Open Access Research Article Association of Obesity Risk Snps in Pcsk1 with Insulin Sensitivity and Proinsulin Conversion

article distributed under the terms of the Creative Commons At-tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Prohormone convertase 1 is involved in maturation of peptides. Rare mutations in gene PCSK1, encoding this enzyme, cause childhood obesity and abnormal glucose homeostasis with elevated proinsulin concentrations. Common single nucleotide polymorphisms (SNPs) within this gene, rs6232 and rs6235, are associated with obesity. We studied whether these SNPs influence the prediabetic traits insulin resistance, β-cell dysfunction, or glucose intolerance. Methods: We genotyped 1498 German subjects for SNPs rs6232 and rs6235 within PCSK1. The subjects were metabolically characterized by oral glucose tolerance test with glucose, insulin, proinsulin, and C-peptide measurements. A subgroup of 512 subjects underwent a hyperinsulinemic-euglycemic clamp. Results: The minor allele frequencies were 25.8% for SNP rs6235 and 6.0% for rs6232. After adjustment for sex and age, we found no association of SNPs rs6235 and rs6232 with BMI or other weight-related traits (all p ≥ 0.07). Both minor alleles, adjusted for sex, age, BMI and insulin sensitivity were associated with elevated AUC proinsulin and AUC proinsulin / AUC insulin (rs6235: p additive model ≤ 0.009, effect sizes 8/8%, rs6232: p dominant model ≤ 0.01, effect sizes 10/21%). Insulin secretion was not affected by the variants (different secretion parameters, all p ≥ 0.08). The minor allele of SNP rs6232 was additionally associated with 15% higher OGTT-derived and 19% higher clamp-derived insulin sensitivity (p dom ≤ 0.0047), 4.5% lower HOMA IR (p dom = 0.02) and 3.5% lower 120-min glucose (p dom = 0.0003) independently of BMI and proinsulin conversion. SNP rs6235 was not associated with parameters of glucose metabolism. Conclusions: Like rare mutations in PCSK1, the more common variants tested determine glucose-stimulated proinsulin conversion, but not insulin secretion. In addition, rs6232, encoding the amino acid exchange N221D, influences insulin sensitivity and glucose homeostasis. Background Proteolytic cleavage is an important step in the matura-tion of several hormones that are derived from inactive precursors [1,2]. One important enzyme that catalyzes this processing is prohormone convertase 1. This pro-tease [3] is selectively expressed in neuronal and endo-crine tissues [4] and is active within dense core secretory granules [2,3]. Among its numerous substrates [3,5] are

Since prohormone convertase 1 is involved in proinsulin conversion and thereby in insulin maturation as well as in body weight regulation, the present study examined the association of the putatively obesity-related SNPs rs6232 and rs6235 in PCSK1 with the prediabetic traits insulin resistance, β-cell dysfunction, and glucose intolerance.

Participants
We studied 1498 non-diabetic subjects from southwestern Germany. The participants were selected from the ongoing Tübingen Family Study which currently includes 2000 individuals with an increased risk for type 2 diabetes mellitus [17]. Selection was done based on the availability of all phenotype data used for the analyses. Especially proinsulin levels for all time points during the OGTT were not available for all subjects in the cohort. Most (68.5%) of these subjects had a family history of diabetes, i.e., at least one second-degree relative with type 2 diabetes. Informed written consent was obtained from all participants and the local Ethics Committee approved the protocol. The OGTT revealed that 73.0% of the non-diabetic participants had normal glucose tolerance, 10.0% had impaired fasting glycaemia, 9.6% had impaired glucose tolerance and 7.3% had impaired fasting glycaemia and impaired glucose tolerance.

Genotyping
DNA from whole blood was isolated using a commercial DNA isolation kit (NucleoSpin, Macherey & Nagel, Düren, Germany). Genotyping was performed using the TaqMan assay (Applied Biosystems, Forster City, CA, USA). The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 7000 and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems). The genotyping success rates were 99.0% for rs6232 and 99.1% for rs6235. The genotypes were verified in 50 randomly selected subjects by bidirectional sequencing, and both methods resulted in 100% identical results. Both SNPs were in Hardy-Weinberg equilibrium (rs6232 p = 0.1 and rs6235 p = 0. 7). The SNPs were incompletely linked (D' = 0.887, r 2 = 0.145).

OGTT
After an overnight fast, subjects ingested a solution containing 75 g glucose at 08:00 hours. Venous blood samples were obtained at 0, 30, 60, 90 and 120 min, and plasma glucose, insulin, C-peptide and proinsulin concentrations were determined.

