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 (Cav)1.2 and Cav1.3 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 Cav1.3). 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 Cav1.2) 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 Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.

Common variant in the HMGA2 gene increases susceptibility to nephropathy in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is a chronic metabolic disorder associated with devastating microvascular complications. Genome-wide association studies have identified more than 60 genetic variants associated with type 2 diabetes and/or glucose and insulin traits, but their role in the progression of diabetes is not established. The aim of this study was to explore whether these variants were also associated with the development of nephropathy in patients with type 2 diabetes. METHODS: We studied 28 genetic variants in 2,229 patients with type 2 diabetes from the local Malmö Scania Diabetes Registry (SDR) published during 2007-2010. Diabetic nephropathy (DN) was defined as micro- or macroalbuminuria and/or end-stage renal disease. Estimated glomerular filtration rate (eGFR) was assessed using the MDRD-4 formula. Replication genotyping of rs1531343 was performed in diabetic (Steno type 2 diabetes [n = 345], Genetics of Diabetes Audit and Research in Tayside Scotland [Go-DARTS] [n = 784]) and non-diabetic (Malmö Preventive Project [n = 2,523], Botnia study [n = 2,247]) cohorts. RESULTS: In the SDR, HMGA2 single-nucleotide polymorphism rs1531343 was associated with DN (OR 1.50, 95% CI 1.20, 1.87, p = 0.00035). In the combined analysis totalling 3,358 patients with type 2 diabetes (n = 1,233 cases, n = 2,125 controls), carriers of the C-allele had a 1.45-fold increased risk of developing nephropathy (95% CI 1.20, 1.75, p = 0.00010). Furthermore, the risk C-allele was associated with lower eGFR in patients with type 2 diabetes (n = 2,499, ß ± SEM, -3.7 ± 1.2 ml/min, p = 0.002) and also in non-diabetic individuals (n = 17,602, ß ± SEM, -0.008 ± 0.003 ml/min (log( e )), p = 0.006). CONCLUSIONS/INTERPRETATION: These data demonstrate that the HMGA2 variant seems to be associated with increased risk of developing nephropathy in patients with type 2 diabetes and lower eGFR in both diabetic and non-diabetic individuals and could thus be a common denominator in the pathogenesis of type 2 diabetes and kidney complications.

Family history of Type 1 diabetes affects insulin secretion in patients with 'Type 2' diabetes.

AIMS: The aim was to evaluate the impact of family history of diabetes on the phenotype of patients diagnosed with Type 2 diabetes and the frequency of susceptibility genotypes. METHODS: Patients with Type 2 diabetes with family history for both Type 1 and Type 2 diabetes (FH(MIX, n) = 196) or Type 2 diabetes only (FH(T2), n = 139) matched for age, sex, BMI and age at diagnosis, underwent an oral glucose tolerance test and a combined glucagon test and insulin tolerance test. Glutamic acid decarboxylase (GAD) antibodies and major Type 1 and Type 2 diabetes susceptibility gene variants were analysed. Patients were stratified into groups according to family history or GAD antibody positivity (GADA+, GADA-) or a combination of these (GADA+/FH(MIX), GADA+/FH(T2), GADA-/FH(MIX), GADA-/FH(T2)). RESULTS: Compared with other patients, those with FH(MIX) more often had GAD antibodies (14.3 vs. 4.3%, P = 0.003), and those with both FH(MIX) and GAD antibodies had the highest frequency of insulin deficiency (stimulated serum C-peptide < 0.7 nmol/l, GADA+/FH(MIX) 46.4% vs. GADA-/FH(MIX) 9.5% (P < 0.00001), GADA-/FH(T2) 4.5% (P < 0.00001), GADA+/FH(T2) 0%). Patients with GADA+/FH(MIX) more often had HLA-DQB1 risk genotypes compared with patients with GADA-/FH(MIX) or GADA-/FH(T2D) (47 vs. 23 or 14%, P = 0.05 and P < 0.00001, respectively). In logistic regression analyses, FH(MIX), GAD antibody positivity and HLA risk genotypes were independently associated with insulin deficiency. CONCLUSION: A family history for both type 1 and type 2 diabetes was associated with higher prevalence of GAD antibodies and HLA-DQB1 risk genotypes than a family history of type 2 diabetes only, and was associated with earlier and more severe development of insulin deficiency, which was only partially explained by GAD antibodies and HLA.

