The influence of high-density lipoprotein (HDL) and HDL subfractions on insulin secretion and cholesterol efflux in pancreatic derived ß-cells.

BACKGROUND: High-density lipoprotein (HDL) is considered a complex plasma-circulating particle with subfractions that vary in function, size, and chemical composition. We sought to test the effects of HDL, and HDL subfractions on insulin secretion and cholesterol efflux in the ß-cell line MIN-6. METHODS: We used total HDL and HDL subfractions 2a, 2b, 3a, 3b, and 3c, isolated from human plasma, to test insulin secretion under different glucose concentrations as well as insulin content and cholesterol efflux in the insulinoma MIN-6 cell line. RESULTS: Incubation of MIN-6 cells with low glucose and total HDL increased insulin release two-fold. Meanwhile, when high glucose and HDL were used, insulin release increased more than five times. HDL subfractions 2a, 2b, 3a, 3b, and 3c elicited higher insulin secretion and cholesterol efflux than their respective controls, at both low and high glucose concentrations. The insulin content of the MIN-6 cells incubated with low glucose and any of the five HDL subclasses had a modest reduction compared with their controls. However, there were no statistically significant differences between each HDL subfraction on their capacity of eliciting insulin secretion, insulin content, or cholesterol efflux. CONCLUSIONS: HDL can trigger insulin secretion under low, normal, and high glucose conditions. We found that all HDL subfractions exhibit very similar capacity to increase insulin secretion and cholesterol efflux. This is the first report demonstrating that HDL subfractions act both as insulin secretagogues (under low glucose) and insulin secretion enhancers (under high glucose) in the MIN-6 cell line.

The -514C>T polymorphism in the LIPC gene modifies type 2 diabetes risk through modulation of HDL-cholesterol levels in Mexicans.

PURPOSE: Both type 2 diabetes (T2D) and low levels of high-density lipoprotein cholesterol (HDL-C) are very prevalent conditions among Mexicans. Genetic variants in the LIPC gene have been associated with both conditions. This study aimed to evaluate the association of the -514C < T (rs1800588) LIPC gene polymorphism with different metabolic traits, particularly the effects of this polymorphism on HDL-C plasma levels and T2D risk. METHODS: Mediation analysis was used to assess the direct and indirect effects of the -514C>T LIPC gene variant on HDL-C levels, T2D risk, and body mass index (BMI), in 2105 Mexican mestizo participants. We also assessed the functional effect of the -514C>T LIPC variant on the promoter activity of a reporter gene in the HepG2 cell line. RESULTS: Direct effects show that the -514C>T LIPC polymorphism is significantly associated with increased HDL-C plasma levels (ß = 0.03; p < 0.001). The -514C>T variant resulted in an indirect protective effect on T2D risk through increasing HDL-C levels (ß = - 0.03; p < 0.001). Marginal direct association between -514C>T and T2D was found (ß = 0.08; p = 0.06). Variables directly influencing T2D status were European ethnicity (ß = - 7.20; p < 0.001), age (ß = 0.04; p < 0.001), gender (ß = - 0.15; p = 0.017) and HDL-C (ß = - 1.07; p < 0.001). In addition, we found that the -514C>T variant decreases the activity of LIPC promoter by 90% (p < 0.001). CONCLUSIONS: The -514C>T polymorphism was not directly associated with T2D risk. HDL-C acts as a mediator between -514C>T LIPC gene variant and T2D risk in the Mexican population.

R230C but not - 565C/T variant of the ABCA1 gene is associated with type 2 diabetes in Mexicans through an effect on lowering HDL-cholesterol levels.

