Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 -LRB- TCF7L2 -RRB- gene polymorphisms Aims/hypothesis Polymorphisms in the transcription factor 7-like 2 -LRB- TCF7L2 -RRB- gene are associated with type 2 diabetes and reduced insulin secretion . The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 -LRB- GLP-1 -RRB- secretion from intestinal L cells . We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms . Introduction Genome-wide scans for diabetes susceptibility genes have been performed in various populations . In early 2006 , it was reported that variants in the transcription factor 7-like 2 -LRB- TCF7L2 -RRB- gene were strongly associated with an increased risk of type 2 diabetes in an Icelandic , a Danish and a US population -LSB- 1 -RSB- . The estimated population attributable risk of type 2 diabetes of this variation lies between 10 and 25 % , which is in the range of the peroxisome proliferator-activated receptor γ gene -LRB- PPARγ -RRB- Pro12Ala -LRB- 25 % -LSB- 2 -RSB- -RRB- and the potassium inwardly-rectifying channel , subfamily J , member 11 gene -LRB- KCNJ11 -RRB- Glu23Lys -LRB- 15 % -LSB- 3 -RSB- -RRB- polymorphisms . In the initial report of Grant et al. -LSB- 1 -RSB- , five single nucleotide polymorphisms -LRB- SNPs -RRB- within introns 3 and 4 of the TCF7L2 gene -LRB- rs12255372 , rs7903146 , rs7901695 , rs11196205 , rs7895340 -RRB- were identified to associate with an increased risk of type 2 diabetes . This finding initiated a series of reports in various populations confirming the effect of these polymorphisms within the TCF7L2 gene on the type 2 diabetes risk -LSB- 4 -- 12 -RSB- . Recently , the T allele of rs7903146 was identified as the variant that most strongly determines the risk of type 2 diabetes -LSB- 13 -RSB- . In carriers of the risk alleles for SNPs within TCF7L2 several studies additionally showed an impaired insulin secretion estimated from an OGTT or IVGTT -LSB- 6 , 7 , 9 , 10 -RSB- . These studies indicate that progressive loss of insulin secretion might be the essential component of the phenotype which predisposes carriers of the TCF7L2 variant to develop type 2 diabetes . The pathogenic mechanism of the impaired insulin secretion due to polymorphisms in the TCF7L2 gene is not yet clear . It could be the consequence of a reduction in beta cell mass or could reflect a dysfunction of pancreatic beta cells . Furthermore , a defect in incretin-induced stimulus secretion coupling could mediate a reduction of insulin secretion in carriers of the TCF7L2 polymorphisms . The latter hypothesis is supported by the molecular mode of action of the transcription factor TCF7L2 . TCF7L2 is an essential component of the wingless-type MMTV integration site family , member 1 -LRB- WNT -RRB- signalling pathway , which is crucial for the regulation of the glucagon gene -LRB- GCG -RRB- expression and the secretion of its product GLP-1 by the intestinal endocrine L cells -LSB- 14 -RSB- . Therefore , an alteration in the WNT signalling pathway through polymorphisms in the TCF7L2 gene might result in an altered GLP-1 response , which in turn could lead to a lower postprandial insulin secretion . To test this hypothesis we analysed the associations of the above-described TCF7L2 polymorphisms with basal GLP-1 secretion and glucose-induced GLP-1 response during an OGTT in non-diabetic participants . We further studied the influence of the TCF7L2 SNPs on insulin secretion kinetics to i.v. administered glucose during an IVGTT and a hyperglycaemic clamp . In addition , we particularly investigated the influence on GLP-1-induced insulin secretion using a combined hyperglycaemic clamp with additional GLP-1 infusion and arginine bolus -LSB- 15 -RSB- . Methods Participants We studied 1,110 non-diabetic participants by an OGTT -LRB- Table 1 -RRB- . Secretion patterns of GLP-1 were obtained in a subgroup of 155 participants . The anthropometric characteristics of this subgroup are shown in Table 2 . All 1,110 participants were also genotyped for the following five SNPs in the TCF7L2 gene : rs7903146 , rs12255372 , rs7901695 , rs11196205 and rs7895340 . Table 1Anthropometric and metabolic data from all 1,110 participants who underwent an OGTT rs7903146p value -LRB- ANOVA -RRB- p value -LRB- CC vs XT -RRB- GenotypeC/CC/TT / Tn54647490NGT/IGT461/85386 / 8863/270 .0070.03 Sex -LRB- female/male -RRB- 346/200315/15950 / 400.130.64 Age -LRB- years -RRB- 39 ± 140 ± 141 ± 10.390.67 BMI -LRB- kg/m2 -RRB- 29.5 ± 0.428.9 ± 0.428.2 ± 1.00.510.24 Plasma glucose -LRB- mmol/l -RRB- Fasting 5.1 ± 0.025.1 ± 0.025.2 ± 0.070.250.39 2 h 6.1 ± 0.076.2 ± 0.076.7 ± 0.20.060.17 Plasma insulin -LRB- pmol/l -RRB- Fasting62 .0 ± 2.153.4 ± 1.749.8 ± 3.10.0040.001 2 h442 ± 19356 ± 17372 ± 340.120.04 Insulin sensitivityOGTT -LRB- arbitrary units -RRB- 16.8 ± 0.518.2 ± 0.517.8 ± 1.20.02 / 0.02 a0 .005 / 0.006 aInsulin secretionOGTT -LRB- pmol/mmol -RRB- 319 ± 5301 ± 5292 ± 100.003 / 0.04 b0 .0009 / 0.02 bInsulin/glucose ratio -LRB- pmol/mmol -RRB- 143 ± 5127 ± 5124 ± 130.003 / 0.03 b0 .001 / 0.01 bData are means ± SEMp values were obtained using χ2 test or ANOVAAnalysis for rs12255372 and rs7901695 also revealed a significant difference in insulin secretion -LRB- p = 0.003 and p = 0.05 -RRB- , whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected -LRB- p = 0.53 and 0.45 -RRB- ap values are derived from multivariate linear regression models : adjusted for age , sex and BMIbp values are derived from multivariate linear regression models : adjusted for age , sex , BMI and insulin sensitivityTable 2Anthropometric and metabolic data of the subgroup of 155 participants with additional measurements of GLP-1 rs7903146p value -LRB- ANOVA -RRB- p value -LRB- CC vs XT -RRB- GenotypeC/CC/TT / Tn736715NGT/IGT64/948 / 1910/50 .030.009 Sex -LRB- female/male -RRB- 50/2342/258 / 70.500.33 Age -LRB- years -RRB- 46 ± 147 ± 147 ± 30.990.96 BMI -LRB- kg/m2 -RRB- 29.8 ± 0.730.3 ± 0.727.1 ± 1.10.150.88 Plasma glucose -LRB- mmol/l -RRB- Fasting5 .2 ± 0.055.3 ± 0.075.6 ± 0.20.170.21 2 h6 .5 ± 0.17.0 ± 0.27.2 ± 0.50.220.10 Insulin sensitivityOGTT -LRB- arbitrary units -RRB- 12.8 ± 0.713.6 ± 1.015.7 ± 2.60.74 / 0.92 a0 .78 / 0.75 aInsulin secretionOGTT -LRB- pmol/mmol -RRB- 304 ± 12287 ± 13235 ± 190.01 / 0.02 b0 .02 / 0.01bGLP-1 -LRB- pmol/l -RRB- 0 min 16.1 ± 0.917.3 ± 1.317.3 ± 2.00.910.88 30 min34 .1 ± 2.138.8 ± 4.038.1 ± 3.50.450.36 120 min28 .9 ± 1.529.0 ± 1.728.9 ± 2.40.870.88 Fold increase 0 -- 30 min2 .5 ± 0.22.7 ± 0.32.4 ± 0.40.770.84 Data are means ± SEMp values were obtained using χ2 test or ANOVAAnalysis for rs7901695 also revealed a