The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes

Aims/hypothesis

Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes.

Methods

Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Ca_v)1.2 and Ca_v1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Ca_v1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes.

Results

In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Ca_v1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes.

Conclusion/interpretation

We conclude that the L-type calcium channel Ca_v1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.     

Evaluation of the driving ability in disabled persons: A practitioners' view

Purpose and methods.?The purpose of this paper is to present, on the basis of four genuine cases from the Rehabilitation Research Unit of Oulu University, the theoretical frame in which evaluations of driving ability of disabled persons can be made.

Results.?First, it is not the operations with the control devices but the correct mental actions which the driver carries out with the help of the control devices which are crucial for safe driving. Second, driving ability is only partly a biomedical object of research and one ought to avoid an excessive medicalisation of an evaluation of driving ability. Third, the driver meets traffic situations not by his or her separate biological or psychological functions, such as vision, attention, memory, thinking, motives, but as an integrated whole, as a personality.

Conclusions.?By its complexity an evaluation of driving ability can be compared to an evaluation of working capacity where often a multidisciplinary team is needed. When evaluating driving ability we have to take a step from low-level motor operations towards high-level mental actions, from the measurement of acuity of eyesight towards the testing of the flexibility of perception, from the diagnosis-based evaluation to the patient-based evaluation, from using the common pencil?–?paper tests towards the traffic-related task-specific tests and from the testing of separate single general non-driving-related factors towards an evaluation of the theoretically based driving performance as whole.     

Ranibizumab combined with verteporfin photodynamic therapy in neovascular age-related macular degeneration (FOCUS): year 2 results

PURPOSE: To assess the efficacy and adverse-events profile of combined treatment with ranibizumab and verteporfin photodynamic therapy (PDT) in patients with predominantly classic choroidal neovascularization (CNV) secondary to neovascular age-related macular degeneration. DESIGN: Two-year, multicenter, randomized, single-masked, controlled study. METHODS: Patients received monthly intravitreal injections of ranibizumab 0.5 mg (n = 106) or sham injections (n = 56). All patients received PDT on day zero, then quarterly as needed. Efficacy assessment included changes in visual acuity (VA) and lesion characteristics and PDT frequency. Adverse events were summarized by incidence and severity. RESULTS: At month 24, 88% of ranibizumab + PDT patients had lost <15 letters from baseline VA (vs 75% for PDT alone), 25% had gained >or=15 letters (vs 7% for PDT alone), and the two treatment arms differed by 12.4 letters in mean VA change (P < .05 for all between-group differences). The VA benefit of adding ranibizumab to PDT in year one persisted through year two. On average, ranibizumab + PDT patients exhibited less lesion growth and greater reduction of CNV leakage and subretinal fluid accumulation, and required fewer PDT retreatments, than PDT-alone patients (mean = 0.4 vs 3.0 PDT retreatments). Endophthalmitis and serious intraocular inflammation occurred, respectively, in 2.9% and 12.4% of ranibizumab + PDT patients and 0% of PDT-alone patients. Incidences of serious nonocular adverse events were similar in the two treatment groups. CONCLUSIONS: Through two years, ranibizumab + PDT was more effective than PDT alone and had a low rate of associated adverse events.     

Common missense variant in the glucokinase regulatory protein gene is associated with increased plasma triglyceride and C-reactive protein but lower fasting glucose concentrations

