencing of the ABCD1, ABCD3, CYP7A1, CYP7B1, CYP27A1, HSD3B7, AKR1D1, and SLC27A5 genes in the X-ALD family. OUTCOMES: In the Polish family affected with AMN a dysregulation of the primary BA synthesis pathway was found. LESSONS: We have demonstrated the coincidence of the adult form of X-ALD with abnormalities in BA synthesis. We suggest that decreased synthesis of BAs may be an additional dysfunction as a consequence of the ABCD1 c.659T>C, p.(Leu220Pro) mutation and may be further evidence that disturbed cholesterol metabolism is important in the pathology of ALD.