Kisspeptin-10 and the G protein-coupled receptor 54 are differentially expressed in the canine pregnant uterus and trophoblast cells.

Uterine tissue was collected from bitches after ovariohysterectomy at different times after ovulation. Samples were assigned to four groups: metestrous non-pregnant, day 10-12, n = 4; pre-implantation, day 10-12, n = 9; post-implantation, day 18-25, n = 13; mid-gestation, day 30-40, n = 7. RT-qPCR detection was performed for kiss1 and the G protein-coupled receptor 54 (GPR54, specific receptor for kisspeptin). In addition, immunohistochemistry was performed for detection of kisspeptin-10 (KP-10), GPR54, as well as pan-cytokeratin and vimentin. The latter two were included to differentiate the different placental cell types. The percentage of positive stained cells was evaluated, and an immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0-3) with the percentage of immunolabelled cells (range: 0-300). In non-pregnant and pre-implantation tissues, gene expression was highly variable for kiss1 and GPR54. Expression of GPR54 was higher before embryo adhesion than during post-implantation and mid-gestation (p < .05), whereas there was no difference found between groups for kiss1. Except during the pre-implantation period, KP-10 expression was higher in the non-pregnant uterus compared to all gestational periods investigated, indicating a pregnancy-related downregulation. In the pre-implantation period, KP-10 was present in larger vessels only, whereas the presence of GPR54 in vessels was found in all samples, with most labelling in the post-implantation period. KP-10 was present in superficial uterine glands, GPR54 in superficial and deep uterine glands of the post-implantation uterus. In myocytes, the highest staining for KP-10 was seen in the non-pregnant uterus, whereas the highest staining for GPR54 was seen in post-implantation and mid-gestation. Syncytiotrophoblast cells stained for both KP-10 and GPR54 in post-implantation and mid-gestation, with maximum intensity for GPR54 in the latter. We conclude that KP-10 and GPR54 are expressed in the canine uterus and trophoblast cells. However, during pregnancy, expression of both proteins seems to be differentially regulated.

Determining the association between adipokine expression in multiple tissues and phenotypic features of non-alcoholic fatty liver disease in obesity.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is an obesity-associated disease, and in obesity adipokines are believed to be involved in the development of NAFLD. However, it is still not clear whether adipokines in the liver and/or adipose tissues can be related to the development of specific characteristics of NAFLD, such as steatosis and inflammation. We aimed to address this question by simultaneously examining the adipokine expression in three tissue types in obese individuals. METHODS: We enrolled 93 severely obese individuals with NAFLD, varying from simple steatosis to severe non-alcoholic steatohepatitis. Their expression of 48 adipokines in the liver, visceral and subcutaneous adipose tissue (SAT) was correlated to their phenotypic features of NAFLD. We further determined whether the correlations were tissue specific and/or independent of covariates, including age, sex, obesity, insulin resistance and type 2 diabetes (T2D). RESULTS: The expression of adipokines showed a liver- and adipose tissue-specific pattern. We identified that the expression of leptin, angiopoietin 2 (ANGPT2) and chemerin in visceral adipose tissue (VAT) was associated with different NAFLD features, including steatosis, ballooning, portal and lobular inflammation. In addition, the expression of tumor necrosis factor (TNF), plasminogen activator inhibitor type 1 (PAI-1), insulin-like growth factor 1 (somatomedin C) (IGF1) and chemokine (C-X-C motif) ligand 10 (CXCL10) in the liver tissue and the expression of interleukin 1 receptor antagonist (IL1RN) in both the liver and SAT were associated with NAFLD features. The correlations between ANGPT2 and CXCL10, and NAFLD features were dependent on insulin resistance and T2D, but for the other genes the correlation with at least one NAFLD feature remained significant after correcting for the covariates. CONCLUSIONS: Our results suggest that in obese individuals, VAT-derived leptin and chemerin, and hepatic expression of TNF, IGF1, IL1RN and PAI-1 are involved in the development of NAFLD features. Further, functional studies are warranted to establish a causal relationship.