OBJECTIVE—Using the genome-wide association approach, we recently identified the glucokinase regulatory protein gene (GCKR, rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought to study the association of GCKR variants with metabolic phenotypes, including measures of glucose homeostasis, to evaluate the GCKR locus in samples of non-European ancestry and to fine- map across the associated genomic interval.RESEARCH DESIGN AND METHODS—We performed association studies in 12 independent cohorts comprising >45,000 individuals representing several ancestral groups (whites from Northern and Southern Europe, whites from the U.S., African Americans from the U.S., Hispanics of Caribbean origin, and Chinese, Malays, and Asian Indians from Singapore). We conducted genetic fine-mapping across the ∼417-kb region of linkage disequilibrium spanning GCKR and 16 other genes on chromosome 2p23 by imputing untyped HapMap single nucleotide polymorphisms (SNPs) and genotyping 104 SNPs across the associated genomic interval.RESULTS—We provide comprehensive evidence that GCKR rs780094 is associated with opposite effects on fasting plasma triglyceride (P_meta = 3 × 10⁻⁵⁶) and glucose (P_meta = 1 × 10⁻¹³) concentrations. In addition, we confirmed recent reports that the same SNP is associated with C-reactive protein (CRP) level (P = 5 × 10⁻⁵). Both fine-mapping approaches revealed a common missense GCKR variant (rs1260326, Pro446Leu, 34% frequency, r² = 0.93 with rs780094) as the strongest association signal in the region.CONCLUSIONS—These findings point to a molecular mechanism in humans by which higher triglycerides and CRP can be coupled with lower plasma glucose concentrations and position GCKR in central pathways regulating both hepatic triglyceride and glucose metabolism.Recently, in the genome-wide association Diabetes Genetics Initiative (DGI) Study for 19 traits, including plasma lipids, we provided evidence that the glucokinase (GCK) regulatory protein gene (GCKR) region was a novel quantitative trait locus associated with plasma triglyceride concentration (1). Of all single nucleotide polymorphisms (SNPs) tested, an intronic SNP at GCKR (rs780094) explained the greatest proportion of interindividual variability in plasma triglycerides (1).GCKR regulates GCK, which functions as a glucose sensor responsible for glucose phosphorylation in the first step of glycolysis. The discoveries that inactivating mutations in GCK cause maturity onset diabetes of the young type 2 (2) and activating GCK mutations lead to permanent hyperinsulinemic hypoglycemia (3) emphasize that GCK plays a major role in glucose metabolism. GCKR-deficient mice have reduced GCK expression but maintain nearly normal GCK activity and show impaired glucose clearance (4). Furthermore, adenoviral-mediated overexpression of GCKR in mouse liver increased GCK activity and lowered fasting blood glucose (5) and overexpression of GCK in liver led to lowered blood glucose and increased triglyceride concentrations (6,7). Thus, experimental evidence suggests that perturbation of the GCKR pathway has opposing effects of triglyceride and glucose metabolism.In our original report, SNP rs780094 in GCKR was associated with fasting triglyceride levels in two independent samples, each of Northern European ancestry (P = 3.7 × 10⁻⁸ and 8.7 × 10⁻⁸, respectively) (1). After initial identification and replication of a chromosomal region associated with a trait, key next steps include extension of the association finding to related phenotypes, validation of the association finding in different ethnicities, and fine- mapping to identify the putative causal variant. Recently, our initial finding was replicated in a Danish study in which a strong association was found between the rs780094 T allele and elevated fasting triglyceride levels but also lower insulin levels, better insulin sensitivity, and a moderately decreased risk of type 2 diabetes (8). In addition, recent genome-wide association studies identified an association between the same GCKR intronic SNP and C-reactive protein (CRP) levels (9,10).Hereby, we sought to examine the effect of SNP rs780094 on triglycerides and related metabolic traits, including fasting glucose concentrations, in 12 samples representing a range of ancestral groups and including a large prospective study with a mean follow-up time of 23 years. In addition, we performed fine-mapping in one of these samples to identify the strongest association signal in the region.     

Occupational exposure to asbestos as evaluated from work histories and analysis of lung tissues from patients with mesothelioma.

The past occupational exposure to asbestos of 23 patients with mesothelioma (21 men and two women) has been evaluated by a personal interview of their work history and by determination of the fibre burden in their lung tissue with scanning electron microscopy (SEM) and x ray microanalysis. According to the work history, nine patients (39%) had definitely been or probably been exposed to asbestos, six patients (26%) had had possible exposures, and eight patients (35%) unlikely or unknown exposure to asbestos. The two female patients were in the unknown exposure category. The fibre concentrations in the patients' lung tissue ranged from less than 0.1 million to 370 million fibres (f) per g dry tissue. Concentrations of over one million f per g dry tissue were found in 15 patients (65%). The lung fibre concentrations of all nine male office workers analysed for reference were less than one million f per g dry tissue. Seventy eight per cent of the patients with mesothelioma had at least possible exposure according to their history of work or concentrations of more than one million f per g dry tissue.     