significant difference in insulin secretion -LRB- p = 0.02 -RRB- , whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected -LRB- p = 0.73 and 0.76 -RRB- ap values were derived from multivariate linear regression models : adjusted for age , sex and BMIbp values were derived from multivariate linear regression models : adjusted for age , sex , BMI and insulin sensitivityA subset of 210 participants were studied by an IVGTT combined with a euglycaemic -- hyperinsulinaemic clamp to determine insulin secretion capacity and insulin sensitivity in one test -LSB- 16 -RSB- -LRB- Table 3 -RRB- . Table 3Anthropometric and metabolic data of 210 participants who underwent a combined IVGTT and hyperinsulinaemic -- euglycaemic clamp rs7903146p value -LRB- CC vs XT -RRB- GenotypeC/CX/Tn97113NGT / IGT80/1781/320 .06 Sex -LRB- female/male -RRB- 59/3864/490 .58 Age -LRB- years -RRB- 44 ± 145 ± 10.29 BMI -LRB- kg/m2 -RRB- 29.0 ± 0.528.8 ± 0.50.82 Insulin sensitivityclamp -LRB- μmol kg − 1 min − 1 -LSB- pmol/l -RSB- − 1 -RRB- 0.07 ± 0.010.07 ± 0.010.77 Insulin secretion indexa IVGTT C-peptide secretion -LRB- pmol/l -RRB- 7,904 ± 3067,679 ± 3230.40 IVGTT insulin secretion -LRB- pmol/l -RRB- 2,059 ± 1311,924 ± 1290.25 OGTT insulin secretion -LRB- pmol/mmol -RRB- 314 ± 8292 ± 80.04 Data are means ± SEMp values were obtained using χ2 test or unpaired t test or multivariate linear regression modelsNone of the four other SNPs in TCF7L2 -LRB- rs12255372 , rs7901695 , rs11196205 and rs7895340 -RRB- affected significantly insulin secretion in the IVGTT -LRB- all p > 0.2 -RRB- aAdjusted for age , sex and BMI , and insulin sensitivityAdditionally , 233 participants were studied by a hyperglycaemic clamp -LSB- 17 -RSB- . The participants were selected from three independent studies in the Netherlands and Germany . The German and the Dutch cohort from Utrecht consisted of NGT and IGT participants , the Dutch cohort from Hoorn consisted of IGT participants only -LRB- Table 4 -RRB- . All participants in our studies underwent a standard OGTT to define their glucose tolerance status . Details of the study groups have been described previously -LSB- 17 -- 19 -RSB- . In the German subgroup -LRB- n = 73 -RRB- , the hyperglycaemic clamp was continued with an additional GLP-1 and arginine administration -LSB- 15 , 20 -- 22 -RSB- . Table 4Anthropometric and metabolic data of 233 participants who underwent a hyperglycaemic clamp and of 73 participants who underwent a modified hyperglycaemic clamp with additional GLP-1 and arginine administration GermanyThe Netherlands -LRB- Utrecht -RRB- The Netherlands -LRB- Hoorn -RRB- rs7903146p valuers7903146p valuers7903146p valueGenotypeC/CX/TC / CX/TC/CX / TSex -LRB- female/male -RRB- 15/2024/140 .085 / 3122/11 < 0.0117 / 1130/330 .27 NGT/IGT30/527 / 110.1331 / 526/70 .530 / 280/66 -- Age -LRB- years -RRB- 38 ± 240 ± 20.6647 ± 147 ± 10.9456 ± 158 ± 10.43 BMI -LRB- kg/m2 -RRB- 26.1 ± 0.925.1 ± 0.60.4125.6 ± 0.726.2 ± 0.70.4929.2 ± 0.827.9 ± 0.40.13 Plasma glucose -LRB- mmol/l -RRB- Fasting5 .1 ± 0.15.0 ± 0.10.724.6 ± 0.14.8 ± 0.10.186.6 ± 0.16.6 ± 0.10.75 2 h glucose5 .9 ± 0.36.5 ± 0.30.215.7 ± 0.35.6 ± 0.30.749.5 ± 0.19.5 ± 0.10.84 Insulin sensitivity -LRB- μmol kg − 1 min − 1 -LSB- pmol/l -RSB- − 1 -RRB- 0.15 ± 0.020.15 ± 0.010.900.17 ± 0.020.18 ± 0.020.600.11 ± 0.010.14 ± 0.010.21 Insulin secretion index -LRB- pmol/l -RRB- a First phase -LRB- sum 0 -- 10 min -RRB- 1,067 ± 158815 ± 1050.18963 ± 75891 ± 960.76699 ± 70679 ± 720.68 Second phase308 ± 47291 ± 410.80389 ± 42376 ± 470.71304 ± 45303 ± 380.18 First phase GLP-12,049 ± 2581,466 ± 1750.03 n.a.n.a.n.a.n.a. Second phase GLP-14,567 ± 5682,881 ± 3410.006 n.a.n.a.n.a.n.a. Arginine2 ,680 ± 2262,252 ± 1810.40 n.a.n.a.n.a.n.a.Data are means ± SEp values were obtained using χ2 test , unpaired t test or multivariate linear regression modelsCarriers of the risk allele for SNP rs12255372 showed reduced first phase -LRB- p = 0.02 -RRB- and second phase -LRB- p = 0.005 -RRB- GLP-1-induced insulin secretionCarriers of the risk allele for SNP rs7901695 showed reduced second phase -LRB- p = 0.02 -RRB- GLP-1-induced insulin secretionn.a . Not availableaAdjusted for age , sex and BMI , and insulin sensitivityThe participants were not related to each other . Informed written consent for all studies was obtained from all participants , and the local ethics committee approved the protocols . Genotyping Detection of the TCF7L2 polymorphisms described by Grant et al. -LSB- 1 -RSB- was done using the TaqMan assay -LRB- Applied Biosystems , Forster City , CA , USA -RRB- in the German population . The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 7000 , and fluorescence was detected on an ABI PRISM 7000 sequence detector -LRB- Applied Biosystems -RRB- . As a quality standard , we randomly included six positive -LRB- two homozygous wild-type allele carriers , two heterozygous and two homozygous risk allele carriers -RRB- and two negative -LRB- all components excluding DNA -RRB- sequenced controls in each TaqMan reader plate . Because all controls were correctly identified , we assumed that the genotyping error rate of this method did not exceed 0.3 % -LSB- 23 -RSB- . In the Dutch population , only rs7903146 was determined using an ABI PRISM 7900HT sequence detector -LRB- Applied Biosystems -RRB- . OGTT At 08:00 hours , participants ingested a solution containing 75 g glucose . Venous blood samples were obtained at 0 , 30 , 60 , 90 and 120 min for determination of plasma glucose , insulin and C-peptide concentrations and 0 , 30 and 120 min for determination of GLP-1 concentrations . The participants did not take any medication known to affect glucose tolerance or insulin sensitivity . Tests were performed after an overnight fast of 12 h. Combined IVGTT and hyperinsulinaemic -- euglycaemic clamp After an overnight fast and after baseline samples had been obtained , 0.3 g/kg bodyweight of a 20 % -LRB- vol . / vol . -RRB- glucose solution was given at time 0 . Blood samples for the measurement of plasma glucose , plasma insulin and C-peptide were obtained at 2 , 4 , 6 , 8 , 10 , 20 , 30 , 40 , 50 and 60 min . After 60 min , a priming dose of insulin was given followed by an infusion -LRB- 40 mU/m2 -RRB- of short-acting human insulin for 120 min . A variable infusion of 20 % glucose was started to maintain the plasma glucose concentration at 5.5 mmol/l . Blood samples for the measurement of plasma glucose were obtained at 5 min intervals throughout the clamp . Hyperglycaemic clamp Hyperglycaemic clamps were performed at 10 mmol/l in all participants . The Dutch NGT and IGT participants