Sex-specific effects of naturally occurring variants in the dopamine receptor D2 locus on insulin secretion and type 2 diabetes susceptibility.

AIMS: Modulation of dopamine receptor D2 (DRD2) activity affects insulin secretion in both rodents and isolated pancreatic ß-cells. We hypothesized that single nucleotide polymorphisms in the DRD2/ANKK1 locus may affect susceptibility to type 2 diabetes in humans. METHODS: Four potentially functional variants in the coding region of the DRD2/ANKK1 locus (rs1079597, rs6275, rs6277, rs1800497) were genotyped and analysed for type 2 diabetes susceptibility in up to 25 000 people (8148 with type 2 diabetes and 17687 control subjects) from two large independent Dutch cohorts and one Danish cohort. In addition, 340 Dutch subjects underwent a 2-h hyperglycaemic clamp to investigate insulin secretion. Since sexual dimorphic associations related to DRD2 polymorphisms have been previously reported, we also performed a gender-stratified analysis. RESULTS: rs1800497 at the DRD2/ANKK1 locus was associated with a significantly increased risk for type 2 diabetes in women (odds ratio 1.14 (1.06-1.23); P = 4.1*104) but not in men (odds ratio 1.00 (95% CI 0.93-1.07); P = 0.92) or the combined group. Although rs1800497 was not associated with insulin secretion, we did find another single nucleotide polymorphism in this locus, rs6275, to be associated with increased first-phase glucose-stimulated insulin secretion in women (P = 5.5*104) but again not in men (P = 0.34). CONCLUSION: The present data identify DRD2/ANKK1 as a potential sex-specific type 2 diabetes susceptibility gene.

Glucocorticoid receptor gene polymorphisms are associated with reduced first-phase glucose-stimulated insulin secretion and disposition index in women, but not in men.

AIM: Glucocorticoids are efficacious anti-inflammatory agents, but, in susceptible individuals, these drugs may induce glucose intolerance and diabetes by affecting ß-cell function and insulin sensitivity. We assessed whether polymorphisms in the glucocorticoid receptor gene NR3C1 associate with measures of ß-cell function and insulin sensitivity derived from hyperglycaemic clamps in subjects with normal or impaired glucose tolerance. METHODS: A cross-sectional cohort study was conducted in four academic medical centres in the Netherlands and Germany. Four hundred and forty-nine volunteers (188 men; 261 women) were recruited with normal glucose tolerance (n=261) and impaired glucose tolerance (n=188). From 2-h hyperglycaemic clamps, first- and second-phase glucose-stimulated insulin secretion, as well as insulin sensitivity index and disposition index, were calculated. All participants were genotyped for the functional NR3C1 polymorphisms N363S (rs6195), BclI (rs41423247), ER22/23EK (rs6189/6190), 9ß A/G (rs6198) and ThtIIII (rs10052957). Associations between these polymorphisms and ß-cell function parameters were assessed. RESULTS: In women, but not in men, the N363S polymorphism was associated with reduced disposition index (P=1.06 10(-4) ). Also only in women, the ER22/23EK polymorphism was associated with reduced first-phase glucose-stimulated insulin secretion (P=0.011) and disposition index (P=0.003). The other single-nucleotide polymorphisms were not associated with ß-cell function. Finally, none of the polymorphisms was related to insulin sensitivity. CONCLUSION: The N363S and ER22/23EK polymorphisms of the NR3C1 gene are negatively associated with parameters of ß-cell function in women, but not in men.

Waist circumference and metabolic risk factors have separate and additive effects on the risk of future Type 2 diabetes in patients with vascular diseases. A cohort study.

