Variants in Pharmacokinetic Transporters and Glycemic Response to Metformin: A Metgen Meta-Analysis.

Therapeutic response to metformin, a first-line drug for type 2 diabetes (T2D), is highly variable, in part likely due to genetic factors. To date, metformin pharmacogenetic studies have mainly focused on the impact of variants in metformin transporter genes, with inconsistent results. To clarify the significance of these variants in glycemic response to metformin in T2D, we performed a large-scale meta-analysis across the cohorts of the Metformin Genetics Consortium (MetGen). Nine candidate polymorphisms in five transporter genes (organic cation transporter [OCT]1, OCT2, multidrug and toxin extrusion transporter [MATE]1, MATE2-K, and OCTN1) were analyzed in up to 7,968 individuals. None of the variants showed a significant effect on metformin response in the primary analysis, or in the exploratory secondary analyses, when patients were stratified according to possible confounding genotypes or prescribed a daily dose of metformin. Our results suggest that candidate transporter gene variants have little contribution to variability in glycemic response to metformin in T2D.

Trefoil factor 3 is required for differentiation of thyroid follicular cells and acts as a context-dependent tumor suppressor.

Trefoil factor 3 (TFF3) is overexpressed in a variety of solid epithelial cancers, where it has been shown to promote migration, invasion, proliferation, survival and angiogenesis. On the contrary, in the majority of thyroid tumors, it is downregulated, yet its role in the development of thyroid cancer remains unknown. Here we show that TFF3 exhibits strong cytoplasmic staining of normal thyroid follicular cells and colloid and the staining is increased in hyperfunctioning thyroid nodules, while it is decreased in all thyroid cancers of follicular cell origin. By meta-analysis of gene expression datasets, we found that in the thyroid cancer, conversely to the breast cancer, the expression of TFF3 mRNA was downregulated by estrogen signaling and confirmed this by treating thyroid cancer cells with estradiol. Forced expression of TFF3 in anaplastic thyroid cancer cells resulted in decreased cell proliferation, clonal spheroid formation and entry into the S phase. Furthermore, it induced acquisition of epithelial-like cell morphology and expression of the differentiation markers of thyroid follicular cells and transcription factors implicated in the thyroid morphogenesis and function. Taken together, this study provides the first evidence that TFF3 may act as a tumor suppressor or an oncogene depending on the cellular context.

Survey of autoantibody responses against tumor-associated antigens in thyroid cancer.

BACKGROUND: Autoantibodies against tumor-associated antigens (TAAs) have been shown to serve as highly specific serological biomarkers for the diagnosis of various solid cancers. Although the autoimmunity against thyroid tissue specific antigens has been studied extensively, so far, the autoantibody responses against common TAAs such as cancer-testis antigens (CTAs), mutated or differentiation antigens have not been comprehensively analyzed in patients with thyroid cancer. OBJECTIVE: The current study aims to characterize the frequency of autoantibody responses against common TAAs in patients with thyroid cancer and benign thyroid nodules. METHODS: A phage-displayed antigen microarray comprising 65 TAAs was produced and tested with sera from 53 patients with thyroid cancer, 90 patients with benign thyroid nodules and 96 cancer-free individuals, 100 melanoma, 54 breast cancer and 14 lung cancer patients as controls. RESULTS: A panel of 6 TAAs was identified that preferentially reacted with sera from patients with thyroid cancer. The top ranked antigen in this panel was GAGE1 eliciting autoantibody response in 6% of patients with thyroid cancer but not with benign nodules, whereas no reactivity to other CTAs was detected in the sera from patients with thyroid cancer. CONCLUSIONS: Although six TAAs, including one CTA, showed thyroid cancer-associated reactivity, overall, spontaneous humoral immune responses against TAAs are rare in thyroid cancer and their utility for the development of non-invasive assay for the differential diagnosis of thyroid nodules is limited.

The efficacy and safety of ezetimibe/simvastatin combination compared with intensified lipid-lowering treatment strategies in diabetic subjects with and without metabolic syndrome.