PURPOSE: Type 2 diabetes (T2D) and low serum concentration of high-density lipoprotein cholesterol (HDL-c) are common coexisting metabolic disorders. ABCA1 variants have been shown to be associated to these conditions. We sought to test the combined effect of two ABCA1 gene common variants, rs2422493 (- 565C > T) and rs9282541 (R230C) on HDL-c levels and T2D risk. METHODS: Path analysis was conducted in 3,303 Mexican-mestizos to assess the specific contributions of rs2422493 and rs9282541 ABCA1 variants, insulin resistance, waist-to-height ratio (WHtR), and age on HDL-c levels and T2D risk. Participants were classified into four groups according to their ABCA1 variants carrier status: (i) the reference group carried wild type alleles for both ABCA1 variants (-/-), (ii) +/- were carriers of rs2422493 but non-carriers of rs9282541, (iii) -/+ for carriers of rs9282541 but not carriers of rs2422493 and (iv) carriers of minor alleles for both SNPs (+/+). Principal components from two previous genome-wide association studies were used to control for ethnicity. RESULTS: We identified significant indirect effects on T2D risk mediated by HDL-c in groups -/+ and +/+ (ß = 0.04; p = 0.03 and ß = 0.06; p < 0.01, respectively) in comparison to the -/- reference group. Low concentrations of HDL-c were directly and significantly associated with increased T2D risk (ß = -0.70; p < 0.01). WHtR, male gender, age, and insulin resistance were also associated with T2D risk (p < 0.05). There was no significant direct effect for any of the ABCA1 groups on T2D risk: p = 0.99, p = 0.58, and p = 0.91 for groups +/-, -/+, and +/+ respectively. CONCLUSIONS: The ABCA1 rs9282541 (R230C) allele is associated with T2D in Mexicans through its effect on lowering HDL-c levels. This is the first report demonstrating that HDL-c levels act as an intermediate factor between an ABCA1 variant and T2D.

Strong association of socioeconomic status with genetic ancestry in Latinos: implications for admixture studies of type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is more prevalent in US American minority populations of African or Native American descent than it is in European Americans. However, the proportion of this epidemiological difference that can be ascribed to genetic or environmental factors is unknown. To determine whether genetic ancestry is correlated with diabetes risk in Latinos, we estimated the proportion of European ancestry in case-control samples from Mexico and Colombia in whom socioeconomic status had been carefully ascertained. METHODS: We genotyped 67 ancestry-informative markers in 499 participants with type 2 diabetes and 197 controls from Medellín (Colombia), as well as in 163 participants with type 2 diabetes and 72 controls from central Mexico. Each participant was assigned a socioeconomic status scale via various measures. RESULTS: Although European ancestry was associated with lower diabetes risk in Mexicans (OR [95% CI] 0.06 [0.02-0.21], p = 2.0 x 10(-5)) and Colombians (OR 0.26 [0.08-0.78], p = 0.02), adjustment for socioeconomic status eliminated the association in the Colombian sample (OR 0.64 [0.19-2.12], p = 0.46) and significantly attenuated it in the Mexican sample (OR 0.17 [0.04-0.71], p = 0.02). Adjustment for BMI did not change the results. CONCLUSIONS/INTERPRETATION: The proportion of non-European ancestry is associated with both type 2 diabetes and lower socioeconomic status in admixed Latino populations from North and South America. We conclude that ancestry-directed search for genetic markers associated with type 2 diabetes in Latinos may benefit from information involving social factors, as these factors have a quantitatively important effect on type 2 diabetes risk relative to ancestry effects.

Association of PPARG2 Pro12Ala variant with larger body mass index in Mestizo and Amerindian populations of Mexico.

Previous studies have sought to associate the Pro12Ala variant of the peroxisome proliferator-activated receptor gamma2 (PPARG2) gene with type 2 diabetes, insulin resistance, and obesity, with controversial results. We have determined the Pro12Ala variant frequency in 370 nondiabetic Mexican Mestizo subjects and in five Mexican Amerindian groups and have investigated its possible association with lipid metabolism, insulin serum levels, and obesity in three of these populations. Two independent case-control studies were conducted in 239 nondiabetic individuals: 135 case subjects (BMI > or = 25 kg/m2) and 104 control subjects (BMI < 25 kg/m2). The PPARG2 Ala12 allele frequency was higher in most Amerindian populations (0.17 in Yaquis, 0.16 in Mazahuas, 0.16 in Mayans, and 0.20 in Triquis) than in Asians, African Americans, and Caucasians. The Pro12Ala and Ala12Ala (X12Ala) genotypes were significantly associated with greater BMI in Mexican Mestizos and in two Amerindian groups. X12Ala individuals had a higher risk of overweight or obesity than noncarriers in Mestizos (OR = 3.67; 95% CI, 1.42-9.48; p = 0.007) and in Yaquis plus Mazahuas (OR = 3.21; 95% CI, 1.27-8.11; p = 0.013). Our results provide further support of the association between the PPARG2 Ala12 allele and risk of overweight or obesity in Mestizos and two Amerindian populations from Mexico.

