Genetics of Plasma Bilirubin and Associations between Bilirubin and Cardiometabolic Risk Profiles in Danish Children and Adolescents.

Bilirubin is the end product of heme catabolism, mainly produced by the breakdown of mature red blood cells. Due to its anti-inflammatory, antioxidant, antidiabetic, and antilipemic properties, circulating bilirubin concentrations are inversely associated with the risk of cardiovascular disease, type 2 diabetes, and all-cause mortality in adults. Some genetic loci associated with circulating bilirubin concentrations have been identified by genome-wide association studies in adults. We aimed to examine the relationship between circulating bilirubin, cardiometabolic risk factors, and inflammation in children and adolescents and the genetic architecture of plasma bilirubin concentrations. We measured fasting plasma bilirubin, cardiometabolic risk factors, and inflammatory markers in a sample of Danish children and adolescents with overweight or obesity (n = 1530) and in a population-based sample (n = 1820) of Danish children and adolescents. Linear and logistic regression analyses were performed to analyze the associations between bilirubin, cardiometabolic risk factors, and inflammatory markers. A genome-wide association study (GWAS) of fasting plasma concentrations of bilirubin was performed in children and adolescents with overweight or obesity and in a population-based sample. Bilirubin is associated inversely and significantly with a number of cardiometabolic risk factors, including body mass index (BMI) standard deviation scores (SDS), waist circumference, high-sensitivity C-reactive protein (hs-CRP), homeostatic model assessment for insulin resistance (HOMA-IR), hemoglobin A1c (HbA1c), low-density lipoprotein cholesterol (LDL-C), triglycerides, and the majority of measured inflammatory markers. In contrast, bilirubin was positively associated with fasting plasma concentrations of alanine transaminase (ALT), high-density lipoprotein cholesterol (HDL-C), systolic blood pressure (SDS), and the inflammatory markers GH, PTX3, THBS2, TNFRSF9, PGF, PAPPA, GT, CCL23, CX3CL1, SCF, and TRANCE. The GWAS showed that two loci were positively associated with plasma bilirubin concentrations at a p-value threshold of <5 × 10-8 (rs76999922: ß = -0.65 SD; p = 4.3 × 10-8, and rs887829: ß = 0.78 SD; p = 2.9 × 10-247). Approximately 25% of the variance in plasma bilirubin concentration was explained by rs887829. The rs887829 was not significantly associated with any of the mentioned cardiometabolic risk factors except for hs-CRP. Our findings suggest that plasma concentrations of bilirubin non-causally associates with cardiometabolic risk factors in children and adolescents.

Type 2 Diabetes risk alleles in Peptidyl-glycine Alpha-amidating Monooxygenase influence GLP-1 levels and response to GLP-1 Receptor Agonists.

Patients with type 2 diabetes vary in their response to currently available therapeutic agents (including GLP-1 receptor agonists) leading to suboptimal glycemic control and increased risk of complications. We show that human carriers of hypomorphic T2D-risk alleles in the gene encoding peptidyl-glycine alpha-amidating monooxygenase (PAM), as well as Pam-knockout mice, display increased resistance to GLP-1 in vivo. Pam inactivation in mice leads to reduced gastric GLP-1R expression and faster gastric emptying: this persists during GLP-1R agonist treatment and is rescued when GLP-1R activity is antagonized, indicating resistance to GLP-1's gastric slowing properties. Meta-analysis of human data from studies examining GLP-1R agonist response (including RCTs) reveals a relative loss of 44% and 20% of glucose lowering (measured by glycated hemoglobin) in individuals with hypomorphic PAM alleles p.S539W and p.D536G treated with GLP-1R agonist. Genetic variation in PAM has effects on incretin signaling that alters response to medication used commonly for treatment of T2D.

Physical Activity and Insulin Sensitivity Independently Attenuate the Effect of FTO rs9939609 on Obesity.

