AGR2 silencing contributes to metformin-dependent sensitization of colorectal cancer cells to chemotherapy.

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells.

Differences in food intake and genetic variability in taste receptors between Czech pregnant women with and without gestational diabetes mellitus.

PURPOSE: Gestational diabetes mellitus (GDM) represents the most frequent metabolic disorder in pregnancy. Since dietary intake plays an important role in obesity and type 2 diabetes development, it is likely to be for the susceptibility to GDM too. Food preferences, driving partly the diet composition, are changing during pregnancy. Taste and genetic variability in taste receptors is an important factor in determining food preferences. Aims of our study were (1) to characterize dietary habits of pregnant women and to find possible differences in food preferences between healthy pregnant women and those with GDM and (2) to ascertain possible association of several single nucleotide polymorphisms (SNPs) in taste receptor (TR) genes with GDM. METHODS: A total of 363 pregnant women (293 with GDM and 70 with physiologic pregnancy) were included in the study. Dietary pattern spanning the period of approx. 6 months preceding the time of GDM screening was assessed using a semi-quantitative food frequency questionnaire. A total of five SNPs in TR genes were selected for genotyping based on their functionality or previous associations. RESULTS: Women with GDM exhibited significantly more frequent meat consumption (esp. poultry, pork and smoked meat), dairy products and sweet beverages consumption. The legumes consumption was found to be inversely correlated with fasting glycaemia (P = 0.007, Spearman). CC genotype in TAS2R9 gene (SNP rs3741845) was significantly associated with GDM (P = 0.0087, Chi-square test). CONCLUSIONS: Our study showed differences in dietary intake of selected food items between healthy pregnant women and those with GDM and genetic association of bitter taste receptor allele with GDM.

Hyperuricemia contributes to the faster progression of diabetic kidney disease in type 2 diabetes mellitus.

AIMS: The aims of the study were (i) to ascertain prognostic value of serum uric acid (SUA) for diabetic kidney disease (DKD) progression and major adverse cardiovascular event (MACE) in a cohort of T2DM patients, (ii) to ascertain eventual protective effect of allopurinol treatment, (iii) to determine the effect of genetic variability in UA transporters on DKD progression, and (iv) to define optimal cut-off values for SUA in patients with DKD. METHODS: Study comprised 422 subjects with diabetes duration at least 15years followed-up for a median of 43 [IQR 22-77] months. Participants were categorized into stable or progressors according to their change in albuminuria or chronic kidney disease (CKD) stage. At baseline, 68% patients had hyperuricemia (SUA≥420µmol/l for men and ≥360µmol/l for women and/or allopurinol treatment). Five SNPs in the SLC2A9 and ABCG2 genes were determined by PCR. RESULTS: Time-to-event analysis with subgroups defined by the presence/absence of initial hyperuricemia revealed significant differences in all three end-points (P<0.0001 for DKD progression, P=0.0022 for MACE and P=0.0002 for death, log-rank test). Subjects with normal SUA not requiring allopurinol had median time to DKD progression 49months compared with remaining subjects (32months, P=0.0002, log-rank test). Multivariate Cox regression model revealed hyperuricemia (i.e. high SUA and/or allopurinol treatment) significant predictor of DKD progression independent of baseline CKD stage. Optimal cut-off values identified by ROC analysis for T2DM subjects were ≤377.5µmol/l for men and ≤309.0µmol/l for women. We found no differences in allele or genotype frequencies in selected SNPs between patients with and without hyperuricemia (all P>0.05). CONCLUSIONS: Our study demonstrated that initial hyperuricemia or need for allopurinol is an independent risk factor for DKD progression and that SUA levels in diabetic subjects conferring protection against DKD progression might be lower than current cut-offs for general population.

Dysfunctional protection against advanced glycation due to thiamine metabolism abnormalities in gestational diabetes.