underwent a 3 h clamp . In the German NGT participants , the clamp lasted for 2 h followed by the GLP-1 and arginine stimulation -LRB- see below -RRB- . After an overnight fast , the participants received an i.v. glucose bolus to acutely raise glucose levels to 10 mmol/l . Plasma glucose levels were measured at the appropriate intervals to maintain a constant plasma glucose during the clamp . Blood samples for insulin were drawn at 2.5 min intervals during the first 10 min of the clamp and at 10 -- 20 min intervals during the remainder . Exact details of the clamping procedures in the different study groups have been described previously -LSB- 15 , 18 , 19 -RSB- . Combined hyperglycaemic clamp This hyperglycaemic clamp combined with GLP-1 and arginine administration was performed as previously described -LSB- 15 , 20 -- 22 -RSB- . After 120 min of hyperglycaemic clamp at 10 mmol/l , a bolus of GLP-1 -LRB- 0.6 pmol/kg -RRB- was given -LRB- human GLP-1 -LRB- 7-36 -RRB- amide ; Poly Peptide , Wolfenbüttel , Germany -RRB- followed by a continuous GLP-1 infusion -LRB- 1.5 pmol kg − 1 min − 1 -RRB- during the next 80 min . At 180 min , a bolus of 5 g arginine hydrochloride -LRB- Pharmacia & Upjohn , Erlangen , Germany -RRB- was injected over 45 s while the GLP-1 infusion was continued . Blood for the measurement of glucose , insulin , proinsulin and C-peptide was obtained during the time-points shown in Fig. 1 . This clamp allows measurement of different aspects of stimulus secretion coupling : first and second phases of glucose-induced insulin secretion , GLP-1-induced insulin secretion , and the response to additional arginine administration . Fig. 1Associations between the genotypes of rs7903146 polymorphism in the TCF7L2 gene with insulin secretion during a hyperglycaemic clamp in 73 German participants . Open circles CC ; closed circles CT and TT . AIR Acute insulin response . Arrow Administration of 5 g arginine . The p values show the differences for first and second phases of glucose-induced insulin secretion , first and second phases of GLP-1-induced insulin secretion and acute insulin secretory response to arginine -LRB- AIR -RRB- -LRB- for calculation see ` Methods ' ; insulin secretion is adjusted for insulin sensitivity -RRB- Analytical procedures Plasma glucose was determined as previously described -LSB- 14 , 17 , 20 , 21 -RSB- . GLP-1 was determined during the OGTT at baseline , 30 and 120 min . GLP-1 immunoreactivity was determined using an RIA specific for the C-terminus of the peptide -LSB- 24 -RSB- . Samples were stored at − 80 °C . The assay measures the sum of the intact peptide plus the primary metabolite , GLP-1 -LRB- 9 -- 36 -RRB- amide using the polyclonal anti-GLP-1 antibody 89390 and synthetic GLP-1 -LRB- 7 -- 36 -RRB- amide as standard.Plasma insulin and C-peptide concentrations in the German cohort were measured by a microparticle enzyme immunoassay -LRB- Abbott , Wiesbaden , Germany -RRB- and an RIA -LRB- Byk-Santec , Dietzenbach , Germany -RRB- . In the participants from the Netherlands , insulin was measured using an in-house competitive RIA employing a polyclonal anti-insulin antibody -LRB- Caris 46 -RRB- , 125I-labelled insulin -LRB- IM 166 ; Amersham , Roosdaal , the Netherlands -RRB- as a tracer and regular insulin -LRB- Humulin ; Eli Lilly , Indianapolis , IN , USA -RRB- as a standard . Calculations Insulin secretion in the OGTT was assessed by calculating the AUC for C-peptide divided by the AUC for glucose -LRB- AUCCP/AUCGlc -RRB- . AUCs were determined by the trapezoidal method . Furthermore , insulin secretion was calculated as insulin : glucose ratio by dividing -LRB- insulin at 30 min -- insulin at 0 min -RRB- by -LRB- glucose at 30 min -- glucose at 0 min -RRB- . Insulin sensitivity during the OGTT was estimated from glucose and insulin values as proposed by Matsuda and DeFronzo -LSB- 25 -RSB- . Insulin secretion during the IVGTT was calculated as the sum of C-peptide levels during the first 10 min after glucose administration . Insulin sensitivity during the hyperinsulinaemic -- euglycaemic clamp was calculated by dividing the average glucose infusion rate during the last 40 min of the clamp by the average plasma insulin concentration during the same time interval.Insulin secretion during the hyperglycaemic clamp was calculated using insulin levels determined during the clamp . The first phase of insulin secretion was defined as the sum of the insulin levels during the first 10 min of the clamp . The second phase of insulin secretion was defined as the mean of the insulin values during the last 40 min -LRB- 80 -- 120 min , NGT group , Germany -RRB- or the last 30 min -LRB- 150 -- 180 min , NGT and IGT groups , the Netherlands -RRB- of the clamp.In the combined hyperglycaemic clamp with GLP-1 and arginine administration , first-phase GLP-1-induced insulin secretion was defined as the mean of the 125 and 130 min insulin levels , second-phase GLP-induced insulin secretion -LRB- plateau -RRB- was defined as the mean of the 160 -- 180 min insulin levels . The acute insulin response to arginine was calculated as the mean of 182.5 and 185 min minus 180 min insulin levels -LSB- 21 -RSB- . The insulin sensitivity index was determined by relating the glucose infusion rate to the plasma insulin concentration during the last 40 min -LRB- NGT , Germany -RRB- or 30 min of the clamp . Statistical analysis Data are given as means ± SEM . Non-normally distributed parameters were logarithmically transformed to approximate linearity for statistical comparison . Distribution was tested for normality using the Shapiro -- Wilk W test . Differences in anthropometrics and metabolic characteristics between genotypes were tested using ANOVA for the three genotype groups . The secretion indices were compared using multivariate regression models . In these models the trait was the dependent variable whereas age , sex , insulin sensitivity and genotype were the independent variables . Due to the relatively low number of participants who were homozygous for the rare alleles in the hyperglycaemic clamp group , a dominant model was used . A p value of < 0.05 was considered to be statistically significant . The statistical software package JMP -LRB- SAS Institute , Cary , NC , USA -RRB- was used . Results Genetic variants in the TCF7L2 gene The SNPs described in the paper of Grant et al. -LSB- 1 -RSB- had an allelic frequency of the minor allele of 30 % -LRB- rs7903146 -RRB- , 28 % -LRB- rs12255372 -RRB- , 31 % -LRB- rs7901695 -RRB- , 46 % -LRB- rs11196205 -RRB- and 45 % -LRB- rs7895340 -RRB- in