AIMS: To assess the effect of various measures of adiposity and of metabolic risk factors, both separately and in combination, on the risk of future Type 2 diabetes in patients with manifest vascular diseases. METHODS: This was a prospective cohort study in 2924 patients (mean age 59 ± 12 years) with manifest atherosclerosis. Metabolic risk factors were defined according to National Cholesterol Education Program criteria for the metabolic syndrome. Incidence of Type 2 diabetes was assessed by questionnaire and subsequent verification. RESULTS: During a median follow-up of 4.9 years (range 3.0-7.6 years) there were 178 cases (6.1%) of incident Type 2 diabetes. An increase with 1 sd waist circumference showed a strong association with incident Type 2 diabetes in both men (hazard ratio 2.45, 95% CI 1.97-3.04) and women (hazard ratio 1.77, 95% CI 1.38-2.26). Compared with patients with normal (i.e. below the National Cholesterol Education Program criteria for abdominal adiposity) waist circumference and < 3 metabolic risk factors, both patients with normal waist circumference and ≥ 3 metabolic risk factors and patients with high (i.e. above the National Cholesterol Education Program criteria for abdominal adiposity) waist circumference and < 3 metabolic risk factors had an increased risk of Type 2 diabetes (hazard ratio 2.44, 95% CI 1.37-4.36 and hazard ratio 3.61, 95% CI 2.23-5.85, respectively). Patients with both high waist circumference and ≥ 3 metabolic risk factors had the highest risk of developing Type 2 diabetes (hazard ratio 10.76, 95% CI 6.95-16.64). CONCLUSIONS: In patients with manifest atherosclerosis, both presence of ≥ 3 metabolic risk factors and presence of a high waist circumference alone are associated with increased risk for developing Type 2 diabetes. The combined presence of ≥ 3 metabolic risk factors and high waist circumference, which is present in 15% of patients, is associated with a 10-fold increased risk of future Type 2 diabetes. To identify patients with manifest atherosclerosis at the highest risk of developing Type 2 diabetes, fat distribution in combination with metabolic risk factors should be considered.

Clustering of additional autoimmunity behaves differently in Hashimoto's patients compared with Graves' patients.

OBJECTIVE: It is unclear whether Hashimoto's thyroiditis and Graves' disease (often referred to as autoimmune thyroid disease, AITD) cluster to the same extent with other autoimmune disorders. METHODS: We assessed adrenal, ß-cell, celiac and gastric antibodies in a cohort of 523 adult patients with Graves' disease and 359 patients with Hashimoto's disease and compared their clustering. RESULTS: Adrenal autoimmunity associated more often with Hashimoto's disease (9.0%) than with Graves' disease (3.3%, P=0.001). ß-cell autoimmunity was seen more frequently in Hashimoto's disease (25.4%) than in Graves' disease (15.6%, P=0.001) patients. We found low prevalences of celiac autoimmunity (1.2% for Graves' and 1.2% for Hashimoto's disease). Celiac and gastric autoimmunity were not statistically different in Hashimoto's and Graves' disease patients. Although gastric autoimmunity itself was equally prevalent (around 20%), Hashimoto's disease often showed significantly more clustering of adrenal autoimmunity with gastric autoimmunity (5.3%) than Graves' disease (1.2%, P=0.001). Similarly, clustering of adrenal autoimmunity was seen with ß-cell autoimmunity in Hashimoto's patients (3.2%), while such clustering was much less encountered in 359 Graves' patients (0.9%, P=0.029). CONCLUSION: In conclusion, Hashimoto's disease shows a markedly higher clustering of additional autoimmunity, especially with adrenal and ß-cell autoimmunity. Combined clustering of gastric and adrenal autoimmunity and combined clustering of adrenal and ß-cell autoimmunity were both seen more often in Hashimoto's patients. Clustering with celiac disease appears to be low. These findings indicate that Hashimoto's and Graves' disease differ in their clinical expression regarding additional autoimmunity, which argues against the indiscriminate use of AITD as an entity.

A method for immediate comparative assessment of microinjected mammalian oocytes.