OBJECTIVE: The association between FTO rs9939609 and obesity is modified by physical activity (PA) and/or insulin sensitivity (IS). We aimed to assess whether these modifications are independent, to assess whether PA and/or IS modify the association between rs9939609 and cardiometabolic traits, and to elucidate underlying mechanisms. RESEARCH DESIGN AND METHODS: Genetic association analyses comprised up to 19,585 individuals. PA was self-reported, and IS was defined based on inverted HOMA insulin resistance index. Functional analyses were performed in muscle biopsies from 140 men and in cultured muscle cells. RESULTS: The BMI-increasing effect of the FTO rs9939609 A allele was attenuated by 47% with high PA (ß [SE], -0.32 [0.10] kg/m2, P = 0.0013) and by 51% with high IS (-0.31 [0.09] kg/m2, P = 0.00028). Interestingly, these interactions were essentially independent (PA, -0.20 [0.09] kg/m2, P = 0.023; IS, -0.28 [0.09] kg/m2, P = 0.0011). The rs9939609 A allele was also associated with higher all-cause mortality and certain cardiometabolic outcomes (hazard ratio, 1.07-1.20, P > 0.04), and these effects tended to be weakened by greater PA and IS. Moreover, the rs9939609 A allele was associated with higher expression of FTO in skeletal muscle tissue (0.03 [0.01], P = 0.011), and in skeletal muscle cells, we identified a physical interaction between the FTO promoter and an enhancer region encompassing rs9939609. CONCLUSIONS: Greater PA and IS independently reduced the effect of rs9939609 on obesity. These effects might be mediated through altered expression of FTO in skeletal muscle. Our results indicated that PA and/or other means of increasing insulin sensitivity could counteract FTO-related genetic predisposition to obesity.

High Plasma Levels of Soluble Lectin-like Oxidized Low-Density Lipoprotein Receptor-1 Are Associated With Inflammation and Cardiometabolic Risk Profiles in Pediatric Overweight and Obesity.

Background Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 is a scavenger receptor for oxidized low-density lipoprotein. In adults, higher soluble lectin-like ox-LDL receptor-1 (sLOX-1) levels are associated with cardiovascular disease, type 2 diabetes, and obesity, but a similar link in pediatric overweight/obesity remains uncertain. Methods and Results Analyses were based on the cross-sectional HOLBAEK Study, including 4- to 19-year-olds from an obesity clinic group with body mass index >90th percentile (n=1815) and from a population-based group (n=2039). Fasting plasma levels of sLOX-1 and inflammatory markers were quantified, cardiometabolic risk profiles were assessed, and linear and logistic regression analyses were performed. Pubertal/postpubertal children and adolescents from the obesity clinic group exhibited higher sLOX-1 levels compared with the population (P<0.001). sLOX-1 positively associated with proinflammatory cytokines, matrix metalloproteinases, body mass index SD score, waist SD score, body fat %, plasma alanine aminotransferase, serum high-sensitivity C-reactive protein, plasma low-density lipoprotein cholesterol, triglycerides, systolic and diastolic blood pressure SD score, and inversely associated with plasma high-density lipoprotein cholesterol (all P<0.05). sLOX-1 positively associated with high alanine aminotransferase (odds ratio [OR], 1.16, P=4.1 E-04), insulin resistance (OR, 1.16, P=8.6 E-04), dyslipidemia (OR, 1.25, P=1.8 E-07), and hypertension (OR, 1.12, P=0.02). Conclusions sLOX-1 levels were elevated during and after puberty in children and adolescents with overweight/obesity compared with population-based peers and associated with inflammatory markers and worsened cardiometabolic risk profiles. sLOX-1 may serve as an early marker of cardiometabolic risk and inflammation in pediatric overweight/obesity. Registration The HOLBAEK Study, formerly known as The Danish Childhood Obesity Biobank, ClinicalTrials.gov identifier number NCT00928473, https://clinicaltrials.gov/ct2/show/NCT00928473 (registered June 2009).

The Arg82Cys Polymorphism of the Protein Nepmucin Implies a Role in HDL Metabolism.