While the pathogenic role of dicarbonyl stress and accelerated formation of advanced glycation end products (AGEs) to glucose intolerance and to the development of diabetic complications is well established, little is known about these processes in gestational diabetes mellitus (GDM), a condition pathogenically quite similar to type 2 diabetes. The aims of the present study were (i) to determine plasma thiamine and erythrocyte thiamine diphosphate (TDP) and transketolase (TKT) activity in pregnant women with and without GDM, (ii) to assess relationships between thiamine metabolism parameters and selected clinical, biochemical and anthropometric characteristics and, finally, (iii) to analyse relationship between variability in the genes involved in the regulation of transmembrane thiamine transport (i.e. SLC19A2 and SLC19A3) and relevant parameters of thiamine metabolism. We found significantly lower plasma BMI adjusted thiamine in women with GDM (P = 0.002, Mann-Whitney) while levels of erythrocyte TDP (an active TKT cofactor) in mid-trimester were significantly higher in GDM compared to controls (P = 0.04, Mann-Whitney). However, mid-gestational TKT activity - reflecting pentose phosphate pathway activity - did not differ between the two groups (P > 0.05, Mann-Whitney). Furthermore, we ascertained significant associations of postpartum TKT activity with SNPs SLC19A2 rs6656822 and SLC19A3 rs7567984 (P = 0.03 and P = 0.007, resp., Kruskal-Wallis). Our findings of increased thiamine delivery to the cells without concomitant increase of TKT activity in women with GDM therefore indicate possible pathogenic role of thiamine mishandling in GDM. Further studies are needed to determine its contribution to maternal and/or neonatal morbidity.

Effect of glucose variability on pathways associated with glucotoxicity in diabetes: Evaluation of a novel in vitro experimental approach.

AIMS: Glycaemic variability (GV) has been hypothesized to increase the risk of diabetes complications; however, results of clinical studies are contradictory. The effect of GV on cell phenotypes has been investigated in vitro showing that GV may have more deleterious effect on cells that high glucose itself. However, methodology used to study GV in vitro differs significantly between studies and does not reflect in vivo situation. Therefore we aimed to establish clinically relevant an in vitro experimental approach for the study of GV that reflects intra-day glucose fluctuations of subjects with type 1 diabetes mellitus (T1DM) and of healthy subjects and to test how low and high GV affect expression of genes that protects cells from hyperglycaemia-induced damage. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured 24h in medium with different glucose profiles: high GV, low GV and GV of healthy subjects-profiles created according to CGM of T1DM patients and healthy subjects. These profiles were compared to commonly used 5.5 and 25mmol/l glucose concentrations. Gene expression was determined using quantitative PCR. RESULTS: Our results showed general down-regulation of enzymes that are involved in the protection against hyperglycaemia-induced intracellular changes in both low and high GV compared to normal glycaemia similarly to the decrease induced by continuous hyperglycaemia. Gene expressions did not differ between high and low GV. CONCLUSION: Our data indicate that GV may have similar or even greater effect than continuous hyperglycaemia on the expression of several genes relevant to pathogenesis of diabetes microvascular complications.

1,25-Dihydroxyvitamin D increases the gene expression of enzymes protecting from glucolipotoxicity in peripheral blood mononuclear cells and human primary endothelial cells.

Besides its classical function as an orchestrator of calcium and phosphorus homeostasis, vitamin D also affects insulin secretion and tissue efficiency. A number of studies have consistently reported the inverse relationship between vitamin D deficiency and type 2 diabetes. Activation of certain metabolic pathways and down-stream transcription factors may protect from glucolipotoxicity and their targeted activation -e.g. by vitamin D - might explain the detrimental role of vitamin D deficiency in diabetes. The aim of the study was to quantify gene and protein expression of selected enzymes involved in the protection from glucolipotoxicity, specifically glyoxalase 1 (GLO1), and other enzymes with antioxidant activity - hemoxygenase (HMOX), thiamin pyrophosphokinase (TPK1) and transketolase (TKT), under normo- and hyperglycemic conditions and upon addition of vitamin D in peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). The results of our study indicate that the active form of vitamin D regulates gene expression of enzymes opposing the harmful effect of glucolipotoxicity whose activities appear to be suppressed by hyperglycemia. However, we were unable to confirm this effect on protein expression. While we cannot speculate on the effect of vitamin D on diabetes itself our results support its role in the protection against existing glucolipotoxicity therefore possibly translating into the prevention of development of diabetic complications.

Serum carboxymethyl-lysine, a dominant advanced glycation end product, is increased in women with gestational diabetes mellitus.