Hyperinsulinemic-euglycemic clamp
Clamp-derived insulin sensitivity was determined in a subgroup of 512 subjects by a hyperinsulinemic-eugylcemic clamp with measurement of glucose and insulin. The test was performed as described earlier [18].

Analytical procedures
Plasma insulin and proinsulin were determined by commercial chemiluminescence assays for ADVIA Centaur (Siemens Medical Solutions, Fernwald, Germany). Blood glucose was measured using a bedside glucose analyzer (glucose oxidase method; Yellow Springs Instruments, Yellow Springs, OH, USA).

Statistical analyses
Unless otherwise stated, data are given as arithmetic means ± SEM. Data were logarithmically transformed prior to statistical analysis. Hardy-Weinberg equilibrium was tested using χ 2 test. Differences in anthropometrics and metabolic parameters between genotypes were tested using multivariate linear regression analysis. PCSK1 SNP rs6235 and SNP rs6232 were examined under both an additive (add) and a dominant inheritance model (dom). However, only the dominant inheritance model was interpreted for SNP rs6232 because of this SNP's low MAF of 6% (only 5 participants were homozygous for the minor allele). All data were adjusted for gender and age. The glucose concentrations and insulin sensitivity indices were additionally adjusted for BMI, and proinsulin concentrations and insulin secretion parameters were additionally adjusted for OGTT-derived insulin sensitivity index and BMI.
Differences with a p-value < 0.05 were considered to indicate nominal associations. After correction for the two unlinked SNPs tested and the endpoints anthropometrics, insulin secretion, and insulin sensitivity (according to Bonferroni), results with values of p < 0.0085 were considered statistically significant. The JMP 7.0 (SAS Institute, Cary, NC, USA) statistical software package was used.

Power calculation
Using two tailed t-test, the study was sufficiently powered (1-beta ≥ 0.8) to detect an effect size of 15% for SNP rs6235 and 22% for SNP rs6232 (dominant inheritance model, p = 0.05). Power calculations were performed using G*power 3.0 software available at http://www.psycho.uni-duesseldorf.de/aap/projects/gpower/.
After appropriate adjustment, both minor alleles of the PCSK1 SNPs rs6232 and rs6235 were neither associated with BMI nor with weight-related traits, like waist circumference and total body fat (all p ≥ 0.07, tables 1 + 2). The frequency of obesity (BMI < 30 versus BMI ≥ 30 kg/ m 2 ) in our cohort was not influenced by the risk alleles (all p ≥ 0.4).
None of the SNPs revealed association with fasting proinsulin concentrations (all p ≥ 0.1, tables 1 + 2). However, upon glucose ingestion, subjects carrying the minor alleles showed an association with higher proinsulin concentrations (rs6232 nominally associated, p dom = 0.0104; rs6235 significantly associated with AUC Proins , p add = 0.0018; adjusted effect sizes 10/8%; tables 1 + 2) and nominally lower proinsulin-to-insulin conversion (AUC Proins / AUC ins ) compared to those with the major allele (rs6232, p dom = 0.0094; rs6235, p add = 0.0090; adjusted effect sizes 21/8%; tables 1 + 2, figure 1A+1D) and this finding was consistent when adjusting for insulin sensitivity or not. Both variants were not associated with differences in insulin secretion (all indices used p ≥ 0.08, tables 1 + 2), and SNP rs6235 was furthermore neither associated with glucose nor with insulin levels during the OGTT (all p ≥ 0.2, figure 1B and 1C).
In addition to these findings, the minor allele of SNP rs6232, but not that of SNP rs6235, was significantly associated with decreased 120-min blood glucose (p dom = 0.0003, table 1, figure 2) and nominally associated with decreased fasting insulin levels (p dom = 0.0297, table 1). Insulin sensitivity derived from the OGTT was significantly increased (p dom = 0.0010, adjusted effect size 15%, table 1, figure 2) and insulin resistance derived from fasting state (HOMA IR ) was accordingly nominally decreased in carriers of the minor allele (p dom = 0.0232, table 1 and figure 2). The effect on OGTT-derived insulin sensitivity remained significant even after adjustment for gender, age, BMI, AUC Proins /AUC Ins , and first-phase insulin secretion (p dom = 0.0033).
To confirm that the association with insulin sensitivity did not solely reflect altered insulin levels due to impaired prohormone processing, we analyzed insulin sensitivity measured by exogenous administration of insulin during hyperinsulinemic-euglycemic clamp. Again, SNP rs6232, but not SNP rs6235 was associated with significantly increased insulin sensitivity (p dom = 0.0047; adjusted effect size 19%; table 1).
To analyze subjects with low risk of type 2 diabetes mellitus versus those with a high risk, we stratified for family history of diabetes. This stratification did not produce consistent results, probably due to the limited power of the subgroups.