Context: Blood lipid levels are linked to the risk of cardiovascular disease and regulated by genetic factors. A low-frequency polymorphism Arg82Cys (rs72836561) in the membrane protein nepmucin, encoded by CD300LG, is associated with lower fasting concentration of high-density lipoprotein cholesterol (HDLc) and higher fasting triglycerides. However, whether the variant is linked to postprandial lipids and glycemic status remains elusive. Objective: Here, we augment the genetic effect of Arg82Cys on fasting plasma concentrations of HDL subclasses, postprandial lipemia after a standardized high-fat meal, and glycemic status to further untangle its role in HDL metabolism. Methods: We elucidated fasting associations with HDL subclasses in a population-based cohort study (Oxford BioBank, OBB), including 4522 healthy men and women. We investigated fasting and postprandial consequences on HDL metabolism in recall-by-genotype (RbG) studies (fasting: 20 carrier/20 noncarrier; postprandial: 7 carrier/17 noncarrier), and shed light on the synergistic interaction with glycemic status. Results: A lower fasting plasma concentration of cholesterol in large HDL particles was found in healthy male carriers of the Cys82 polymorphism compared to noncarriers, both in the OBB (P = .004) and RbG studies (P = .005). In addition, the Cys82 polymorphism was associated with low fasting plasma concentrations of ApoA1 (P = .008) in the OBB cohort. On the contrary, we did not find differences in postprandial lipemia or 2-hour plasma glucose levels. Conclusion: Taken together, our results indicate an association between the Arg82Cys variant and a lower concentration of HDL particles and HDLc, especially in larger HDL subclasses, suggesting a link between nepmucin and HDLc metabolism or maturation.

Hyperglucagonemia in Pediatric Adiposity Associates With Cardiometabolic Risk Factors but Not Hyperglycemia.

CONTEXT: In adults, hyperglucagonemia is associated with type 2 diabetes, impaired glucose tolerance, and obesity. The role of glucagon in pediatric overweight/obesity remains unclear. OBJECTIVE: We examined whether fasting concentrations of glucagon are elevated in youth with overweight/obesity and whether this associates with cardiometabolic risk profiles. METHODS: Analyses were based on the cross-sectional HOLBAEK study, including children and adolescents 6 to 19 years of age, with overweight/obesity from an obesity clinic group (n = 2154) and with normal weight from a population-based group (n = 1858). Fasting concentrations of plasma glucagon and cardiometabolic risk outcomes were assessed, and multiple linear and logistic regressions models were performed. RESULTS: The obesity clinic group had higher glucagon concentrations than the population-based group (P < 0.001). Glucagon positively associated with body mass index (BMI) standard deviation score (SDS), waist, body fat %, liver fat %, alanine transaminase (ALT), high-sensitivity C-reactive protein, homeostasis model assessment of insulin resistance, insulin, C-peptide, LDL-C, triglycerides, SDS of diastolic and systolic blood pressure, and was inversely associated with fasting glucose. The inverse relationship between glucagon and glucose was attenuated in individuals with high BMI SDS and high fasting insulin. Glucagon was associated with a higher prevalence of insulin resistance, increased ALT, dyslipidemia, and hypertension, but not with hyperglycemia. Glucagon was positively associated with fasting total glucagon-like peptide-1. CONCLUSION: Compared with normal weight peers, children and adolescents with overweight/obesity had elevated concentrations of fasting glucagon, which corresponded to worsened cardiometabolic risk outcomes, except for hyperglycemia. This suggests hyperglucagonemia in youth may precede impairments in glucose regulation.

Fasting Plasma GLP-1 Is Associated With Overweight/Obesity and Cardiometabolic Risk Factors in Children and Adolescents.