Occupational exposure to asbestos as evaluated from work histories and analysis of lung tissues from patients with mesothelioma

The past occupational exposure to asbestos of 23 patients with mesothelioma (21 men and two women) has been evaluated by a personal interview of their work history and by determination of the fibre burden in their lung tissue with scanning electron microscopy (SEM) and x ray microanalysis. According to the work history, nine patients (39%) had definitely been or probably been exposed to asbestos, six patients (26%) had had possible exposures, and eight patients (35%) unlikely or unknown exposure to asbestos. The two female patients were in the unknown exposure category. The fibre concentrations in the patients' lung tissue ranged from less than 0.1 million to 370 million fibres (f) per g dry tissue. Concentrations of over one million f per g dry tissue were found in 15 patients (65%). The lung fibre concentrations of all nine male office workers analysed for reference were less than one million f per g dry tissue. Seventy eight per cent of the patients with mesothelioma had at least possible exposure according to their history of work or concentrations of more than one million f per g dry tissue.     

A closed unventilated chamber for the measurement of transepidermal water loss

Background/aims Open chamber systems for measuring transepidermal water loss (TEWL) have limitations related to ambient and body‐induced airflows near the probe, probe size, measurement sites and angles, and measurement range. The aim of the present investigation was to develop a closed chamber system for the TEWL measurement without significant blocking of normal evaporation through the skin. Additionally, in order to use the evaporimeter to measure evaporation rates through other biological and non‐biological specimens and in the field applications, a small portable, battery‐operated device was a design criteria. Methods A closed unventilated chamber (inner volume 2.0 cm³) was constructed. For the skin measurement, the chamber with one side open (open surface area 1.0 cm²) is placed on the skin. The skin application time was investigated at low and high evaporation rates in order to assess the blocking effect of the chamber on normal evaporation. From the rising linear part of the relative humidity (RH) in the chamber the slope was registered. The slope was calibrated into a TEWL value by evaporating water at different temperatures and measuring the water loss of heated samples with a laboratory scale. The closed chamber evaporation technique was compared with a conventional evaporimeter based on an open chamber method (DermaLab^®, Cortex Technology, Hadsund, Denmark). The reproducibility of the closed chamber method was measured with the water samples and with volar forearm and palm of the hand in 10 healthy volunteers. Results The skin application time varied between 7 and 9 s and the linear slope region between 3 and 5 s at the evaporation rates of 3–220 g/m² h. A correlation coefficient between the TEWL value from the closed chamber measurements and the readings of the laboratory scale was 0.99 (P < 0.001). The reproducibility of the evaporation measurements with the water samples was 4.0% at the evaporation rate of 40 g/m² h. A correlation coefficient of the TEWL values between the closed chamber and open chamber measurements was 0.99 (P < 0.001) in the range where the response of a conventional evaporimeter was linear (until 120 g/m² h). With volar forearm and palm of the hand of 10 healthy volunteers the reproducibility of the measurements was 8.0 and 10.1%. Conclusion The closed chamber technique solves the drawbacks related to open chamber evaporimeters. Especially, it extends the measurement range to high evaporation rates and TEWL measurements can be performed practically at any anatomical sites and measurement angle. By the use of a closed chamber the disturbance related to external or body‐induced air flows on the measurement can be avoided.     

Musculoskeletal symptoms of dentists assessed by a multidisciplinary approach

Musculoskeletal health was studied as part of a comprehensive health examination in 131 professionally active dentists. 42% of dentists had experienced pain and disability (interference with daily activities) by neck-shoulder problems during the preceding year, with a tendency to greater prevalence in salaried dentists than in private practitioners. For the lower back, this percentage was 37. Somatic symptoms of stress, perceiving dentistry as physically too heavy or mentally too straining and a poorer general health status rating were all associated with a greater 1-yr prevalence of neck-shoulder and lower back pain and disability and with poorer general physical fitness. Age, weekly work hours, working posture, use of an assistant, or radiographic degenerative changes in the dentist's skeleton were not associated with 1-yr prevalence of neck-shoulder or lower back pain and disability. The results provide evidence that physical exercise should be recommended to dentists and might also be applicable to subjects in other occupations with similar requirements.