AIMS: The objective was to assess the consistency of effect of switching to ezetimibe/simvastatin 10/20 mg versus doubling the baseline statin dose (to simvastatin 40 mg or atorvastatin 20 mg) or switching to rosuvastatin 10 mg across subgroups of subjects with (n = 617) and without (n = 191) metabolic syndrome (MetS). METHODS: This was a post hoc analysis of a randomized, double-blind, 6-week study of adults 18-79 years with cardiovascular disease and diabetes mellitus with low-density lipoprotein cholesterol (LDL-C) ≥70 and ≤160 mg/dl. The percent change in LDL-C and other lipids was estimated within each subgroup separately. Safety and tolerability were assessed. RESULTS: In subjects with MetS, percent changes in LDL-C and other lipids were greater with ezetimibe/simvastatin versus doubling baseline statin or numerically greater versus switching to rosuvastatin, except high-density lipoprotein cholesterol and apolipoprotein (Apo) AI (mean percent changes in LDL-C were: -22.49% ezetimibe/simvastatin, -9.64% doubled baseline statin and -19.20% rosuvastatin). In subjects without MetS, percent changes in LDL-C, total cholesterol and Apo B were greater with ezetimibe/simvastatin versus doubling baseline statin or numerically greater versus switching to rosuvastatin (mean percent changes in LDL-C were: -25.14% ezetimibe/simvastatin, -4.75% doubled baseline statin and -19.75% rosuvastatin). Safety profiles were generally similar. CONCLUSION: These results showed that switching to ezetimibe/simvastatin 10/20 mg was more effective at reducing LDL-C, total cholesterol and Apo B versus doubling the baseline statin dose to simvastatin 40 mg or atorvastatin 20 mg or switching to rosuvastatin 10 mg regardless of MetS status. These results were generally similar to those of the full cohort.

Low risk of severe hypoglycaemia in patients with type 2 diabetes mellitus starting insulin therapy with premixed insulin analogues BID in outpatient settings.

AIMS: The choice of insulin at initiation in type 2 diabetes remains controversial. The aim of this study was to assess the occurrence of self-reported severe hypoglycaemia associated with premixed insulin analogues in routine clinical care. METHODS: A 12-month, prospective, observational, multicentre study in patients starting a commonly prescribed premixed insulin analogue (either insulin lispro 25/75 or biphasic insulin aspart 30/70, twice daily) after suboptimal glycaemic control on oral antidiabetic agents. Treatment decisions were made solely in the course of usual practice. RESULTS: Study follow-up was completed by 991 (85.5%) of the 1150 patients enrolled. At baseline, mean (SD) age was 57.9 (10.1) years; mean diabetes duration was 9.2 (5.9) years; mean haemoglobin A(1c) (HbA(1c)) was 9.9 (1.8) % and the rate of severe hypoglycaemia was 0.03 episode/patient-year. At 12 months, the rate of severe hypoglycaemia was 0.04 episode/patient-year (95% CI 0.023, 0.055 episode/patient-year) and mean insulin dose was 41.5 (19.4) units. Changes from baseline to 12 months for mean fasting plasma glucose and HbA(1c) were -5.1 mmol/l and -2.5%, respectively. CONCLUSIONS: After initiation of premixed insulin analogues in patients with type 2 diabetes in real-world settings, the incidence of severe hypoglycaemia was lower than expected from previously reported studies.

Imeglimin, a novel glimin oral antidiabetic, exhibits a good efficacy and safety profile in type 2 diabetic patients.

AIMS: Imeglimin is the first in a new tetrahydrotriazine-containing class of oral antidiabetic agents, the glimins. It has been shown to act on the liver, muscle and pancreatic ß-cells to uniquely target the key defects of type 2 diabetes. Two studies were performed to compare the safety and efficacy of imeglimin with metformin and placebo on glycaemic control in type 2 diabetes patients. METHODS: In a 4-week phase IIa, three-arm parallel group study, patients were randomized to imeglimin 2000 mg once daily (od), imeglimin 1000 mg twice daily (bid) or metformin 850 mg bid and responses to an oral glucose tolerance test (OGTT) were measured. In an 8-week phase IIa, four-arm controlled multi-centre study, patients were randomized to imeglimin 500 mg bid, imeglimin 1500 mg bid, metformin 850 mg bid or placebo. Glycaemic assessments included area under the curve (AUC) up to 6 h (AUC(0-6h)) for glucose during a prolonged meal, fasting plasma glucose (FPG) and HbA1c. Safety and tolerability were assessed in both studies through adverse event recording and laboratory parameters, vital signs and electrocardiogram. RESULTS: Imeglimin was found to be as effective as metformin at reducing the AUC(PG) and AUC(0-6h) , FPG and HbA1c. Imeglimin exhibited a favourable tolerability profile in comparison to metformin. CONCLUSIONS: The results from both studies confirm that imeglimin displays a superior benefit : risk profile compared with metformin in type 2 diabetes patients. The encouraging tolerability profile of imeglimin could make it suitable for combination with other classes of antidiabetic agents and may increase availability to a wider patient population.