Manipulation of mammalian oocytes at the molecular level is hampered by low transcriptional activity and the presence of large stores of mRNA and protein. Microinjection of interfering macromolecules has become an important tool in studying oocyte maturation, although injection success, final concentrations of injected substances and viability after injection remain difficult to assess with current techniques. To address these problems, we developed an epifluorescence microscopy based technique to evaluate oocytes directly after (co-)injection of green fluorescent protein (GFP).

Identification of genes targeted by FSH and oocytes in porcine granulosa cells.

In the mammalian ovarian follicle maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa cells and the cumulus cells. These cells are regulated by follicle-stimulating hormone (FSH), originating from the pituitary, and paracrine factors derived from the oocyte. To gain insight into the mechanisms involved in the regulation of granulosa cell function, this study aimed to identify genes in mural granulosa cells that are regulated by FSH and oocyte secreted factors using the pig as a model organism. Mural granulosa cells were collected from 3-6 mm follicles from sow ovaries and cultured in serum free medium in the presence or absence of FSH and/or isolated cumulus oocyte complexes (COCs). FSH significantly increased both the metabolic activity and progesterone production of granulosa cells, while the presence of COCs reversed these FSH effects. Expression levels of mRNA in the absence/presence of FSH and COCs were analyzed on porcine specific microarrays representing 11,300 genes. Both previously identified and novel FSH target genes as well as some oocyte affected genes were found. Expression of inhibitor of DNA binding protein 2 and 3, ID2 and ID3, was decreased by FSH but increased by COCs, as validated by quantitative PCR. These proteins function as dominant negative basic helix loop helix (bHLH) transcription factors and since all regulated genes contain the consensus E-box sequence that can bind bHLH factors, our data suggest that FSH and COCs may regulate granulosa cell function by tuning the activity of bHLH factors, through ID2 and ID3.

Sex hormone-binding globulin concentrations before conception as a predictor for gestational diabetes in women with polycystic ovary syndrome.

BACKGROUND: Low plasma sex hormone-binding globulin (SHBG) concentrations during pregnancy have been associated with the risk of developing gestational diabetes mellitus (GDM). Women presenting with polycystic ovary syndrome (PCOS) often exhibit low plasma SHBG concentration and are at increased risk of developing GDM. In this study, we investigate whether SHBG levels before conception are predictive of GDM in women with PCOS. METHODS: A total of 50 women with PCOS were enrolled and followed up during pregnancy. Initial endocrine, metabolic and physical features were assessed according to a standardized preconception screening program. At 24-26 weeks of gestational age a 100-g glucose tolerance test was performed to screen for GDM. RESULTS: Of the 50 women, 21 (42%) were diagnosed with GDM by a 100-g glucose tolerance test. Waist circumference, BMI, blood pressure, plasma glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR) and SHBG levels before conception were significantly different between women who did and did not develop GDM. Stepwise logistic regression analysis showed that SHBG was the most significant predictive parameter for GDM (odds ratio 0.92; 95% confidence interval 0.87-0.97), without significant contribution of waist circumference and HOMA-IR. Receiver operator characteristic (ROC) analysis indicated that plasma SHBG (area under the curve 0.86) had the highest predictive value for subsequent development of GDM, however, the limited group size did not allow for calculation of a threshold value of SHBG. CONCLUSIONS: In women with PCOS, preconception SHBG levels are strongly associated with subsequent development of GDM. Regression and ROC analysis show that preconception SHBG levels may be a better predictor for GDM in PCOS women compared with waist circumference or HOMA-IR. CLINICAL TRIAL REGISTRATION NUMBER: NCT00821379.

Genetic association analysis of LARS2 with type 2 diabetes.