CONTEXT: The importance of fasting glucagon-like peptide-1 (GLP-1) in altered metabolic outcomes has been questioned. OBJECTIVE: This work aimed to assess whether fasting GLP-1 differs in children and adolescents with overweight/obesity compared to a population-based reference, and whether concentrations predict cardiometabolic risk (CMR) factors. METHODS: Analyses were based on The Danish Childhood Obesity Data- and Biobank, a cross-sectional study including children and adolescents, aged 6 to 19 years, from an obesity clinic group (n = 1978) and from a population-based group (n = 2334). Fasting concentrations of plasma total GLP-1 and quantitative CMR factors were assessed. The effects of GLP-1 as a predictor of CMR risk outcomes were examined by multiple linear and logistic regression modeling. RESULTS: The obesity clinic group had higher fasting GLP-1 concentrations (median 3.3 pmol/L; interquartile range, 2.3-4.3 pmol/L) than the population-based group (2.8 pmol/L; interquartile range, 2.1-3.8 pmol/L; P < 2.2E-16). Body mass index SD score (SDS), waist circumference, and total body fat percentage were significant predictors of fasting GLP-1 concentrations in boys and girls. Fasting GLP-1 concentrations were positively associated with homeostasis model assessment of insulin resistance, fasting values of insulin, high-sensitivity C-reactive protein, C-peptide, triglycerides, alanine transaminase (ALT), glycated hemoglobin A1c, and SDS of diastolic and systolic blood pressure. A 1-SD increase in fasting GLP-1 was associated with an increased risk of insulin resistance (odds ratio [OR] 1.59), dyslipidemia (OR 1.16), increased ALT (OR 1.14), hyperglycemia (OR 1.12) and hypertension (OR 1.12). CONCLUSION: Overweight/obesity in children and adolescents is associated with increased fasting plasma total GLP-1 concentrations, which was predictive of higher CMR factors.

No detectable effect of a type 2 diabetes-associated TCF7L2 genotype on the incretin effect.

The T allele of TCF7L2 rs7903146 is a common genetic variant associated with type 2 diabetes (T2D), possibly by modulation of incretin action. In this study, we evaluated the effect of the TCF7L2 rs7903146 T allele on the incretin effect and other glucometabolic parameters in normal glucose tolerant individuals (NGT) and participants with T2D. The rs7903146 variant was genotyped in cohorts of 61 NGT individuals (23 were heterozygous (CT) or homozygous (TT) T allele carriers) and 43 participants with T2D (20 with CT/TT). Participants were previously examined by an oral glucose tolerance test (OGTT) and a subsequent isoglycemic intravenous glucose infusion (IIGI). The incretin effect was assessed by quantification of the difference in integrated beta cell secretory responses during the OGTT and IIGI. Glucose and hormonal levels were measured during experimental days, and from these, indices of beta cell function and insulin sensitivity were calculated. No genotype-specific differences in the incretin effect were observed in the NGT group (P = 0.70) or the T2D group (P = 0.68). NGT T allele carriers displayed diminished glucose-dependent insulinotropic polypeptide response during OGTT (P = 0.01) while T allele carriers with T2D were characterized by lower C-peptide AUC after OGTT (P = 0.04) and elevated glucose AUC after OGTT (P = 0.04). In conclusion, our findings do not exclude that this specific TCF7L2 variant increases the risk of developing T2D via diminished incretin effect, but genotype-related defects were not detectable in these cohorts.

Greater glucagon-like peptide-1 responses to oral glucose are associated with lower central and peripheral blood pressures.