our population . These polymorphisms were all in Hardy -- Weinberg equilibrium and in linkage disequilibrium -LRB- Electronic supplementary material Table 1 -RRB- . OGTT : glucose tolerance , insulin secretion , insulin sensitivity and GLP-1 levels The percentage of participants with IGT was significantly higher in carriers of the risk allele for rs7903146 -LRB- p = 0.007 -RRB- , rs12255372 -LRB- p = 0.02 -RRB- and rs7901695 -LRB- p = 0.02 -RRB- . Insulin secretion assessed as AUCCP/AUCGlc during the OGTT was significantly reduced in participants with the risk alleles for rs7903146 and rs12255372 in the additive and dominant model both unadjusted and adjusted for relevant covariates . Participants with the risk allele for rs7901695 had a significantly decreased insulin secretion in the additive model -LRB- p = 0.02 -RRB- -LRB- Table 1 -RRB- . Insulin sensitivity , estimated by the index of Matsuda and DeFronzo -LSB- 25 -RSB- , was significantly higher in participants carrying the risk alleles for rs7903146 and rs1255372 in TCF7L2 -LRB- Table 1 -RRB- . In a subgroup of 155 participants we measured GLP-1 concentrations at baseline , at 30 min and 120 min after oral ingestion of 75 g glucose . The anthropometric characteristics are shown in -LRB- Table 1 -RRB- . Neither basal GLP-1 plasma levels nor GLP-1 levels during the OGTT significantly differed between the genotypes in any of the above-described SNPs in the TCF7L2 gene -LRB- Table 2 -RRB- . Combined IVGTT and hyperinsulinaemic -- euglycaemic clamp : glucose-induced insulin secretion and insulin sensitivity C-peptide and insulin values during the IVGTT were not significantly different between the genotypes in any of the above-described SNPs in the TCF7L2 gene . Table 3 shows the results for rs7903146 in the additive and dominant model adjusted for relevant covariates . Including the glucose tolerance status as an additional co-variate in the model did not affect the results . Interestingly , a lower insulin secretion in this subgroup of participants could be demonstrated when insulin secretion was calculated from the OGTT as described above -LRB- Table 3 -RRB- . Insulin sensitivity measured with the clamp technique was not affected by any of the genotypes -LRB- all p > 0.4 -RRB- . Hyperglycaemic clamp : glucose - , GLP-1 - and arginine-induced insulin secretion and insulin sensitivity First - and second-phase insulin secretion during the hyperglycaemic clamp were not significantly different between carriers and non-carriers of the risk allele for rs7903146 -LRB- Table 4 -RRB- in any of the three populations from the Netherlands and from Germany . The other four SNPs in TCF7L2 which were described above were determined in the German population only and were not associated with glucose-induced insulin secretion -LRB- data not shown -RRB- during the hyperglycaemic clamp.The first phase of GLP-1-induced insulin secretion was significantly reduced in carriers of the risk alleles for rs7903146 -LRB- p = 0.03 -RRB- and rs12255372 -LRB- p = 0.02 -RRB- . In addition , we found significant differences in the second phase of GLP-1-induced insulin secretion for rs7903146 -LRB- p = 0.006 -RRB- , rs12255372 -LRB- p = 0.005 -RRB- and rs7901695 -LRB- p = 0.02 -RRB- with carriers of the risk alleles having a significantly lower second phase of GLP-1-induced insulin secretion . Table 4 and Fig. 1 show the results for SNP rs7903146.In contrast to GLP-1-induced insulin secretion , arginine-induced insulin secretion was not affected by any of the analysed SNPs in TCF7L2 -LRB- Table 4 -RRB- . Discussion The variants in TCF7L2 described by Grant et al. -LSB- 1 -RSB- that mediate an increased risk of type 2 diabetes have been found to be associated with reduced insulin secretion -LSB- 6 , 7 , 9 , 10 -RSB- . We could confirm this reduced insulin secretion in our study in a group of 1,110 German non-diabetic participants using C-peptide levels during an OGTT . Unexpectedly , the i.v. application of glucose during an IVGTT did not affect insulin secretion in carriers of the risk alleles for SNPs in TCF7L2 . The same results were obtained using i.v. glucose challenge during a hyperglycaemic clamp in subgroups of the German population and a population from the Netherlands . The observed different results between an orally and i.v. administered glucose challenge generate the hypothesis that incretin-induced insulin secretion is affected by the variants in the TCF7L2 gene . Possible mechanisms explaining these findings include impaired incretin production or incretin signalling . To address this issue we first measured basal GLP-1 concentrations and GLP-1 response during an OGTT . None of the tested variants that have been shown in the literature to exert a reproducible effect on type 2 diabetes were associated with basal GLP-1 levels or with GLP-1 response during the OGTT . Furthermore , we combined the hyperglycaemic clamp with additional GLP-1 infusion to measure GLP-1-induced insulin secretion . Both acute -LRB- so called first phase -RRB- and prolonged -LRB- so called second phase -RRB- GLP-1-induced insulin secretion during the hyperglycaemic clamp were significantly impaired in carriers of the risk alleles in TCF7L2 . These data suggest that participants carrying the risk alleles have an impaired insulin secretion due to an impairment of the GLP-1 signalling chain . The putative molecular mechanism for this finding is not clear . As the transcription factor TCF7L2 plays an essential role in the WNT signalling pathway -LSB- 14 -RSB- , which on the one hand is required for a normal development of the pancreas and the pancreatic islets -LSB- 26 -RSB- and on the other hand is crucial for the regulation of GLP-1 expression and secretion in intestinal L cells -LSB- 14 -RSB- . In theory , polymorphisms in the TCF7L2 gene might therefore through alteration of the WNT signalling pathway directly affect beta cell growth , beta cell differentiation and beta cell function . Furthermore insulin secretion might also be affected indirectly via the enteroinsular axis either through an impaired overall GLP-1 secretion or through a defective or dysfunctional GLP-1-induced insulin secretion . An impaired or dysfunctional GLP-1 effect might result first in a reduced postprandial insulin secretion and second , might influence stimulation of beta cell growth and beta cell differentiation . Our data show that basal and glucose-induced GLP-1 secretion during the OGTT were not affected by any of the analysed SNPs , whereas the first and second phases of GLP-1-induced insulin secretion were significantly reduced