AIMS/HYPOTHESIS: LARS2 has been previously identified as a potential type 2 diabetes susceptibility gene through the low-frequency H324Q (rs71645922) variant (minor allele frequency [MAF] 3.0%). However, this association did not achieve genome-wide levels of significance. The aim of this study was to establish the true contribution of this variant and common variants in LARS2 (MAF > 5%) to type 2 diabetes risk. METHODS: We combined genome-wide association data (n = 10,128) from the DIAGRAM consortium with independent data derived from a tagging single nucleotide polymorphism (SNP) approach in Dutch individuals (n = 999) and took forward two SNPs of interest to replication in up to 11,163 Dutch participants (rs17637703 and rs952621). In addition, because inspection of genome-wide association study data identified a cluster of low-frequency variants with evidence of type 2 diabetes association, we attempted replication of rs9825041 (a proxy for this group) and the previously identified H324Q variant in up to 35,715 participants of European descent. RESULTS: No association between the common SNPs in LARS2 and type 2 diabetes was found. Our replication studies for the two low-frequency variants, rs9825041 and H324Q, failed to confirm an association with type 2 diabetes in Dutch, Scandinavian and UK samples (OR 1.03 [95% CI 0.95-1.12], p = 0.45, n = 31,962 and OR 0.99 [0.90-1.08], p = 0.78, n = 35,715 respectively). CONCLUSIONS/INTERPRETATION: In this study, the largest study examining the role of sequence variants in LARS2 in type 2 diabetes susceptibility, we found no evidence to support previous data indicating a role in type 2 diabetes susceptibility.

Type 2 Diabetes Mellitus: New Genetic Insights will Lead to New Therapeutics.

Type 2 diabetes is a disorder of dysregulated glucose homeostasis. Normal glucose homeostasis is a complex process involving several interacting mechanisms, such as insulin secretion, insulin sensitivity, glucose production, and glucose uptake. The dysregulation of one or more of these mechanisms due to environmental and/or genetic factors, can lead to a defective glucose homeostasis. Hyperglycemia is managed by augmenting insulin secretion and/or interaction with hepatic glucose production, as well as by decreasing dietary caloric intake and raising glucose metabolism through exercise. Although these interventions can delay disease progression and correct blood glucose levels, they are not able to cure the disease or stop its progression entirely. Better management of type 2 diabetes is sorely needed. Advances in genotyping techniques and the availability of large patient cohorts have made it possible to identify common genetic variants associated with type 2 diabetes through genome-wide association studies (GWAS). So far, genetic variants on 19 loci have been identified. Most of these loci contain or lie close to genes that were not previously linked to diabetes and they may thus harbor targets for new drugs. It is also hoped that further genetic studies will pave the way for predictive genetic screening. The newly discovered type 2 diabetes genes can be classified based on their presumed molecular function, and we discuss the relation between these gene classes and current treatments. We go on to consider whether the new genes provide opportunities for developing alternative drug therapies.

Minimal external masculinization in a SRY-negative XX male Podenco dog.

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5alpha-reductase deficiency.

Variants of CDKAL1 and IGF2BP2 affect first-phase insulin secretion during hyperglycaemic clamps.

AIMS/HYPOTHESIS: Genome-wide association studies have recently identified novel type 2 diabetes susceptibility gene regions. We assessed the effects of six of these regions on insulin secretion as determined by a hyperglycaemic clamp. METHODS: Variants of the HHEX/IDE, CDKAL1, SLC30A8, IGF2BP2 and CDKN2A/CDKN2B genes were genotyped in a cohort of 146 participants with NGT and 126 with IGT from the Netherlands and Germany, who all underwent a hyperglycaemic clamp at 10 mmol/l glucose. RESULTS: Variants of CDKAL1 and IGF2BP2 were associated with reductions in first-phase insulin secretion (34% and 28%, respectively). The disposition index was also significantly reduced. For gene regions near HHEX/IDE, SLC30A8 and CDKN2A/CDKN2B we did not find significant associations with first-phase insulin secretion (7-18% difference between genotypes; all p > 0.3). None of the variants showed a significant effect on second-phase insulin secretion in our cohorts (2-8% difference between genotypes, all p > 0.3). Furthermore, the gene variants were not associated with the insulin sensitivity index. CONCLUSIONS: Variants of CDKAL1 and IGF2BP2 attenuate the first phase of glucose-stimulated insulin secretion but show no effect on the second phase of insulin secretion. Our results, based on hyperglycaemic clamps, provide further insight into the pathogenic mechanism behind the association of these gene variants with type 2 diabetes.