Stronger association of common variants in TCF7L2 gene with nonobese type 2 diabetes in the Latvian population.

Polymorphisms in the gene coding for transcription factor 7 like 2 (TCF7L2) are recognized as the strongest common genetic risk factors for type 2 diabetes (T2D) across multiple ethnicities. This study was conducted to evaluate an association between TCF7L2 variants and diabetes susceptibility in the population of Latvia. We genotyped 4 single nucleotide polymorphisms (SNP) rs7901695, rs7903146, rs11196205 and rs12255372 in 1 093 controls and 1 043 diabetic subjects. Association with T2D was found for 3 SNPs rs7901695, rs7903146 and rs12255372 in the whole sample (under an additive genetic model, the adjusted odds ratios (OR) were 1.26, 95% CI [1.08-1.48], P=0.003; OR=1.32, 95% CI [1.12-1.55], P=0.001 and OR=1.35, 95% CI [1.15-1.60], P=0.0004 respectively). In addition observed effects on T2D susceptibility for analysed SNPs were higher among subjects with BMI under 30 kg/m². The impact of TCF7L2 variation on T2D risk in Latvian population is compatible with that demonstrated by a range of studies conducted in various ethnic groups.

Acute and multiple-dose studies to determine the safety, tolerability, and pharmacokinetic profile of advantame in healthy volunteers.

Advantame, a new, high-intensity sweetener that is thought not to be absorbed from the human gastrointestinal tract in appreciable quantities, was evaluated for safety and tolerability in a total of 48 healthy adult volunteers in 2 studies. In the first study 24 subjects were randomized to receive escalating single doses of 0.1, 0.25, or 0.5mg/kg of body weight, and pharmacokinetic and safety parameters were assessed subsequently for 8 days. In the second study, 24 subjects were randomized to receive over 4 weeks either 30 mg advantame/day (split into 3 doses per day), or placebo. All subjects completed both studies and no significant treatment-related adverse effects were observed in any subjects in either study. There were no clinically relevant changes in laboratory parameters, vital signs, electrocardiogram, or physical examination findings. Plasma concentrations of advantame were mostly below the limit of quantification in all samples taken after a single dose or prior to the dose in the repeat-dose study. The concentrations of the hydrolysis product, advantame-acid, were also below the limit of quantification at 12, 36, and 48 h after a single dose of 0.1, 0.25, and 0.5mg/kg body weight, respectively. These studies demonstrate the safety and tolerability of advantame in healthy subjects at doses far exceeding those likely to be encountered in food and beverage use.

Is the ADA/EASD algorithm for the management of type 2 diabetes (January 2009) based on evidence or opinion? A critical analysis.

The ADA and the EASD recently published a consensus statement for the medical management of hyperglycaemia in patients with type 2 diabetes. The authors advocate initial treatment with metformin monotherapy and lifestyle modification, followed by addition of basal insulin or a sulfonylurea if glycaemic goals are not met (tier 1 recommendations). All other glucose-lowering therapies are relegated to a secondary (tier 2) status and only recommended for selected clinical settings. In our view, this algorithm does not offer physicians and patients the appropriate selection of options to individualise and optimise care with a view to sustained control of blood glucose and reduction both of diabetes complications and cardiovascular risk. This paper critically assesses the basis of the ADA/EASD algorithm and the resulting tiers of treatment options.

Impact of peripheral arterial disease in patients with diabetes--results from PROactive (PROactive 11).