HNF-1alpha G574S is a functional variant with decreased transactivation activity.

AIM: To assess the functional consequence of the hepatocyte nuclear factor 1alpha gene (HNF-1alpha) G574S variant previously proposed as a diabetes susceptibility allele, in a group of Mexican Type 2 diabetic patients with end-stage renal disease (ESRD). METHODS: The transcriptional activity of the HNF-1alpha G574S recombinant protein on the human insulin promoter was assessed by transfection assays in RINm5f and HepG2 cell lines. RESULTS: Two unrelated Mexican diabetic patients with no known African ancestry were found to carry the G574S variant. This substitution was not found among unrelated healthy control subjects. Whereas the G574S HNF-1alpha transcription activation of the human insulin promoter was 40% lower than that of the wild-type protein in RINm5f beta cells, no difference was found in a hepatic cell line (HepG2). CONCLUSIONS: G574S affects the transactivation potential of HNF-1alpha on the insulin promoter in pancreatic beta-cells. Although it has been difficult to prove its role in the development of diabetes in case-control association studies, this variant exhibits functional effects consistent with it being a potential diabetes susceptibility allele.

Contribution of chromosome 1q21-q23 to familial combined hyperlipidemia in Mexican families.

Familial combined hyperlipidemia (FCHL) is the most common familial dyslipidemia, with a prevalence of 1-2% in the general population. A major locus for FCHL has been mapped to chromosome 1q21-q23 in Finnish, Chinese, German and US families. We studied seven extended Mexican families with 153 members, including 64 affected subjects. A total of 11 markers were genotyped, including D1S104 which has been linked to FCHL in other studies. Two point linkage analysis for the FCHL phenotype, and for the elevated triglyceride (TG) trait, allowing for heterogeneity, gave a maximum HLOD of 1.67 (alpha = 0.49) and 1.93 (alpha = 0.43) at D1S2768 (2.69 cM proximal to D1S104) respectively. Heterogeneity and non-parametric (NPL) multipoint analyses for the FCHL phenotype and the TG trait showed maximum HLODs of 1.27 (alpha = 0.46) and 1.64 (alpha = 0.38), and NPLs of 4.00 (P = 0.0001) and 3.68 (P = 0.0003) near D1S2768, respectively. In addition, analysis of four candidate genes putatively involved in the expression of FCHL showed no evidence of linkage for the LCAT gene or the APOA1/C3/A4/A5 gene cluster. However, we cannot exclude the participation of these genes, or the LIPC and LPL genes, as minor susceptibility loci in the expression of FCHL, or the TG or elevated total cholesterol (TC) traits in our families. In conclusion, our data confirm the involvement of a major susceptibility locus on chromosome 1q21-q23 in FCHL Mexican families, consistent with findings in other populations.

Early-onset type 2 diabetes: metabolic and genetic characterization in the mexican population.