AIMS: The objective of the study was to measure one of the circulating Advanced Glycation End Products (AGEs) - Nε-(carboxymethyl)lysine (CML) - in a case-control study (n = 307) of pregnant women with gestational diabetes mellitus (GDM) and physiological pregnancies and to ascertain the factors contributing to CML levels and the potential relevance of CML for selected perinatal and postpartum outcomes. METHODS: All subjects underwent oGTT between 24th and 30th week of gestation and GDM was diagnosed according to WHO criteria. CML was determined by ELISA using commercial kit. RESULTS: Unadjusted and plasma protein adjusted CML levels were significantly higher in women with GDM compared to healthy controls (P = 0.00043 and P = 1x10(-5), respectively, Mann-Whitney). CML was significantly inversely correlated with both pre- and mid-gestational BMI, however, differences between GDM and control group remained significant even after adjustment for BMI. CML levels correlated with 1-h and 2-h post-load glycaemia during oGTT. CONCLUSION: In conclusion, we found statistically significantly higher protein- and BMI-normalised CML levels measured during 24-30th week of gestation in women with GDM compared to healthy pregnant controls. Further studies are warranted to comprehensively asses the spectrum of AGEs in GDM and their relevance to future metabolic health of mother and offspring.

Vitamin D Status in Women with Gestational Diabetes Mellitus during Pregnancy and Postpartum.

Of many vitamin D extraskeletal functions, its modulatory role in insulin secretion and action is especially relevant for gestational diabetes mellitus (GDM). The aims of the present study were to determine midgestational and early postpartum vitamin D status in pregnant women with and without GDM and to describe the relationship between midgestational and postpartum vitamin D status and parallel changes of glucose tolerance. A total of 76 pregnant women (47 GDM and 29 healthy controls) were included in the study. Plasma levels of 25(OH)D were measured using an enzyme immunoassay. Vitamin D was not significantly decreased in GDM compared to controls during pregnancy; however, both groups of pregnant women exhibited high prevalence of vitamin D deficiency. Prevalence of postpartum 25(OH)D deficiency in post-GDM women remained significantly higher and their postpartum 25(OH)D levels were significantly lower compared to non-GDM counterparts. Finally, based on the oGTT repeated early postpartum persistent glucose abnormality was ascertained in 15% of post-GDM women; however, neither midgestational nor postpartum 25(OH)D levels significantly differed between subjects with GDM history and persistent postpartum glucose intolerance and those with normal glucose tolerance after delivery.

Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues.

Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five CHASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1-BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. The identified bacterial artificial chromosome (BAC) clones were fingerprinted and representative clones in five distinct groups were sequenced. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1-BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1-3nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively. Interestingly, the AHK2 homologues could be divided into two subfamilies (BnCHK1/BnCK2 and BnCHK3/BnCHK4) that differ in putative transmembrane domain topology and CK binding specificity, thus implying potential functional divergence.

Evidence for altered thiamine metabolism in diabetes: Is there a potential to oppose gluco- and lipotoxicity by rational supplementation?

Growing prevalence of diabetes (type 2 as well as type 1) and its related morbidity due to vascular complications creates a large burden on medical care worldwide. Understanding the molecular pathogenesis of chronic micro-, macro- and avascular complications mediated by hyperglycemia is of crucial importance since novel therapeutic targets can be identified and tested. Thiamine (vitamin B1) is an essential cofactor of several enzymes involved in carbohydrate metabolism and published data suggest that thiamine metabolism in diabetes is deficient. This review aims to point out the physiological role of thiamine in metabolism of glucose and amino acids, to present overview of thiamine metabolism and to describe the consequences of thiamine deficiency (either clinically manifest or latent). Furthermore, we want to explain why thiamine demands are increased in diabetes and to summarise data indicating thiamine mishandling in diabetics (by review of the studies mapping the prevalence and the degree of thiamine deficiency in diabetics). Finally, we would like to summarise the evidence for the beneficial effect of thiamine supplementation in progression of hyperglycemia-related pathology and, therefore, to justify its importance in determining the harmful impact of hyperglycemia in diabetes. Based on the data presented it could be concluded that although experimental studies mostly resulted in beneficial effects, clinical studies of appropriate size and duration focusing on the effect of thiamine supplementation/therapy on hard endpoints are missing at present. Moreover, it is not currently clear which mechanisms contribute to the deficient action of thiamine in diabetes most. Experimental studies on the molecular mechanisms of thiamine deficiency in diabetes are critically needed before clear answer to diabetes community could be given.