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms.

AIMS/HYPOTHESIS: Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms. METHODS: We genotyped 1,110 non-diabetic German participants for five single nucleotide polymorphisms in TCF7L2. All participants underwent an OGTT; GLP-1 secretion was measured in 155 participants. In 210 participants, an IVGTT combined with a hyperinsulinaemic-euglycaemic clamp was performed. In another 160 participants from the Netherlands and 73 from Germany, a hyperglycaemic clamp (10 mmol/l) was performed. In 73 German participants this clamp was combined with a GLP-1 infusion and an arginine bolus. RESULTS: The OGTT data confirmed that variants in TCF7L2 are associated with reduced insulin secretion. In contrast, insulin secretion induced by an i.v. glucose challenge in the IVGTT and hyperglycaemic clamp was not different between the genotypes. GLP-1 concentrations during the OGTT were not influenced by the TCF7L2 variants. However, GLP-1-infusion combined with a hyperglycaemic clamp showed a significant reduction in GLP-1-induced insulin secretion in carriers of the risk allele in two variants (rs7903146, rs12255372, p < 0.02). CONCLUSIONS/INTERPRETATION: Variants of TCF7L2 specifically impair GLP-1-induced insulin secretion. This seems to be rather the result of a functional defect in the GLP-1 signalling in beta cells than a reduction in GLP-1 secretion. This defect might explain the impaired insulin secretion in carriers of the risk alleles and confers the increased risk of type 2 diabetes.

Insulin sensitivity and beta-cell function in healthy cats: assessment with the use of the hyperglycemic glucose clamp.

A hyperglycemic clamp (HGC) was developed for use in conscious cats. In 21 healthy, normal glucose tolerant cats glucose disposal rate (M), insulin sensitivity (ISI (HGC)), and beta-cell response (I) at arterial plasma glucose of 9 mmol.l (-1) were measured. The HGC was tolerated well and steady state glucose infusion was achieved. Compared to values reported for humans, M values for the cats were low, which appeared to relate to both a low ISI (HGC) and a low I. HGC measures correlated with fasting plasma glucose and insulin concentrations as well as with their HOMA (homeostasis model assessment) and QUICKI (quantitative insulin sensitivity check index) counterparts. Also, I and ISI (HGC) correlated with their counterparts derived from intravenous glucose tolerance tests. In conclusion, this is the first report of hyperglycemic glucose clamping in cats. The procedure (HGC) allows for measurements of glucose disposal, beta-cell response and insulin sensitivity. Compared to human data, both insulin sensitivity and insulin secretion appeared to be low in cats. This is compatible with the carnivorous nature of this species, for which insulin resistance would be advantageous during periods of restricted food availability.

Association of variants of transcription factor 7-like 2 (TCF7L2) with susceptibility to type 2 diabetes in the Dutch Breda cohort.