The objective of this study was to investigate possible defects in the insulin sensitivity and/or the acute insulin response in a group of Mexican patients displaying early-onset type 2 diabetes and to evaluate the contribution of mutations in three of the genes linked to maturity-onset diabetes of the young. We studied 40 Mexican patients with an age of diagnosis between 20 and 40 yr in which the insulin sensitivity as well as the insulin secretory response were measured using the minimal model approach. A partial screening for possible mutations in 3 of the 5 genes linked to maturity-onset diabetes of the young was carried out by PCR-single strand conformation polymorphism analysis. A low insulin secretory capacity (AIRg = 68.5 +/- 5 muU/mL.min) and a near-normal insulin sensitivity (3.43 +/- 0.2 min/muU.mL x 10(4)) were found in these patients. Among this group we found two individuals carrying missense mutations in exon 4 of the hepatocyte nuclear factor-1alpha (HNF-4alpha) gene (Asp(126)-->His/Tyr and Arg(154)-->Gln, respectively) and one carrying a nonsense mutation in exon 7 of the HNF-1alpha gene (Gln(486)-->stop codon); 7.5% had positive titers for glutamic acid decarboxylase antibodies. Thirty-five percent of cases had insulin resistance; these subjects had the lipid abnormalities seen in the metabolic syndrome. A defect in insulin secretion is the hallmark in Mexican diabetic patients diagnosed between 20 and 40 yr of age. Mutations in either the HNF-1alpha or the HNF-4alpha genes are present among the individuals who develop early-onset diabetes in our population. These particular sequence changes have not been previously reported and therefore represent putative new mutations. Even in the absence of endogenous hyperinsulinemia, insulin resistance is associated with an adverse lipid profile.

[Genetics of type 2 diabetes mellitus: genes implicated in early onset diabetes]. / La genética de la diabetes mellitus tipo 2: genes implicados en la diabetes de aparición temprana.

Non-insulin dependent type 2 diabetes (NIDDM) is a chronic and degenerative disease characterized by elevated glucose serum and the predisposition to the development of vascular complications. In Mexico the incidence of the disease reaches 8%, where one in every ten patients are diagnosed before age 40 (early-onset diabetes). NIDDM is a clinically and genetic heterogeneous entity. Mutations in the glucokinase gene and the genes for the transcription factors HNF-1 alpha, HNF-4 alpha, IPF-1, HNF-1 beta y HNF-3 beta have been demonstrated to cause MODY, a subtype of NIDDM characterized by autosomal dominate pattern of inheritance and an early-onset. Mutations in any of these genes result in deficient insulin synthesis and/or secretion. Five of these genes encode transcription factors that activate the transcription of various genes in pancreatic beta cell including, the insulin gene. Mutations in any of the genes associated to MODY may contribute to the insulin secretion deficiency frequently observed in early-onset type 2 diabetic patients. The structural and functional analysis of these genes, as well as other transcription factors expressed in pancreatic beta cell has allowed their recognition as putative candidate genes involved in the susceptibility to develop the disease.

Mutation analysis in patients with congenital adrenal hyperplasia in the Spanish population: identification of putative novel steroid 21-hydroxylase deficiency alleles associated with the classic form of the disease.

Steroid 21-hydroxylase deficiency, due to the genetic impairment of the CYP21 gene, is a major cause of congenital adrenal hyperplasia (CAH). In about 80% of the cases, the defect is related with the transfer of deleterious point mutations from the CYP21P pseudogene to the active CYP21 gene. Sixteen different point mutations have been searched for in 60 Spanish patients with the classic form of CAH and 171 unaffected family members, using selective amplification of the CYP21 gene followed by allele-specific oligonucleotide hybridization (PCR-ASOH) and sequencing analysis. While 31.9% of the disease alleles carry CYP21 deletions or large gene conversions, around 58% of the alleles carry single point mutations. Corresponding segregation of mutations was found in every case indicating that none of them has apparently appeared de novo. The most frequent mutations found in our sample are i2G, V281L, R356W, Q318X, P453S and F306+t, with rates of 30, 14.2, 10, 9.2, 9.2 and 7. 5%, respectively. We found similar frequencies for the A and C polymorphism at position 656 (40 and 31.5%, respectively) in wild-type alleles for the i2G mutation. Around 10% of the alleles, for which no mutations were identified by searching for the sixteen previously known mutations, are currently being sequenced and new possible mutations and polymorphisms have been identified.

Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: evidence of different genetic mechanisms involved in the production of the disease.