Association of PCSK1 with weight-related traits
In our cohort, the reported association of the common variants rs6232 and rs6235 within the PCSK1 gene with BMI and obesity could not be replicated. This is most likely explained by the limited power of our study to detect relatively small effect sizes of the SNPs on BMI and obesity. The effect sizes detectable in our study were as small as 15%, and for the established obesity risk locus FTO an effect size of about 10% could be demonstrated [21].

Association of PCSK1 with proinsulin levels
In the present study, we found that the minor alleles of both candidate SNPs in the PCSK1 gene are associated with significantly higher glucose-stimulated proinsulin levels indicating reduced proinsulin conversion, while insulin secretion was unaffected. These findings were not altered by adjustment for insulin sensitivity.
Basal proinsulin levels were unaffected by the variants in PCSK1. Thus, under basal conditions the enzyme cleaves sufficient amounts of proinsulin. However, in the state of much higher secretory demands following a glucose load, proinsulin levels and proinsulin-to-insulin ratios were increased in carriers of both SNPs and this points to insufficient insulin maturation.
Both SNPs in the PCSK1 gene alter the amino acid sequence of the protein. SNP rs6232 changes asparagine to aspartic acid (N221D) in the catalytic domain and leads to a 10% reduction of activity, while SNP rs6235 substitutes serine by threonine (S690T) without any effect on the enzymatic activity [6]. Reduced enzymatic activity due to SNP rs6232 can plausibly explain this observation, though the mechanism is less clear in the case of the other SNP, rs6235. The substituted amino acid by that variation could possibly influence binding of regulators of the enzyme's activity, its localization within the β-cell, or the available number of prohormone convertase 1 molecules via altered stability. All these effects could theoretically influence prohormone conversion.
Although both PCSK1 SNPs affect proinsulin levels, they do not alter insulin secretion after glucose stimulation. Thus, the enzyme does not represent the rate-limit-  ing step in insulin secretion which is in contrast to SNPs in diabetes risk genes which influence both proinsulin conversion and insulin secretion [22].

Association of PCSK1 SNP rs6232 with insulin sensitivity
Furthermore, the putative obesity risk allele of SNP rs6232 was unexpectedly associated with increased insulin sensitivity (fasting, OGTT-derived, and hyperinsulinemic euglycemic-clamp derived) and reduced 120-min glucose levels independently of BMI and proinsulin conversion. The similar findings with different independent measurements of insulin sensitivity as well as the inverse influence on 120-min blood glucose argue against a mere statistical type 1 error. Since only SNP rs6232 in the PCSK1 gene, the one that decreases the enzyme's catalytic activity [6], influences insulin sensitivity, independently of BMI and proinsulin conversion, and since there is a long list of potential substrates of the prohormone convertases [3][4][5], it seems reasonable to speculate that the enzyme also cleaves factors that modulate insulin sensitivity (e.g. BNDF [23]).
In most cases, obesity is associated with insulin resistance. However, a proportion of overweight/obese persons display a phenotype of obesity with is metabolically benign and characterized by high insulin sensitivity [17]. Since the minor allele of SNP rs6232 was associated with higher insulin sensitivity in our study and with obesity in a large metaanalysis including more then 13,000 subjects [6], we hypothesize that this allele may promote a kind of obesity, which is more benign.
Increased proinsulin levels are reported to be a marker of insulin resistance [24]. Interestingly, for carriers of PCSK1 SNP rs6232 it is the other way round: Those with higher proinsulin concentrations during the OGTT have also increased insulin sensitivity. Similar findings are reported in another publication where insulin resistance was associated with enhanced proinsulin processing in subjects with normal glucose tolerance [25]. Hence, increased proinsulin concentration can not generally be seen as a marker of insulin resistance.

Limitations
Our study was underpowered to detect the previous reported associations of the PCSK1 variants with obesity. The associations with proinsulin conversion and insulin sensitivity found in this study clearly need replication in other cohorts, in particular, the unexpected association of SNP rs6232 with insulin sensitivity. Furthermore, efforts should also be undertaken to identify prohormone convertase 1 substrates that alter insulin sensitivity and to further characterize these mechanisms. Studies on the molecular level could additionally help to answer the question how PCSK1's SNP rs6235 influences proinsulin levels without changing the enzyme's activity.

Conclusions
In conclusion, our data suggest that the common genetic variants rs6232 and rs6235 within PCSK1 determine glucose-stimulated proinsulin conversion, but not insulin secretion. In addition, rs6232, encoding the mutation N221D, influences glucose homeostasis and insulin sensitivity independently of BMI and proinsulin concentrations.