BACKGROUND AND AIM: Cardiovascular diseases (CVDs) are globally the leading cause of death and hypertension is a significant risk factor. Treatment with glucagon-like peptide-1 (GLP-1) receptor agonists has been associated with decreases in blood pressure and CVD risk. Our aim was to investigate the association between endogenous GLP-1 responses to oral glucose and peripheral and central haemodynamic measures in a population at risk of diabetes and CVD. METHODS: This cross-sectional study included 837 Danish individuals from the ADDITION-PRO cohort (52% men, median (interquartile range) age 65.5 (59.8 to 70.7) years, BMI 26.1 (23.4 to 28.5) kg/m2, without antihypertensive treatment and known diabetes). All participants received an oral glucose tolerance test with measurements of GLP-1 at 0, 30 and 120 min. Aortic stiffness was assessed by pulse wave velocity (PWV). The associations between GLP-1 response and central and brachial blood pressure (BP) and PWV were assessed in linear regression models adjusting for age and sex. RESULTS: A greater GLP-1 response was associated with lower central systolic and diastolic BP of - 1.17 mmHg (95% confidence interval (CI) - 2.07 to - 0.27 mmHg, P = 0.011) and - 0.74 mmHg (95% CI - 1.29 to - 0.18 mmHg, P = 0.009), respectively, as well as lower brachial systolic and diastolic BP of - 1.27 mmHg (95% CI - 2.20 to - 0.33 mmHg, P = 0.008) and - 1.00 (95% CI - 1.56 to - 0.44 mmHg, P = 0.001), respectively. PWV was not associated with GLP-1 release (P = 0.3). Individuals with the greatest quartile of GLP-1 response had clinically relevant lower BP measures compared to individuals with the lowest quartile of GLP-1 response (central systolic BP: - 4.94 (95% CI - 8.56 to - 1.31) mmHg, central diastolic BP: - 3.05 (95% CI - 5.29 to - 0.80) mmHg, brachial systolic BP: - 5.18 (95% CI - 8.94 to - 1.42) mmHg, and brachial diastolic BP: - 2.96 (95% CI - 5.26 to - 0.67) mmHg). CONCLUSION: Greater glucose-stimulated GLP-1 responses were associated with clinically relevant lower central and peripheral blood pressures, consistent with beneficial effects on the cardiovascular system and reduced risk of CVD and mortality. Trial registration ClinicalTrials.gov Identifier: NCT00237549. Retrospectively registered 10 October 2005.

Evidence of a liver-alpha cell axis in humans: hepatic insulin resistance attenuates relationship between fasting plasma glucagon and glucagonotropic amino acids.

AIMS/HYPOTHESIS: The secretion of glucagon is controlled by blood glucose and inappropriate secretion of glucagon contributes to hyperglycaemia in diabetes. Besides its role in glucose regulation, glucagon regulates amino acid metabolism in hepatocytes by increasing ureagenesis. Disruption of this mechanism causes hyperaminoacidaemia, which in turn increases glucagon secretion. We hypothesised that hepatic insulin resistance (secondary to hepatic steatosis) via defective glucagon signalling/glucagon resistance would lead to impaired ureagenesis and, hence, increased plasma concentrations of glucagonotropic amino acids and, subsequently, glucagon. METHODS: To examine the association between glucagon and amino acids, and to explore whether this relationship was modified by hepatic insulin resistance, we studied a well-characterised cohort of 1408 individuals with normal and impaired glucose regulation. In this cohort, we have previously reported insulin resistance to be accompanied by increased plasma concentrations of glucagon. We now measure plasma levels of amino acids in the same cohort. HOMA-IR was calculated as a marker of hepatic insulin resistance. RESULTS: Fasting levels of glucagonotropic amino acids and glucagon were significantly and inversely associated in linear regression models (persisting after adjustment for age, sex and BMI). Increasing levels of hepatic, but not peripheral insulin resistance (p > 0.166) attenuated the association between glucagon and circulating levels of alanine, glutamine and tyrosine, and was significantly associated with hyperaminoacidaemia and hyperglucagonaemia. A doubling of the calculated glucagon-alanine index was significantly associated with a 30% increase in hepatic insulin resistance, a 7% increase in plasma alanine aminotransferase levels, and a 14% increase in plasma γ-glutamyltransferase levels. CONCLUSIONS/INTERPRETATION: This cross-sectional study supports the existence of a liver-alpha cell axis in humans: glucagon regulates plasma levels of amino acids, which in turn feedback to regulate the secretion of glucagon. With hepatic insulin resistance, reflecting hepatic steatosis, the feedback cycle is disrupted, leading to hyperaminoacidaemia and hyperglucagonaemia. The glucagon-alanine index is suggested as a relevant marker for hepatic glucagon signalling.