in carriers of the risk alleles in three of the five above-described SNPs in the TCF7L2 gene , among them rs7903146 , which is most strongly associated in the literature with type 2 diabetes -LSB- 1 , 6 , 9 , 12 , 13 -RSB- . The finding that basal and glucose-stimulated GLP-1 secretion were not influenced by the TCF7L2 polymorphisms indicates that a reduced GLP-1 secretion is not likely to explain the impaired insulin secretion in carriers of the risk genotypes , although it can not fully exclude an effect of these SNPs on GLP-1 levels . First , by measuring total GLP-1 levels , we may have missed a subtle defect in GLP-1 secretion , which may have been detected by measuring the active form of GLP-1 . Second , systemic GLP-1 levels may not adequately reflect the level of the active hormone acting in the gut wall on the autonomic nervous system -LSB- 27 -RSB- . Third , impaired TCF7L2 activity might tissue-specifically reduce the GLP-1 levels in the brain , which are believed as well to be important for insulin secretion -LSB- 28 -RSB- . While we have no evidence for reduced GLP-1 secretion we have strong evidence for an impaired GLP-1-induced insulin secretion in carriers of the above-described polymorphisms in TCF7L2 . The data show a reduced efficiency of GLP-1 to stimulate insulin secretion in pancreatic beta cells . Alterations of the GLP-1 signalling pathway in beta cells might thereby play a role . Recently the TCF7L2 gene was also identified in human pancreas -LSB- 14 -RSB- . Therefore variations in this transcription factor could specifically impair the transcription of genes involved in the incretin signalling chain , resulting in resistance of the pancreatic beta cells to GLP-1 . In contrast to the observed reduction in the first and second phases of GLP-1-induced insulin secretion , the arginine-induced insulin secretion was not significantly affected by the analysed SNPs in the TCF7L2 gene . The arginine bolus in the combined hyperglycaemic clamp produces a maximal challenge for the secretory capacity of the beta cell and can be considered as a surrogate for beta cell mass -LSB- 15 , 20 -RSB- . The SNPs do not affect this maximal insulin secretion , suggesting that the variants in TCF7L2 do not influence beta cell mass , at least in the prediabetic state . In addition , impaired beta cell function may also include the efficiency of the conversion from proinsulin to insulin -LSB- 21 -RSB- . However , there was no evidence for this abnormality related to the variants in the TCF7L2 gene during the hyperglycaemic clamp -LRB- data not shown -RRB- . We also found that insulin sensitivity calculated from the OGTT using an established index -LSB- 25 -RSB- was significantly increased in participants carrying the risk alleles for all analysed SNPs in the TCF7L2 gene . This was also described in the study of Florez et al. -LSB- 9 -RSB- . When we measured insulin sensitivity with the gold standard , the euglycaemic -- hyperinsulinaemic clamp and the hyperglycaemic -- hyperinsulinaemic clamp , we found no association of the risk alleles with insulin sensitivity . Taken together , our data suggest that the variants in the TCF7L2 gene do not have a strong effect on insulin sensitivity . In summary , our data show that variations in the TCF7L2 gene are associated with impaired GLP-1-induced insulin secretion . This might be consistent with a state of relative incretin resistance . Increasing the incretin levels by pharmacological tools may thus be a logical therapy to overcome impaired insulin secretion in carriers of the TCF7L2 variants . Electronic supplementary material Below is the link to the electronic supplementary material . ESM 1 Linkage disequilibrium statistics -LRB- D ′ , r2 -RRB- among the five SNPs reported by Grant et al. -LSB- 1 -RSB- in the German population -LRB- n = 1,110 -RRB- -LRB- PDF 17 KB -RRB- .

O O O B I O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O B O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Effect of beta-adrenergic stimulation on whole-body and abdominal subcutaneous adipose tissue lipolysis in lean and obese men Aims/hypothesis Obesity is characterised by increased triacylglycerol storage in adipose tissue . There is in vitro evidence for a blunted beta-adrenergically mediated lipolytic response in abdominal subcutaneous adipose tissue -LRB- SAT -RRB- of obese individuals and evidence for this at the whole-body level in vivo . We hypothesised that the beta-adrenergically mediated effect on lipolysis in abdominal SAT is also impaired in vivo in obese humans . Introduction Obesity is characterised by excess fat storage in adipose tissue , in the form of triacylglycerol -LRB- TAG -RRB- . A blunted fat mobilisation , due to decreased adipose tissue lipolysis , might be an important factor contributing to the development or maintenance of the expanded adipose tissue mass in obesity . Fasting lipolysis per unit of lean body mass has been reported to be increased in obesity , whereas a decrease is reported when expressed per unit of fat mass -LRB- FM -RRB- -LSB- 1 -RSB- . The hormonal regulation of lipolysis is still under debate . There is strong evidence from in vitro and in vivo studies for the existence of lipolytic resistance to catecholamines in obese individuals . Blunted whole-body catecholamine-induced lipolysis has been shown in vivo in obese persons -LSB- 2 , 3 -RSB- . This impaired lipolysis did not improve after weight reduction -LSB- 4 -RSB- . Furthermore , a decreased lipolytic response to catecholamines is a feature of childhood-onset obesity -LSB- 5 , 6 -RSB- and is also present in adipocytes from first-degree relatives of obese persons -LSB- 7 -RSB- . These observations suggest that catecholamine resistance of lipolysis may be an important early , and perhaps primary factor , in the development of obesity . In vitro catecholamine resistance of lipolysis was reported in subcutaneous adipocytes of men and women with upper-body obesity , in relation with a decreased cell surface density of β2-adrenoceptors -LSB- 8 -RSB- . We hypothesised that beta-adrenergically mediated lipolytic response at the whole-body and abdominal subcutaneous adipose tissue -LRB- SAT -RRB- level is impaired in vivo in obese persons . If so , this might contribute to the increased fat storage in this adipose tissue depot . To obtain an accurate estimation of lipolysis , state-of-the-art -LSB- 2H5 -RSB- glycerol tracer methodology in combination with the measurement of arterio -- venous concentration differences across abdominal SAT was used . Obese and lean male control participants