Effect of a common variant of the PCSK2 gene on reduced insulin secretion.

AIM/HYPOTHESIS: Individuals at risk of developing type 2 diabetes show a progressive decline in insulin secretion and increased insulin resistance over time. However, inability of the beta cells to compensate for the increased insulin resistance represents a key defect leading to overt type 2 diabetes. The aims of the present study were to replicate the association between genetic variants of the PCSK2 gene and insulin secretion, and to explore the effect on risk of type 2 diabetes. METHODS: Replication of PCSK2 variants against insulin secretion included 7,682 non-diabetic Scandinavian individuals. Insulin secretion was measured as the corrected insulin response or disposition index, i.e. insulin secretion adjusted for the degree of insulin resistance. Risk of type 2 diabetes was studied in 28,287 Scandinavian individuals. RESULTS: The C-allele of PCSK2 rs2208203 was associated with reduced insulin secretion measured as the corrected insulin response (n = 8,151; ß = -0.112, p = 1.3 × 10(-6)) as well as disposition index (n = 8,078, ß = -0.128, p = 1.6 × 10(-7)). The variant was also associated with lower fasting glucagon levels (ß = -0.084, p = 0.005) in non-diabetic individuals with a fasting plasma glucose of over 5.5 mmol/l. In human pancreatic islets, PCSK2 expression correlated negatively with HbA(1c) (n = 133, r = -0.196, p = 0.038), and showed a tendency to be lower in hyperglycaemic (HbA(1c) ≥6.0% or type 2 diabetes; n = 47, p = 0.13) than normoglycaemic (HbA(1c) >6.0%; n = 66) donors. The presence of the PCSK2 rs2208203 risk allele did not influence gene expression, nor did it show an apparent risk in terms of type 2 diabetes. CONCLUSIONS/INTERPRETATION: A variant of the PCSK2 gene was associated with reduced glucose-stimulated insulin secretion, but also with lower glucagon levels, which could potentially counteract the effects of decreased insulin secretion on the risk of type 2 diabetes.

A common variant upstream of the PAX6 gene influences islet function in man.

AIMS/HYPOTHESIS: Impaired glucose tolerance and impaired insulin secretion have been reported in families with PAX6 mutations and it is suggested that they result from defective proinsulin processing due to lack of prohormone convertase 1/3, encoded by PCSK1. We investigated whether a common PAX6 variant would mimic these findings and explored in detail its effect on islet function in man. METHODS: A PAX6 candidate single nucleotide polymorphism (rs685428) was associated with fasting insulin levels in the Diabetes Genetics Initiative genome-wide association study. We explored its potential association with glucose tolerance and insulin processing and secretion in three Scandinavian cohorts (N = 8,897 individuals). In addition, insulin secretion and the expression of PAX6 and transcriptional target genes were studied in human pancreatic islets. RESULTS: rs685428 G allele carriers had lower islet mRNA expression of PAX6 (p = 0.01) and PCSK1 (p = 0.001) than AA homozygotes. The G allele was associated with increased fasting insulin (p (replication) = 0.02, p (all) = 0.0008) and HOMA-insulin resistance (p (replication) = 0.02, p (all) = 0.001) as well as a lower fasting proinsulin/insulin ratio (p (all) = 0.008) and lower fasting glucagon (p = 0.04) and gastric inhibitory peptide (GIP) (p = 0.05) concentrations. Arginine-stimulated (p = 0.02) insulin secretion was reduced in vivo, which was further reflected by a reduction of glucose- and potassium-stimulated insulin secretion (p = 0.002 and p = 0.04, respectively) in human islets in vitro. CONCLUSIONS/INTERPRETATION: A common variant in PAX6 is associated with reduced PAX6 and PCSK1 expression in human islets and reduced insulin response, as well as decreased glucagon and GIP concentrations and decreased insulin sensitivity. These findings emphasise the central role of PAX6 in the regulation of islet function and glucose metabolism in man.

Telomere length in blood and skeletal muscle in relation to measures of glycaemia and insulinaemia.