We compared cardiovascular disease outcomes according to the presence of peripheral arterial disease (PAD) at baseline in a post hoc analysis from the PROactive study. Of the 5238 patients in PROactive (a study of pioglitazone versus placebo in patients with type 2 diabetes and macrovascular disease; mean follow-up=34.5 months), 1274 had PAD at baseline (619=pioglitazone; 655=placebo). Patients with PAD at baseline showed significantly higher rates of the primary endpoint, main secondary endpoint, all-cause mortality (all P<0.0001), and stroke (P=0.0175) than those with no PAD at baseline. The risk of PAD alone was similar to that of myocardial infarction alone. In patients with no PAD at baseline, the event rates of the primary endpoint (P=0.0160), main secondary endpoint (P=0.0453), and acute coronary syndrome (P=0.0287) were significantly lower with pioglitazone than with placebo. This beneficial effect of pioglitazone was not seen in patients with PAD at baseline. In the total population, there was a higher frequency of leg revascularizations with pioglitazone than placebo-this was wholly due to first events that occurred within the initial 12 months of treatment. The presence of PAD increased the risk of all major cardiovascular events. Those without PAD at baseline seemed to benefit more from pioglitazone treatment than the overall PROactive population.

SNPs of PSMA6 gene--investigation of possible association with myocardial infarction and type 2 diabetes mellitus.

In our preceding studies we have identified microsatellite polymorphisms inside the PSMA6 gene and in its 5' upstream region. Following the observed associations of microsatellite polymorphisms with non-insulin dependent diabetes mellitus and Graves' disease we extended the evaluation of PSMA6 genetic variations to cardiovascular disorders and non-insulin dependent diabetes mellitus. New polymorphisms in the promoter region and exon 6 of the gene were identified by direct sequencing of the promoter region and all seven exons of the gene in 30 individuals of European descent. Two SNPs at positions -110 and -8 from the translation start, in the promoter region and 5'UTR respectively, were analyzed. Neither polymorphism was associated with the risk of myocardial infarction. No significant association of the polymorphisms with plasma lipid levels or BMI was observed. A borderline association of both polymorphisms with diastolic blood pressure was observed in the control group. Genotype -8CG was significantly more frequent in type 2 diabetes patients, and haplotype C-110/G-8, compared to C-110/C-8 was associated with a higher risk of NIDDM.

Association of microsatellite polymorphisms of the human 14q13.2 region with type 2 diabetes mellitus in Latvian and Finnish populations.

A polymorphic microsatellite in intron 6 of the human proteasome core particle PSMA6 gene (HSMS006), and four other microsatellites localized upstream on human chromosome 14q13.2 (HSMS801, HSMS702, HSMS701, HSMS602), were genotyped in 104 type 2 diabetic patients and 129 age-matched control subjects from Latvia and replicated in 91 type 2 diabetic patients and 88 age-matched healthy control subjects from the Botnia Study in Finland. In type 2 diabetic patients from both populations the HSMS006 (TG)22 allele was two times more frequent compared to the control group. In the Latvian population the (CAA)8 allele of the HSMS602 marker was less frequent in the diabetic group, as was the (AC)24 allele of microsatellite HSMS801. Allele frequencies of the HSMS701 and 702 repeats were similar in healthy controls and type 2 diabetic patients. In conclusion, our data suggest that variants in the PSMA6 gene on chromosome 14q13.2 are associated with type 2 diabetes.

Prevalence of nosocomial infections in two Latvian hospitals.

The first point prevalence survey of the nosocomial infection (NI) rate was conducted in two Latvian hospitals. At the time of the survey 17.5% (226/1291) patients had symptoms or were being treated for infection. The overall prevalence rate was 5.6% (72/1291) for NI and 12.7% for community acquired infections (164/1291). Surgical site infection (SSI) was the most common NI (62%) followed by respiratory tract infection (RTI) (7.5%), and urinary tract infection (UTI) (6.4%). NI rate was higher with increasing age of patients, in intensive care units and surgical wards, and among those who had an intravenous device or urinary catheter. Microbiological investigation yielded positive results in 29% (21/72) of patients with NIs. Antibacterial treatment was given to 22.3% (288/1291) of hospitalised patients and in 62% (182/288) of these, cefazolin was prescribed. Results from this study will be used to plan a national prevalence survey.

Two HLA DRB 1 alleles confer independent genetic susceptibility to Graves disease: relevance of cross-population studies.