Congenital adrenal hyperplasia (CAH) is an inherited recessive disorder of adrenal steroidogenesis caused by mutations in the steroid 21-hydroxylase gene (CYP21) in more than 90% of affected patients. The CYP21 gene is located within the HLA complex locus on chromosome 6 (6p21.3). During a molecular characterisation study of a group of 47 Mexican families with 21-hydroxylase deficiency, we identified nine in which the mutation or mutations found in the patient did not appear to originate from one of the parents. Through DNA fingerprinting, paternity was established in all nine families with a probability of non-paternity in the range of 10(-19) to 10(-23). Among these families, we identified one patient with exclusive paternal inheritance of all eight markers tested on chromosome 6p, despite normal maternal and paternal contributions for eight additional markers on three different chromosomes. We did not identify duplication of paternal information for markers in the 6q region, consistent with lack of expression of transient neonatal diabetes owing to genomic imprinting in this patient. Our results substantiate evidence for the existence of different genetic mechanisms involved in the expression of this recessive condition in a substantial portion (approximately 19%) of affected Mexican families. In addition to the identification of a patient with paternal uniparental disomy, the occurrence of germline mutations may explain the unusual pattern of segregation in the majority of the remaining eight families.

Molecular genetic analysis of patients carrying steroid 21-hydroxylase deficiency in the Mexican population: identification of possible new mutations and high prevalence of apparent germ-line mutations.

Steroid 21-hydroxylase deficiency is the underlying cause in over 90% of patients with congenital adrenal hyperplasia, an inherited metabolic disorder of adrenal steroidogenesis. We have characterized 94 mutant alleles from 47 unrelated Mexican patients and the corresponding mutant alleles in their parents by amplification of the functional CYP21 gene by PCR, followed by direct sequence analysis. The study included patients diagnosed with the three clinical forms of the disease. Our results revealed: (1) the presence of relatively few mutations or combinations of mutations associated with particular phenotypes; (2) the presence of putative new mutations; (3) the finding of identical genotypes in patients displaying discordant phenotypes; (4) the identification of patients lacking all previous reported mutations; and (5) an apparent high frequency of germ-line mutations. The absence of previously reported mutations in about 22% of the disease alleles, the finding of putative new mutations in some of the patients lacking previously known mutations, and the apparent high prevalence of germ-line mutations make evident the differences in the genetic background leading to this disorder between the Caucasian and the Mexican populations.

Analysis of the glucokinase gene in Mexican families displaying early-onset non-insulin-dependent diabetes mellitus including MODY families.

Non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes, affecting 5% of the general population. Genetic factors play an important role in the development of the disease. While in other populations NIDDM is usually diagnosed after the fifth decade of life, in Mexico a large proportion of patients develop the disease at an early age (between the third and the fourth decade). In Caucasian population, mutations in the glucokinase gene, the TCF1, and TCF14 genes, have been identified in a subgroup of early-onset NIDDM patients denominated MODY (maturity-onset diabetes of the young), which show an autosomal dominant pattern of inheritance. As a first step in the molecular characterization of Mexican families displaying early-onset NIDDM we searched for mutations in the glucokinase gene through SSCP analysis and/or direct sequencing in 26 individuals from 22 independent families, where at least four can be classified as MODY. No mutations were detected in the exons or the intron-exon boundaries of the gene in any of the screened individuals. The phenotype and clinical profile of some of the studied patients is compatible with that of patients carrying mutations in the TCF1 or TCF14 genes, while others may carry mutations in different loci. Through computer simulation analysis we identified at least four informative families which will be used for further linkage studies.

Low frequency of deletion alleles in patients with steroid 21-hydroxylase deficiency in a Mexican population.

Steroid 21-hydroxylase deficiency is caused by mutations in the CYP21 gene. Approximately 95% of mutant alleles are generated by recombination events between the active gene CYP21 and its highly homologous pseudogene, CYP21P. Deletion alleles are generated by unequal crossing over, while point mutations are the result of gene conversion events. Deletions account for 20-25% of the 21-hydroxylase deficiency alleles in most populations studied. We have looked for deletions among 53 unrelated Mexican patients with steroid 21-hydroxylase deficiency and found that deletions represent less than 1% of the disease alleles. These findings suggest that nearly all mutant alleles in our patient population contain point mutations and that the low representation of deletion alleles among clinically diagnosed patients may be due to missing detection of salt wasters, mainly males, who may die during the neonatal period.

Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over.