AIMS: Skeletal muscle is a major metabolic organ and plays important roles in glucose metabolism, insulin sensitivity and insulin action. Muscle telomere length reflects the myocyte's exposure to harmful environmental factors. Leukocyte telomere length is considered a marker of muscle telomere length and is used in epidemiologic studies to assess associations with ageing-related diseases where muscle physiology is important. However, the extent to which leucocyte and muscle telomere length are correlated is unknown, as are their relative correlations with glucose and insulin concentrations. The purpose of this study was to determine the extent of these relationships. METHODS: Leucocyte and muscle telomere length were measured by quantitative real-time polymerase chain reaction in participants from the Malmö Exercise Intervention (n = 27) and the Prevalence, Prediction and Prevention of Diabetes-Botnia studies (n = 31). Participants in both studies were free from Type 2 diabetes. We assessed the association between leucocyte telomere length, muscle telomere length and metabolic traits using Spearmen correlations and multivariate linear regression. Bland-Altman analysis was used to assess agreement between leucocyte and muscle telomere length. RESULTS: In age-, study-, diabetes family history- and sex-adjusted models, leucocyte and muscle telomere length were positively correlated (r = 0.39, 95% CI 0.15-0.59). Leucocyte telomere length was inversely associated with 2-h glucose concentrations (r = -0.58, 95% CI -1.0 to -0.16), but there was no correlation between muscle telomere length and 2-h glucose concentrations (r = 0.05, 95% CI -0.35 to 0.46) or between leucocyte or muscle telomere length with other metabolic traits. CONCLUSIONS: In summary, the current study supports the use of leucocyte telomere length as a proxy for muscle telomere length in epidemiological studies of Type 2 diabetes aetiology.

Heritability and familiality of type 2 diabetes and related quantitative traits in the Botnia Study.

AIMS/HYPOTHESIS: To study the heritability and familiality of type 2 diabetes and related quantitative traits in families from the Botnia Study in Finland. METHODS: Heritability estimates for type 2 diabetes adjusted for sex, age and BMI are provided for different age groups of type 2 diabetes and for 34 clinical and metabolic traits in 5,810 individuals from 942 families using a variance component model (SOLAR). In addition, family means of these traits and their distribution across families are calculated. RESULTS: The strongest heritability for type 2 diabetes was seen in patients with age at onset 35-60 years (h (2) = 0.69). However, including patients with onset up to 75 years dropped the h (2) estimates to 0.31. Among quantitative traits, the highest h (2) estimates in all individuals and in non-diabetic individuals were seen for lean body mass (h (2) = 0.53-0.65), HDL-cholesterol (0.52-0.61) and suppression of NEFA during OGTT (0.63-0.76) followed by measures of insulin secretion (insulinogenic index [IG(30)] = 0.41-0.50) and insulin action (insulin sensitivity index [ISI] = 0.37-0.40). In contrast, physical activity showed rather low heritability (0.16-0.18), whereas smoking showed strong heritability (0.57-0.59). Family means of these traits differed two- to fivefold between families belonging to the lowest and highest quartile of the trait (p < 0.00001). CONCLUSIONS/INTERPRETATION: To detect stronger genetic effects in type 2 diabetes, it seems reasonable to restrict inclusion of patients to those with age at onset 35-60 years. Sequencing of families with extreme quantitative traits could be an important next step in the dissection of the genetics of type 2 diabetes.

A large multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a clinical biomarker for the diagnosis of diabetes subtypes.

AIMS/HYPOTHESIS: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. METHODS: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n = 457), glucokinase (GCK)-MODY (n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n = 54) and type 2 diabetes (n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. RESULTS: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score -21.8, p < 5 × 10(-105)). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10(-5)). High-sensitivity CRP values between assays were strongly correlated (r (2) ≥ 0.91, p ≤ 1 × 10(-5)). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. CONCLUSIONS/INTERPRETATION: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes.

A family history of diabetes is associated with reduced physical fitness in the Prevalence, Prediction and Prevention of Diabetes (PPP)-Botnia study.