Recent studies of Graves disease (GD) employing genome scanning techniques excluded the major histocompatibility complex as a contributor to disease liability. These findings contradict earlier population association studies. Our own earlier studies have also emphasized that genetic variation in human populations may give novel clues to disease liability and manifestations. To this end, we studied HLA class II alleles in 47 Latvian GD patients and 111 matched healthy controls. As expected, we found that DRB1*03 and DQA1*0501 (OR = 3.6, P = 0.029 and OR 2.35, P = 0.0373, respectively) were associated with GD. Unforeseen, DRB1*04 was found to be significantly increased in the patients compared to controls (OR 3.267, corrected P = 0.0319). The two DRB1 alleles conferred two non-overlapping and independent susceptibilities to GD, in that only three patients were positive for both alleles, and the removal of each allele in turn resulted in only the other DRB1 allele showing significant association with the disease. There was no heterogeneity between the two patient groups (DRB1*03 positive and DRB1*04 positive) in clinical characteristics or disease manifestations. The phenotype DRB1*03 and/or DRB1*04 was found in 34/47 patients compared to 27/111 controls yielding an OR of 7.395 (P corrected = 0.000019). We examined the structural basis of DRB1 susceptibility to GD in light of this and previous studies, showing that DRB1*03, 04, and 08 were positively associated with the disease, whereas DRB1*07 was negatively associated. Differences in protein sequences were noted at residues 54, 57, 59, and 66; positions 54, 57, and 66 are on the same face of the alpha helix. The canonical arginine 54 is replaced by glutamine in DRB1*07. At position 66, asparagine in DRB1*03 and tyrosine in DRB1*04 are replaced by phenylalanine in DRB1*07. Residue 59, likely involved in pocket formation in the antigen binding groove, is modified by replacement of tyrosine in DRB1*03, 08, and 04 and by leucine in DRB1*07. The predicted differences in the shape and charges of the proximal reaches of the antigen binding groove between DRB1*07, and 03, 04, and 08, could determine whether or not a peptide from an auto-antigen would be bound or not. Genetic variation among human populations may yield important clues to specific disease liability.

Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro.

AIMS/HYPOTHESIS: Postprandial hyperglycaemia carries an increased risk of macrovascular disease even without Type II (non-insulin-dependent) diabetes mellitus. Chronic hyperglycaemia activates protein kinase C (PKC) in vitro and in vivo but it is not known whether PKC is regulated by short-term post-prandial hyperglycaemia in vivo in humans. We investigated whether PKC is regulated in vivo in hyperglycaemic and hyperinsulinaemic infusion tests and correlated the results to stimulations in vitro. METHODS: Protein kinase C regulation was measured in platelets obtained from 8 healthy subjects who were infused with glucose and insulin for 2 h attaining peak concentrations of 16 mmol/l glucose and in platelets from 8 healthy young subjects, 8 older subjects without diabetes, and 10 older subjects with Type II diabetes after incubation in vitro with 16 mmol/l glucose or glucose and insulin. For precise quantification, a shortened PKC beta1 standard protein was generated by bacterial expression and PKC alpha, beta1, beta2 and delta isoenzyme values were measured by immunoblot analyses. RESULTS: Hyperglycaemic and hyperinsulinaemic in vivo tests increased the amounts of PKC alpha, beta1 and beta2 in the membrane fraction of platelets to 225 +/- 87 %, 164 +/- 22 % and 302 +/- 135 %, respectively, when compared with the baseline values in young healthy volunteers (n = 8, p < 0.05). The expression of PKC delta did not change. In comparison to the recombinant PKC beta1 standard protein, 5 ng PKC beta1/ microg protein was measured before the test and 2 ng/microg were translocated to the membrane fraction after the infusion. No change in the absolute amount of PKC beta1 was detected. In contrast, after incubation in vitro PKC was not regulated by glucose or glucose and insulin in 8 young healthy subjects (age 26 +/- 0.7 years) and in 8 older, healthy subjects (age 64,8 +/- 4 years) although 100 nmol/l 12-O-tetradecanoylphorbol 13-acetate caused maximal activation. In marked contrast, PKC beta1 and PKC beta2, but not PKC alpha or PKC delta, were increased in vitro in the membrane fraction by 292 +/- 61% and 432 +/- 88% (p < 0.05) in 10 subjects with Type II diabetes mellitus matched for age, sex and BMI. CONCLUSION/INTERPRETATION: We found that short-term hyperglycaemia activates PKC alpha, beta1 and beta2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of postprandial hyperglycaemia. Hyperglycaemia and hyperinsulinaemia did not cause short-term activation of PKC in platelets in vitro suggesting the existence of additional stimuli. Subjects with Type II diabetes showed a markedly altered reactivity of platelet PKC beta in vitro indicating some diabetes-related regulation.

Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.