were investigated after an overnight fast and during catecholamine stimulation using the non-selective beta-adrenergic agonist isoprenaline . The primary outcome measures of the present study were glycerol rate of appearance -LRB- Ra -RRB- and glycerol exchange across abdominal SAT . A methodological issue that arises when determining local adipose tissue glycerol release is the possibility that adipose tissue may also take up small amounts of glycerol -LSB- 9 -RSB- . A pilot study was performed to determine the time period required to obtain a steady-state in glycerol enrichment in both arterialised and venous blood draining from adipose tissue , since a lack of isotopic equilibration may explain previous discrepant findings on glycerol uptake -LSB- 10 -RSB- . Methods Participants Three lean -LRB- two women and one man ; BMI < 25 kg/m2 -RRB- participants took part in a pilot experiment , during which -LSB- 2H5 -RSB- glycerol enrichment was measured for 6 h to determine the time required for obtaining an isotopic steady-state . Thirteen lean -LRB- BMI < 25 kg/m2 -RRB- and ten obese -LRB- BMI ≥ 30 kg/m2 -RRB- non-smoking normotensive men participated in the actual SAT lipolysis study , during which -LSB- 2H5 -RSB- glycerol was infused for a 3 h period . Clinical characteristics of the participants are summarised in Table 1 . Body weight and body density were determined after an overnight fast , as previously described -LSB- 11 -RSB- . All participants were in good health as assessed by their medical history , free of any medication and spent no more than 3 h/week in organised sports activities . The Medical Ethical Committee of Maastricht University approved the study protocol and all participants gave their written informed consent before participating in the study . Table 1Clinical characteristics of the participants Lean -LRB- n = 13 -RRB- Obese -LRB- n = 10 -RRB- Age -LRB- years -RRB- 43 ± 354 ± 3BMI -LRB- kg/m2 -RRB- 23.0 ± 0.531.9 ± 0.6 aFM -LRB- kg -RRB- 15.2 ± 0.832.4 ± 1.2 aWHR0 .91 ± 0.011.01 ± 0.01 aSystolic blood pressure -LRB- mmHg -RRB- 126 ± 3137 ± 4Diastolic blood pressure -LRB- mmHg -RRB- 77 ± 285 ± 3HOMA-IR1.8 ± 0.23.4 ± 0.3 aValues are mean ± SEMap < 0.05 obese vs leanHOMA-IR , homeostatic model assessment of insulin resistance Experimental protocol Pilot study The time course in -LSB- 2H5 -RSB- glycerol enrichment was determined in order to identify when steady-state levels were achieved -LRB- n = 3 -RRB- . Glycerol enrichment was measured in arterialised blood and blood draining the abdominal SAT -LRB- adipose vein -RRB- during primed -LRB- 3 μmol/kg -RRB- constant infusion for 6 h of -LSB- 2H5 -RSB- glycerol -LRB- 0.20 μmol kg − 1 min − 1 -RRB- . Blood samples were taken simultaneously from the two sites , both at baseline before the start of the tracer infusion -LRB- t0 min -RRB- , and at ten time points during glycerol infusion -LRB- t60 , t90 , t120 , t150 , t180 , t210 , t240 , t330 , t345 , t360 min -RRB- . Whole-body and SAT lipolysis study Glycerol enrichment and exchange across abdominal SAT were investigated during primed -LRB- 3 μmol/kg -RRB- constant infusion of -LSB- 2H5 -RSB- glycerol for 3 h -LRB- 0.20 μmol kg − 1 min − 1 -RRB- . Following a 120 min baseline period , isoprenaline was infused at a rate of 20 ng -LSB- kg fat free mass -LRB- FFM -RRB- -RSB- − 1 min − 1 for 60 min . During the experiment , heart rate was recorded continuously by means of a three-lead ECG . When heart rate increased by more than 40 beats per min or in the event of ECG irregularities , isoprenaline infusion was stopped -LRB- n = 2 , one lean and one obese participant -RRB- . Before the start of the tracer infusion an arterialised blood sample was taken for background enrichment . Blood samples were also taken simultaneously from the arterialised and adipose vein at three baseline time points -LRB- t90 , t105 and t120 min -RRB- and at three time points during the last 30 min of isoprenaline infusion -LRB- t150 , t165 and t180 min -RRB- . Adipose tissue blood flow -LRB- ATBF -RRB- was monitored continuously using the 133Xe washout technique -LSB- 12 -RSB- . ATBF results have been published previously in another context -LSB- 13 -RSB- . Clinical methods All participants were asked to refrain from drinking alcohol and to perform no strenuous exercise for 24 h before the study . Participants came to the laboratory by car or bus at 08:00 hours after an overnight fast . Three cannulas were inserted before the start of the experiment . Arterialised venous blood was obtained through a 20-gauge cannula inserted retrogradely into a superficial dorsal hand vein . The hand was warmed in a hotbox , which was maintained at 60 °C to achieve adequate arterialisation -LSB- 14 -RSB- . In the same arm , a second cannula was inserted in a forearm antecubital vein for the infusion of -LSB- 2H5 -RSB- glycerol tracer and the non-selective beta-adrenergic agonist isoprenaline at a rate of 20 ng -LRB- kg FFM -RRB- − 1 min − 1 . At this infusion rate , plasma isoprenaline concentrations are comparable in lean and obese participants -LSB- 3 -RSB- . Finally , the veins on the anterior abdominal wall were identified by means of a fibre-optic light source . In order to obtain adipose tissue venous blood , a 10 cm 22-gauge catheter -LRB- Central venous catheter kit Seldinger technique ; Becton Dickinson , Alphen aan den Rijn , the Netherlands -RRB- was introduced anterogradely over a guide wire into one of the superficial veins and threaded towards the groin , so that its tip lay just superior to the inguinal ligament -LSB- 15 -RSB- . This method provides the drainage from the adipose tissue of the abdomen , uncontaminated by muscle drainage and with only a minor contribution from skin -LSB- 15 -RSB- . The adipose vein was kept patent by continuous saline -LRB- 9 g/l NaCl -RRB- infusion at a rate of 80 ml/h . The participants rested in a supine position for the duration of the study . Analytical methods A small portion of blood was used to measure oxygen saturation -LRB- % HbO2 -RRB- and ensure adequate arterialisation -LRB- ABL510 ; Radiometer , Copenhagen , Denmark -RRB- . Blood was collected in tubes containing EDTA and immediately centrifuged for 10 min at 1,000 g , 4 °C . The plasma was removed for enzymatic colorimetric quantification of NEFA -LRB- NEFA C kit ; Wako , Neuss , Germany -RRB- , glycerol -LRB- Boehringer , Mannheim , Germany -RRB- and TAG -LRB- Sigma , St Louis , MO , USA -RRB- on a centrifugal spectrophotometer -LRB- Cobas Fara ; Roche Diagnostica , Basel , Switzerland -RRB- . Plasma glucose concentration -LRB- ABX Diagnostics , Montpellier , France -RRB- was measured on an automated spectrophotometer -LRB- Cobas Mira ; Roche Diagnostica -RRB- . Plasma insulin was measured with a double antibody radioimmunoassay -LRB- Linco Research , St Charles , MO , USA -RRB- . Insulin sensitivity was assessed by the homeostasis model assessment index for insulin resistance , calculated from fasting glucose and insulin -LSB- 16 -RSB- . Packed cell volume was measured using a microcapillary system -LRB- Hirschmann Laborgeräte , Eberstadt , Germany -RRB- . Isotope enrichment To determine isotopic enrichment of glycerol , samples first were derivatised . Acetone -LRB- 1 ml -RRB- was added to 150 μl plasma and each tube was vortexed for 2 min and centrifuged for 20 min at 17,500 g , 4 °C . The supernatant fraction was transferred to a clean tube and dried under nitrogen at 37 °C and derivatised by adding 80 μl ethyl acetate -LRB- cat . no. 45765 ; Sigma-Aldrich , Seelze , Germany -RRB- and 80 μl heptafluorobutyric acid anhydride -LRB- cat . no. 63164 ; Pierce Biotechnology , Rockford , IL , USA -RRB- . The tubes were vortexed for 2 min and incubated for 1 h at 70 °C . Samples were than rotated end over end for 5 min at 25 °C and evaporated under nitrogen at room temperature . Ethyl acetate -LRB- 70 μl -RRB- was added before injection into the GC-MS -LRB- Finnigan MAT 252 , Bremen , Germany -RRB- for measurement of glycerol enrichment . Stable isotope enrichment was analysed by selectively monitoring the mass to charge ratio -LRB- m/z -RRB- of molecular ions 253 and 257 for glycerol -LSB- 17 -RSB- . Calculations The net exchange -LRB- flux -RRB- of metabolites across abdominal SAT was calculated by multiplying the arterio -- venous concentration difference of metabolites by adipose tissue plasma flow . Plasma flow was calculated as ATBF × -LRB- 1 -- packed cell volume -RRB- , with packed cell volume expressed as a fraction . A positive net flux indicates net uptake from plasma , whereas a negative net flux indicates net tissue release . The expected adipose vein enrichment , in case of no glycerol uptake , was calculated as arterialised enrichment multiplied by arterialised glycerol concentration divided by the measured adipose vein enrichment . The Ra of glycerol was calculated according to the following steady-state equation : where TTR is tracer/tracee ratio and F is the isotope infusion rate -LRB- μmol kg − 1 min − 1 -RRB- . The fractional extraction -LRB- fract -RRB- of glycerol across abdominal SAT was calculated by dividing the arterio -- venous concentration difference of -LSB- 2H5 -RSB- glycerol by the arterialised -LSB- 2H5 -RSB- glycerol concentration . Abdominal SAT total glycerol uptake was calculated as follows : where the units are nmol -LRB- 100 g tissue -RRB- − 1 min − 1 ; -LRB- glycerolart -RRB- is arterialised glycerol concentration -LRB- μmol/l -RRB- ; and ATBF is in ml -LRB- 100 g tissue -RRB- − 1 min − 1 . Abdominal SAT total glycerol release was calculated from the formula : Statistical analysis Baseline fasting values and changes -LRB- Δbeta-adrenergic stimulation to baseline -RRB- were compared between groups -LRB- obese vs lean -RRB- using Student 's unpaired t test . Statistical calculations were performed with SPSS for Macintosh -LRB- version 11.0 ; SPSS , Chicago , IL , USA -RRB- . Data are presented as mean ± SEM . A value of p < 0.05 was considered statistically significant . Results Characteristics Obese participants had significantly higher BMI , FM , waist to hip ratio , fasting circulating TAG and insulin concentrations than their lean counterparts -LRB- Tables 1 and 2 -RRB- . Table 2Circulating -LRB- arterialised -RRB- metabolite levels during baseline -LRB- fasting -RRB- and isoprenaline infusion in lean and obese participants LeanObeseBaseline -LRB- n = 13 -RRB- Isoprenaline -LRB- n = 10 -RRB- Baseline -LRB- n = 10 -RRB- Isoprenaline -LRB- n = 7 -RRB- TAG -LRB- μmol/l -RRB- 701 ± 66648 ± 641,464 ± 190b1 ,667 ± 217aNEFA -LRB- μmol/l -RRB- 661 ± 41942 ± 53638 ± 421,124 ± 82aGlycerol -LRB- μmol/l -RRB- 102 ± 5118 ± 7106 ± 4147 ± 10aGlycerol Ra -LRB- μmol/min -RRB- 199 ± 12311 ± 28220 ± 15391 ± 30Glycerol Ra/FM -LSB- μmol -LRB- kg FM -RRB- − 1 min − 1 -RSB- 13.1 ± 0.920.9 ± 1.67.3 ± 0.6 b12 .9 ± 1.1 aGlucose -LRB- mmol/l -RRB- 5.3 ± 0.15.4 ± 0.15.5 ± 0.25.4 ± 0.1 Insulin -LRB- pmol/l -RRB- 50 ± 474 ± 694 ± 7b167 ± 16aValues are mean ± SEMbp < 0.05 baseline obese vs lean ; ap < 0.05 change -LRB- Δ -RRB- from baseline obese vs lean using unpaired Student 's t test Tracer/tracee ratio In the pilot experiment , the tracer/tracee ratios -LRB- TTR -RRB- obtained during a 6 h -LSB- 2H5 -RSB- glycerol infusion after an overnight fast were examined -LRB- n = 3 -RRB- . Arterialised and adipose vein TTR reached a steady-state after 1 h of infusion -LRB- Fig. 1 -RRB- . Mean values are presented , as all participants -LRB- n = 3 -RRB- showed the same pattern . The measured adipose vein enrichment was consistently lower than the expected enrichment , implying uptake of glycerol by adipose tissue . In the actual SAT lipolysis experiment -LRB- 3 h -LSB- 2H5 -RSB- glycerol infusion -RRB- , TTR also reached a steady-state after 1 h and remained stable during isoprenaline infusion . Data in lean and obese men were comparable -LRB- data not shown -RRB- . Fig. 1Plasma glycerol TTR during 6 h primed constant infusion of -LSB- 2H5 -RSB- glycerol -LRB- n = 3 -RRB- in arterialised blood -LRB- squares -RRB- and blood draining from abdominal SAT -LRB- adipose vein ; black circles -RRB- . White circles , expected adipose vein enrichment . The measured adipose vein enrichment was consistently lower than the expected enrichment Circulating metabolites Beta-adrenergic stimulation with isoprenaline increased arterialised TAG concentrations in obese participants , while in lean participants TAG concentrations decreased during isoprenaline infusion -LRB- Table 2 -RRB- . Thus , the change in arterialised TAG concentrations from baseline to isoprenaline was different between obese and lean participants -LRB- ΔTAG obese vs lean , 100 ± 37 vs − 45 ± 22 μmol/l , p < 0.05 ; Table 2 -RRB- . Furthermore , beta-adrenergic stimulation increased arterialised NEFA and glycerol concentrations in lean and obese participants . The beta-adrenergic mediated increase in arterialised NEFA -LRB- ΔNEFA 454 ± 68 vs 271 ± 46 μmol/l , p < 0.05 -RRB- and glycerol concentration -LRB- Δglycerol 40 ± 8 vs 15 ± 5 μmol/l , p < 0.05 -RRB- was more pronounced in obese than in lean participants -LRB- Table 2 -RRB- , suggesting a higher whole-body lipolytic response