AIMS/HYPOTHESIS: We studied the impact of a family history of type 2 diabetes on physical fitness, lifestyle factors and diabetes-related metabolic factors. METHODS: The Prevalence, Prediction and Prevention of Diabetes (PPP)-Botnia study is a population-based study in Western Finland, which includes a random sample of 5,208 individuals aged 18 to 75 years identified through the national Finnish Population Registry. Physical activity, dietary habits and family history of type 2 diabetes were assessed by questionnaires and physical fitness by a validated 2 km walking test. Insulin secretion and action were assessed based upon OGTT measurements of insulin and glucose. RESULTS: A family history of type 2 diabetes was associated with a 2.4-fold risk of diabetes and lower physical fitness (maximal aerobic capacity 29.2 +/- 7.2 vs 32.1 +/- 7.0, p = 0.01) despite having similar reported physical activity to that of individuals with no family history. The same individuals also had reduced insulin secretion adjusted for insulin resistance, i.e. disposition index (p < 0.001) despite having higher BMI (27.4 +/- 4.6 vs 26.0 +/- 4.3 kg/m(2), p < 0.001). CONCLUSIONS/INTERPRETATION: Individuals with a family history of type 2 diabetes are characterised by lower physical fitness, which cannot solely be explained by lower physical activity. They also have an impaired capacity of beta cells to compensate for an increase in insulin resistance imposed by an increase in BMI. These defects should be important targets for interventions aiming at preventing type 2 diabetes in individuals with inherited susceptibility to the disease.

Assessing the effect of interaction between an FTO variant (rs9939609) and physical activity on obesity in 15,925 Swedish and 2,511 Finnish adults.

AIMS/HYPOTHESIS: Recent reports have suggested that genotypes at the FTO locus interact with physical activity to modify levels of obesity-related traits. We tested this hypothesis in two non-diabetic population-based cohorts, the first from southern Sweden and the second from the Botnia region of western Finland. METHODS: In total 2,511 Finnish and 15,925 Swedish non-diabetic middle-aged adults were genotyped for the FTO rs9939609 variant. Physical activity was assessed by questionnaires and standard clinical procedures were conducted, including measures of height and weight and glucose regulation. Tests of gene x physical activity interaction were performed using linear interaction effects to determine whether the effect of this variant on BMI is modified by physical activity. RESULTS: The minor A allele at rs9939609 was associated with higher BMI in both cohorts, with the per allele difference in BMI being about 0.13 and 0.43 kg/m(2) in the Swedish and Finnish cohorts, respectively (p < 0.0001). The test of interaction between physical activity and the rs9939609 variant on BMI was not statistically significant after controlling for age and sex in either cohort (Sweden: p = 0.71, Finland: p = 0.18). CONCLUSIONS/INTERPRETATION: The present report does not support the notion that physical activity modifies the effects of the FTO rs9939609 variant on obesity risk in the non-diabetic Swedish or Finnish adults studied here.

The use of primary hepatocytes from brown trout (Salmo trutta lacustris) and the fish cell lines RTH-149 and ZF-L for in vitro screening of (anti)estrogenic activity of wood extractives.

Wood extractives are constituents of wood present in pulp and paper mill effluents, which may cause reproductive disturbances in fish. In the present study, we examined three cellular in vitro bioassays in order to assess (anti)estrogenic potencies of the wood extractives dehydroabietic acid (DHAA), isopimaric acid (IPA), betulinol (BET), hydroxymatairesinol (HMR), a phytosterol preparation (ULT), an oxidized phytosterol preparation (OX) and the model estrogen 17beta-estradiol (E2). The test systems used were primary hepatocyte cultures from brown trout and two piscine liver cell lines, RTH-149 and ZF-L. Estrogenicity was measured as vitellogenin (Vtg) secretion in cell culture medium. The primary hepatocytes cultures responded to E2 in a dose-dependent way. Vtg induction was inhibited with a simultaneous exposure to 4-hydroxytamoxifen (4-HT) indicating an estrogen receptor mediated response. DHAA and ULT induced a weak statistically non-significant Vtg production, and weak additive effects were found in some combination treatments of wood extractives and E2. Additionally, a pulp mill effluent tested on primary hepatocytes induced Vtg production when exposed at a 1% dilution. The cell lines secreted negligible amounts of Vtg upon E2 stimulation, which was neither dose-dependent nor inhibited by 4-HT. In conclusion, trout primary hepatocytes could be useful for assessing (anti)estrogenic potencies of compounds, and the wood extractives and a pulp mill effluent showed only weak or no estrogenic activity in this model system.

