Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3' and 5' untranslated regions (P = 1.84×10(-5) and 4.7×10(-4), respectively) and SNPs that are synonymous-coding (P = 9.9×10(-4)). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10(-10)). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Early insulin sensitivity after restrictive bariatric surgery, inconsistency between HOMA-IR and steady-state plasma glucose levels.

BACKGROUND: The low-grade inflammatory condition present in morbid obesity is thought to play a causative role in the pathophysiology of insulin resistance (IR). Bariatric surgery fails to improve this inflammatory condition during the first months after surgery. Considering the close relation between inflammation and IR, we conducted a study in which insulin sensitivity was measured during the first months after bariatric surgery. Different methods to measure IR shortly after bariatric surgery have given inconsistent data. For example, the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) levels have been reported to decrease rapidly after bariatric surgery, although clamp techniques have shown sustained insulin resistance. In the present study, we evaluated the use of steady-state plasma glucose (SSPG) levels to assess insulin sensitivity 2 months after bariatric surgery. METHODS: Insulin sensitivity was measured using HOMA-IR and SSPG levels in 11 subjects before surgery and at 26% excess weight loss (approximately 2 months after restrictive bariatric surgery). RESULTS: The SSPG levels after 26% excess weight loss did not differ from the SSPG levels before surgery (14.3 +/- 5.4 versus 14.4 +/- 2.7 mmol/L). In contrast, the HOMA-IR values had decreased significantly (3.59 +/- 1.99 versus 2.09 +/- 1.02). CONCLUSION: During the first months after restrictive bariatric surgery, we observed a discrepancy between the HOMA-IR and SSPG levels. In contrast to the HOMA-IR values, the SSPG levels had not improved, which could be explained by the ongoing inflammatory state after bariatric surgery. These results suggest that during the first months after restrictive bariatric surgery, HOMA-IR might not be an adequate marker of insulin sensitivity.

Short-term overfeeding induces insulin resistance in weight-stable patients after bariatric surgery.

BACKGROUND: Short time overfeeding of rats rapidly leads to insulin resistance (IR). A study with healthy human volunteers, which we suggest are less susceptible for developing IR after short time overfeeding, did not show these effects on IR. Therefore a study population of weight-stable, former morbidly obese subjects (BMI 31.3 kg/m2), which were treated with bariatric surgery approximately 3 years ago was selected. METHODS: Eleven subjects were submitted to a 7-day overfeeding study, resulting in a 53% increase in caloric intake (1,227 +/- 394.4 to 1,879.2 +/- 298.4 kcal/day). During normal diet and after overfeeding, insulin sensitivity was measured using steady state plasma glucose (SSPG) levels. At these time points, BMI and waist/hip ratio together with plasma levels of inflammatory markers (CRP, AGP, LBP, and TNF-alpha receptors) and plasma leptin values were also measured. RESULTS: SSPG levels after overfeeding increased from 8.2 +/- 3.2 to 10.6 +/- 2.6 mmol/l (P < 0.05), indicating decreased insulin sensitivity after overfeeding. Fasting plasma insulin, glucose, circulating levels of inflammatory markers, BMI, and waist/hip ratio remained unchanged. CONCLUSIONS: This study shows that overfeeding in a group of weight-stable, former morbidly obese subjects 3 years after bariatric surgery results in decreased insulin sensitivity. The mechanisms behind decreased insulin sensitivity induced by overfeeding are poorly understood, but the present results reveal that a unique human model is available to study these mechanisms, leading to a better understanding of the pathophysiology of IR.

Endothelial activation markers and their key regulators after restrictive bariatric surgery.

OBJECTIVE: Increased plasma levels of endothelial activation markers in obese subjects reflect the positive association between cardiovascular diseases and obesity. The pro-inflammatory state associated with obesity is thought to play a major role in endothelial cell activation in severely obese individuals. Previous studies demonstrated that long-term weight loss after bariatric surgery is accompanied by a decreased proinflammatory state. However, little is known about the long-term effects of bariatric surgery on endothelial cell activation. RESEARCH METHODS AND PROCEDURES: Plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble endothelial selectin (sE-selectin), and soluble vascular cell adhesion molecule-1 (sVCAM-1), all markers of endothelial cell activation, and of their regulators adiponectin and resistin were measured at different time-points postoperatively in 26 consecutive patients who underwent restrictive surgery, with a follow-up of 2 years. RESULTS: During the first 6 months after bariatric surgery, sE-selectin levels decreased. Despite substantial weight loss, sICAM-1 and sVCAM-1 plasma levels did not decrease significantly. After 24 months, sICAM-1 levels were significantly decreased, whereas sE-selectin levels were further decreased. However, sVCAM-1 levels remained elevated. Adiponectin levels did not change significantly during the first 6 months after bariatric surgery, whereas resistin levels increased. After 24 months, adiponectin levels were similar to normal-weight controls, but resistin levels remained high. DISCUSSION: Reductions in plasma levels of different markers of endothelial activation after bariatric surgery show different temporal patterns, suggesting that distinct mechanisms are involved in their regulation. Although not all endothelial activation markers normalize after bariatric surgery, our findings suggest that bariatric surgery can reduce endothelial activation in the long term.

Contribution of serum response factor and myocardin to transcriptional regulation of smoothelins.

OBJECTIVE: Smoothelin-A and -B isoforms are highly restricted to contractile smooth muscle cells (SMCs). Serum response factor (SRF) and myocardin are essential for contractile SMC differentiation. We evaluated the contribution of SRF/myocardin to transcriptional regulation of smoothelins. METHODS: Rat vascular SMCs were transfected with smoothelin-A and smoothelin-B promoter reporter constructs and promoter activity was analyzed. The effects of mutations in the smoothelin-A promoter CArG-boxes and co-transfections with a myocardin expression plasmid were assessed. Electrophoretic mobility shift assays and chromatin immunoprecipitations were performed to investigate SRF-binding to the smoothelin-A CArG-boxes. RESULTS: Smoothelin promoter activity was detected in vascular SMCs. Comparative sequence analysis revealed two conserved CArG elements in the smoothelin-A promoter that bind SRF as shown by chromatin immunoprecipitation. The proximal CArG-near bound SRF stronger than CArG-far in gel shift assays. Mutagenesis studies also indicated that CArG-near is more important than CArG-far in regulating smoothelin-A promoter activity. Myocardin augmented smoothelin-A promoter activity 2.5-fold in a CArG-near-dependent manner. In contrast, myocardin had little effect on the smoothelin-B promoter. CONCLUSION: Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF. Our data show a role for SRF/myocardin in regulating smoothelin-A whereas the higher smoothelin-B expression appears to be SRF/myocardin-independent.