Genetic variability in enzymes of metabolic pathways conferring protection against non-enzymatic glycation versus diabetes-related morbidity and mortality.

BACKGROUND: We hypothesized that genetic variability in genes encoding enzymes metabolizing glycolytic intermediates produced in excess under hyperglycemic conditions [i.e., transketolase (TKT), transaldolase, TKT-like protein 1, fructosamine 3-kinase (FN3K), glyoxalase 1 and glucose-6-phosphate dehydrogenase] could influence progression of diabetic nephropathy (DN) and diabetes-related morbidity and mortality. METHODS: A total of 19 single nucleotide polymorphisms (SNPs) in six candidate genes were studied in 314 type 2 diabetic subjects with variable stage of kidney disease (normo- and microalbuminuria, proteinuria, end-stage renal disease). SNP selection criteria were based on known functional effect and gene coverage. SNPs were detected using polymerase chain reaction based methods. Subjects were followed up for median of 38 months. Time-to-event analysis considered three end-points: 1) DN progression by at least one stage; 2) major cardiovascular event; and 3) all-cause mortality. RESULTS: We found combined effect of TKT SNP rs11130362 and FN3K SNP rs1056534 on DN progression (p<0.01). Additionally, TKT rs3736156 alone and also in combination with the previous two SNPs exhibited significant effect on incidence of major cardiovascular events (p<0.01 and p=0.01, respectively). CONCLUSIONS: Genetic variability in rate-limiting enzymes of pathways proposed to confer hypothetical protection against hyperglycemia might act as an important determinant of hyperglycemia toxicity in long-standing diabetes.

NOS3 894G>T polymorphism is associated with progression of kidney disease and cardiovascular morbidity in type 2 diabetic patients: NOS3 as a modifier gene for diabetic nephropathy?

BACKGROUND/AIMS: We have previously associated SNP 894G>T in the NOS3 gene with diabetic nephropathy (DN) using multi-locus analysis. Variant 894G>T has been widely studied as a DN susceptibility factor with contradictory results. In the present study we genotyped 894G>T in the cohort of prospectively followed type 2 diabetics with the aim to investigate its possible role in the progression of DN and development of morbidity and mortality associated with diabetes. METHODS: 311 subjects with defined stage of DN were enrolled in the study and followed up for a median of 38 months. We considered three end-points: progression of DN, major cardiovascular event and all-cause mortality. RESULTS: Considering baseline GFR, age at enrolment and diabetes duration as confounders, Cox regression analysis identified 894GT genotype as a risk factor for DN progression (HR = 1.843 [95% CI 1.088 - 3.119], P = 0.023) and 894TT genotype as a risk factor for major cardiovascular event (HR = 2.515 [95% CI 1.060 - 5.965], P = 0.036). CONCLUSION: We ascertained the significant effect of the NOS3 894G>T variant on DN progression and occurrence of major cardiovascular event in T2DM subjects. Based on these results NOS3 can be considered a modifier gene for DN.

ADMA, SDMA and L-arginine/ADMA ratio but not DDAH genetic polymorphisms are reliable predictors of diabetic nephropathy progression as identified by competing risk analysis.

BACKGROUND/AIMS: Complex interplay of genetic and (patho)physiological factors influence availability of nitric oxide during the development and progression of diabetic complications. We assessed predictive value of commonly studied methylated asymmetric and symmetric dimethylarginines (ADMA and SDMA) and selected single nucleotide polymorphisms (SNPs) in dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 genes for the progression of diabetic nephropathy (DN). METHODS: A total of 341 type 1 and type 2 diabetes patients with variable degree of kidney disease were included at baseline. Plasma levels of ADMA, SDMA and L-arginine were measured and six tagging SNPs in DDAH1 and 2 were determined. Progression of DN was defined as a transition from any given stage to a more advanced stage of albuminuria. Competing risk analysis was applied. RESULTS: Plasma levels of ADMA and SDMA significantly correlated with GFR. No significant genotype-phenotype relationship was ascertained for ADMA and DDAH variants, but SNP rs805304 exhibited marginally significant association with DN. ADMA, SDMA and L-arginine/ADMA ratio standardised to GFR were identified as significant predictors of DN progression but not GFR decline using multivariate competing risk analysis. CONCLUSIONS: In our study we confirmed potentially significant role of ADMA and SDMA for the assessment of risk of DN progression in European diabetic population.