Characterization of the MODY3 phenotype. Early-onset diabetes caused by an insulin secretion defect.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.

Morphological characterization of exovesicles and endovesicles released from human erythrocytes following treatment with amphiphiles.

In order to morphologically characterize exo- and endovesicles released during treatment of erythrocytes with amphiphiles and to look for possible amphiphile-specific effects on the vesiculation pattern, human erythrocytes were treated at 37 degrees C with amphiphiles at concentrations where they exhibit maximum protection against hypotonic haemolysis (cAHmax). Released exo-and endovesicles and treated cells were studied by means of transmission (TEM) and scanning (SEM) electron microscopy. All sphero-echinocytogenic amphiphiles induced a release of both spherical and tubular exovesicles. Dodecyl maltoside, a nonionic amphiphile with a bulky polar head, induced a release of predominantly tubular exovesicles, while all other sphero-echinocytogenic amphiphiles induced a release of predominantly spherical exovesicles. Some branched tubular exovesicles were released by a double-chained cationic amphiphile. Tail- and tongue-like structures were often seen on the exovesicles. Spherical exovesicles were frequently invaginated. Stomatocytogenic amphiphiles induced endovesiculation. In erythrocytes treated with most of the stomatocytogenic amphiphiles the endovesicles were clustered, but with some amphiphiles the endovesicles were randomly distributed. Large ringformed endovesicles (octaethyleneglycol alkyl ethers) and endovesicles in chains (octyl and decyl glucopyranoside) also occurred. The endovesicle membrane was often budding onto the lumen of the vesicle and in some cases this could ultimately lead to a vesicle inside the endovesicle. We conclude that amphiphiles do not only trigger vesiculation, but may also specifically affect the vesiculation processes.

Vesiculation induced by amphiphiles in erythrocytes.

The ability of shape-transforming cationic, anionic, zwitterionic, and nonionic amphiphiles to induce vesiculation in human erythrocytes was studied. At concentrations where they exhibit maximum protection against hypotonic haemolysis (CAHmax) echinocytogenic amphiphiles induced a rapid release of exovesicles. Following 5 min of incubation, the vesicle release (acetylcholinesterase release) amounted from 4% (sodium alkyl sulphates) to 13% (zwittergents) of the total acetylcholinesterase activity of the erythrocytes. At concentrations corresponding to CAH50 the vesicle release was less than 15% of that released at CAHmax. The size and the appearance of the vesicles varied with the type of amphiphile. Stomatocytogenic amphiphiles which do not pass the erythrocytes through echinocytic stages, did not induce release of exovesicles. Electron and fluorescence microscopic observations of erythrocytes treated with stomatocytogenic amphiphiles strongly indicated that an endovesiculation had occurred. Amphiphiles which pass the erythrocytes through echinocytic stages before stomatocytic shapes are attained, induced a release of both exo- and endovesicles.

Shape and volume changes in rat erythrocytes induced by surface-active alkyltrimethylammonium salts and sodium dodecyl sulphate.

Surface-active alkyltrimethylammonium salts (C12, C14 and C16) and sodium dodecyl sulphate (SDS) caused shape alterations and a volume increase in rat erythrocytes. The alkyltrimethylammonium salts caused echinocytic shapes at both prelytic and lytic concentrations during the first minutes of incubation at 37 degrees C but as the incubation proceeded some of the echinocytes were transformed into stomatocytes. This transformation developed through the normal discocyte shape and it occurred only above certain concentrations. With C14 and C16 the concentration at which stomatocytic shapes appeared coincided with those at which the volume increase began. With the C12 homologue stomatocytic shapes did not appear until lytic concentrations were reached, whereas the volume increase began at prelytic concentrations. SDS caused only echinocytic shapes at 37 degrees C and these appeared at prelytic concentrations, whereas the volume increase was associated with lytic concentrations. When erythrocytes crenated by SDS were cooled to room temperature they were transformed into stomatocytes and discocytes. Our results indicate that (a) even though ionic surfactants induce both swelling and shape alterations in erythrocytes these two changes are not necessarily connected, and that (b) the different shapes induced by cationic and anionic surfactants cannot be due to differences in the distribution of the surfactant molecules within the lipid bilayer of the erythrocyte membrane alone.