AIM/HYPOTHESIS: A strong association between susceptibility to type 2 diabetes and common variants of transcription factor 7-like 2 (TCF7L2), encoding an enteroendocrine transcription factor involved in glucose homeostasis, has been reported in three different populations (Iceland, Denmark and USA) by Grant et al. We aimed to replicate these findings in a Dutch cohort. METHODS: We analysed the genotypes of two intronic single nucleotide polymorphisms (SNPs) in TCF7L2 gene in 502 unrelated type 2 diabetes patients and in a set of healthy controls (n = 920). The two SNPs showed almost complete linkage disequilibrium (D' = 0.91). RESULTS: We were able to replicate the previously reported association in our Breda cohort. The minor alleles of both variants were significantly over-represented in cases (odds ratio [OR] 1.29, 95% CI 1.09-1.52, [Formula: see text] for rs12255372; OR 1.41, 95% CI 1.19-1.66, [Formula: see text] for rs7903146). In addition, TCF7L2 haplotypes were analysed for association with the disease. The analysis of haplotypes did not reveal any strong association beyond that expected from analysing individual SNPs. The TT haplotype carrying the minor alleles was more frequent among cases (OR 1.38, [Formula: see text]). CONCLUSIONS/INTERPRETATION: Our data strongly confirm that variants of the TCF7L2 gene contribute to the risk of type 2 diabetes. The population-attributable risk from this factor in the Dutch type 2 diabetes population is 10%.

Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods.

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in "warming solution" consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM(+)) followed by washes in MEM(+) with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM(+) plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM(+) (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as "live" and "dead" markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.

Preservation of caprine preantral follicle viability after cryopreservation in sucrose and ethylene glycol.

Caprine preantral follicles within ovarian fragments were cryopreserved in the absence or presence of 0.5 M sucrose with or without 1 M dimethyl sulfoxide and/or 1 M ethylene glycol (EG). After being thawed, they were washed in minimum essential medium with or without 0.3 M sucrose. Histological analysis of follicle integrity immediately after cryopreservation showed consistent beneficial effects of including sucrose in the three cryoprotectant solutions analyzed when tissue was thawed without sucrose (53.9+/-14.8-82.4+/-3.2% normal vs 27.6+/-1.6-36.6+/-6.5%, P<0.05). However, in further studies, the addition of sucrose to the thaw solutions proved detrimental or of no benefit. An analysis of the cryopreserved material with calcein-AM and ethidium homodimer (markers for living and dead cells, respectively) gave comparable results to those obtained by histology. Follicles cryopreserved in EG, EG plus sucrose, or sucrose alone were cultured in vitro for 24 h following warming. During this culture period, viability fell most rapidly in material cryopreserved in sucrose alone and was no longer correlated with either the viability or integrity estimates made immediately after warming. By contrast, the viability of follicles cryopreserved in EG with sucrose and then cultured for 24 h was not significantly different from the cultured non-frozen controls. These results indicate that cryopreservation in 1 M EG plus 0.5 M sucrose combined with thawing without sucrose is effective for caprine ovarian tissue.

Effect of octreotide on plasma concentrations of glucose, insulin, glucagon, growth hormone, and cortisol in healthy dogs and dogs with insulinoma.

The inhibitory effect of the somatostatin analogue octreotide on the secretion of insulin could be used in the treatment of insulinoma. However, current information on the effectiveness of octreotide in dogs is conflicting. Therefore, the endocrine effects of a single subcutaneous dose of 50 microg octreotide were studied in healthy dogs in the fasting state (n=7) and in dogs with insulinoma (n=12). Octreotide did not cause any adverse effects. In healthy dogs in the fasting state, both plasma insulin and glucagon concentrations declined significantly. Basal (non-pulse related) GH and ACTH concentrations were not affected. A slight but significant decrease in the plasma glucose concentrations occurred. Dogs with insulinoma had significantly higher baseline insulin concentrations and lower baseline glucose concentrations than healthy dogs in the fasting state. Plasma glucagon, GH, ACTH, and cortisol concentrations did not differ from those in healthy dogs. Baseline plasma insulin concentrations decreased significantly in dogs with insulinoma after octreotide administration, whereas plasma concentrations of glucagon, GH, ACTH, and cortisol did not change. In contrast to the effects in the healthy dogs, in the dogs with insulinoma plasma glucose concentrations increased. Thus, the consistent suppression of plasma insulin concentrations in dogs with insulinoma, in the absence of an suppressive effect on counter-regulatory hormones, suggests that further studies on the effectiveness of slow-release preparations in the long-term medical treatment of dogs with insulinoma are warranted.