Lipid and protein composition of exovesicles released from human erythrocytes following treatment with amphiphiles.

Human erythrocytes were treated with different water-soluble amphiphiles (detergents) at sublytic concentrations, whereafter released exovesicles and treated cells were isolated. Lipid analyses showed that exovesicles had a lower cholesterol/phospholipid ratio and a higher phosphatidylserine/phospholipid ratio compared to parent cells. Protein analyses revealed that exovesicles were, relative to their total protein content, depleted in spectrin, actin and band 6 protein and enriched in band 3 protein and acetylcholinesterase. Exovesicles contained all major glycoproteins. By using a radiolabeled amphiphile ([14C]cetyltrimethylammonium bromide) it was shown that the amphiphile/phospholipid ratio was similar in the vesicle membrane and in the parent cell membrane. This indicates that no significant segregation of the intercalated amphiphile between the exovesicle membrane and the parent cell membrane occurs during the vesiculation process. It is suggested that the redistributions of membrane lipids and proteins during the vesiculation process are secondary to the detachment of the cytoskeletal network from the membrane.

Shape transformations induced by amphiphiles in erythrocytes.

Shape alterations induced in human erythrocytes by cationic, anionic, zwitterionic and nonionic amphiphiles (C10-C16) at antihaemolytic concentrations (CAH50 and CAHmax) and at a slightly lytic concentration (2-10% haemolysis) were studied. Anionic (sodium alkyl sulphates) and zwitterionic amphiphiles (3-(alkyldimethylammonio)-1-propanesulfonates) proved to be potent echinocytogenic agents. Among the nonionic amphiphiles there were potent stomatocytogenicagents (octaethyleneglycol alkyl ethers, pentaethyleneglycol dodecyl ether), one potent echinocytogenic agent (dodecyl D-maltoside) and one weak echinocytogenic agent (decyl beta-D-glucopyranoside). Shape alterations induced by cationic amphiphiles (alkyltrimethylammonium bromides, cetylpyridinium chloride and dodecylamine hydrochloride) showed a strong time-dependence. These amphiphiles immediately induced strongly crenated erythrocytes which during incubation shifted to less crenated erythrocytes or to stomatocytes. All of the echinocytogenic amphiphiles induced echinocytes immediately, and there were only small alterations of the induced shape during incubation. Among the stomatocytogenic amphiphiles there were some that induced stomatocytes immediately or after a short lag time while others first passed the erythrocytes through echinocytic stages before stomatocytic shapes were attained. Erythrocytes treated with amphiphiles did not recover their normal discoid shape following repeated washing and reincubation for 1 h in amphiphile-free medium. Our study shows that shape alterations induced by amphiphiles in erythrocytes cannot be explained solely by assuming a selective intercalation of differently charged amphiphiles into the monolayers of the lipid bilayer as suggested in the bilayer couple hypothesis (Sheetz, M.P. and Singer, S.J. (1976) J. Cell Biol. 70, 247-251). We suggest that amphiphiles, when intercalated into the lipid bilayer, trigger a rapid formation of intrabilayer non-bilayer phases which protect the bilayer against a collapse and bring about a transbilayer redistribution of intercalated amphiphiles as well as of bilayer lipids.

Is calmodulin inhibition involved in shape transformations induced by amphiphiles in erythrocytes?

Potent antihaemolytic and shape transforming amphiphilic compounds were studied for their ability to inhibit calmodulin-activated phosphodiesterase activity. Some echinocytogenic and stomatocytogenic amphiphiles were potent calmodulin inhibitors. The most potent echinocytogenic and stomatocytogenic amphiphiles, however, had no or only weak inhibitory effect. Our results show that there is no causal relationship between the ability of amphiphiles to induce antihaemolysis or shape transformations in erythrocytes and their ability to inhibit calmodulin-activated phosphodiesterase activity, and it is suggested that calmodulin is not involved in shape transformations induced by amphiphiles.

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa.

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes.

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C10-C16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic greater than zwitterionic, non-ionic greater than cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.