in the former . Indeed , beta-adrenergic stimulation increased whole-body glycerol Ra in lean and obese participants -LRB- Table 2 -RRB- , this increase tending to be higher in the obese group -LRB- Δglycerol Ra obese vs lean : 172 ± 19 vs 109 ± 13 μmol/min , p = 0.07 ; Table 2 -RRB- . Expressed per unit of FM , fasting glycerol Ra was significantly reduced in obese compared with lean participants -LRB- p < 0.05 ; Table 2 -RRB- . Interestingly , the beta-adrenergically mediated increase in glycerol Ra per unit of FM was significantly blunted in obese participants -LSB- Δglycerol Ra per unit FM : 5.4 ± 0.9 vs 7.7 ± 1.5 μmol -LRB- kg FM -RRB- − 1 min − 1 , p < 0.05 -RSB- -LRB- Table 2 -RRB- , suggesting a blunted lipolytic response per unit of FM in obese participants . Finally , beta-adrenergic stimulation increased arterialised insulin concentrations in lean and obese participants -LRB- Table 2 -RRB- . This increase in circulating insulin levels was significantly higher in obese than in lean participants -LRB- Δinsulin 62 ± 13 vs 25 ± 4 pmol/l , p < 0.05 ; Table 2 -RRB- . Abdominal SAT lipolysis Glycerol uptake by abdominal SAT was observed in lean and obese participants after an overnight fast -LRB- Fig. 2a -RRB- . Fractional extraction of -LSB- 2H5 -RSB- glycerol from the circulation -LRB- lean vs obese , 16.6 ± 4.5 vs 13.9 ± 6.7 % -RRB- and total glycerol uptake expressed relative to total glycerol release were very small -LRB- lean vs obese , 9.7 ± 3.4 vs 9.3 ± 2.5 % of total release -RRB- with no significant difference between lean and obese participants -LRB- p = 0.74 and p = 0.92 , respectively -RRB- . Adipose tissue total glycerol uptake increased during beta-adrenergic stimulation in lean and obese participants , but this increase was not significantly different between groups -LSB- Δ total glycerol uptake obese vs lean , 4 ± 9 vs 21 ± 5 nmol -LRB- 100 g tissue -RRB- − 1 min − 1 , p = 0.15 -RSB- -LRB- Fig. 2a -RRB- . The increased total glycerol uptake during beta-adrenergic stimulation appeared to be partly explained by the increase in ATBF -LRB- r = 0.633 , p < 0.05 -RRB- . Fig. 2Total glycerol uptake -LRB- a -RRB- and release -LRB- b -RRB- across abdominal SAT after an overnight fast -LRB- black bars -RRB- and during beta-adrenergic stimulation -LRB- white bars -RRB- in obese vs lean participants during 3 h -LSB- 2H5 -RSB- glycerol infusion . Values are mean ± SEM . * p < 0.05 for change -LRB- Δ -RRB- from baseline obese vs lean Fasting net glycerol and NEFA release across abdominal SAT were comparable between lean and obese participants -LRB- Table 3 -RRB- . Beta-adrenergic stimulation increased net NEFA and glycerol release across abdominal SAT to a greater extent in lean than in obese participants , although changes were not significantly different between groups -LRB- Table 3 -RRB- . In line with these findings , the beta-adrenergically mediated increase in total glycerol release across abdominal SAT was blunted in the obese group -LSB- Δ total glycerol release obese vs lean , 140 ± 71 vs 394 ± 112 nmol -LRB- 100 g tissue -RRB- − 1 min − 1 , p < 0.05 -RSB- -LRB- Fig. 2b -RRB- , suggesting a blunted lipolytic response per unit of abdominal SAT in obese participants . Finally , obese men tended to show an increased net TAG flux across abdominal SAT during beta-adrenergic stimulation -LSB- Δ net TAG flux obese vs lean , 75 ± 32 vs 16 ± 11 nmol -LRB- 100 g tissue -RRB- − 1 min − 1 , p = 0.06 -RSB- -LRB- Table 3 -RRB- . Table 3Blood flow and net SAT fluxes during baseline -LRB- fasting -RRB- and isoprenaline infusion in lean and obese participants LeanObeseBaseline -LRB- n = 13 -RRB- Isoprenaline -LRB- n = 10 -RRB- Baseline -LRB- n = 10 -RRB- Isoprenaline -LRB- n = 7 -RRB- ATBF -LSB- ml -LRB- 100 g tissue -RRB- − 1 min − 1 -RSB- 2.2 ± 0.26.3 ± 1.21.4 ± 0.2 a3 .6 ± 0.6 Net SAT fluxes -LSB- nmol -LRB- 100 g tissue -RRB- − 1 min − 1 -RSB- TAG25 ± 843 ± 1734 ± 29113 ± 62NEFA − 780 ± 160 − 2,101 ± 371 − 486 ± 101 − 1,824 ± 667Glycerol − 229 ± 49 − 640 ± 148 − 211 ± 51 − 486 ± 128Glucose53 ± 49 − 69 ± 151 − 143 ± 96 − 677 ± 632Values are mean ± SEMA positive net flux indicates net uptake from plasma , whereas a negative net flux indicates net tissue releaseap < 0.05 baseline obese vs lean using unpaired Student 's t test Discussion The present study was designed to investigate in vivo whole-body and abdominal SAT lipolysis in obese and lean men . To our knowledge , this is the first study to show in vivo that obese participants have a blunted beta-adrenergically mediated lipolytic response per unit of adipose tissue . Methodological considerations A point of discussion with studies on glycerol uptake and release using tracer methodology is the infusion time of the labelled glycerol . In previous studies , the infusion time was relatively short -LRB- 1 -- 3 h -RRB- , raising the question of whether equilibration between labelled glycerol and the adipose tissue glycerol pool is complete or not -LSB- 9 , 10 -RSB- . We investigated glycerol enrichment during a 3 h and 6 h period of -LSB- 2H5 -RSB- glycerol infusion . Steady-state levels in labelled -LSB- 2H5 -RSB- glycerol were achieved in arterialised and adipose vein enrichment after 1 h and remained constant for the subsequent 5 h. Thus , our data support the use of a relatively short infusion time -LRB- 1 h -RRB- for study of glycerol metabolism . Glycerol uptake The present data show a slight glycerol uptake by abdominal SAT of lean and obese participants . Glycerol uptake was not significantly different between lean and obese participants . Uptake and dilution of -LSB- 2H5 -RSB- glycerol across abdominal SAT has been shown previously during 1 h -LSB- 9 -RSB- of tracer infusion . In this study , the dilution of the labelled glycerol was consistently greater than expected from the measured net release of glycerol , indicating significant uptake of glycerol by adipose tissue . In line with this study , we observed a two - to fivefold higher glycerol enrichment in arterialised than in venous blood draining adipose tissue . The observed enrichment in venous blood was universally lower than that predicted from the net addition of glycerol to venous blood . This indicates that an exchange must occur between enriched glycerol in the blood and the unenriched non-esterified glycerol pool in adipose tissue . In contrast , some studies were unable to detect significant uptake of glycerol by adipose tissue after 1 h of tracer infusion -LSB- 10 -RSB- . The reason for this apparent discrepancy remains to be elucidated . It should be mentioned that glycerol uptake is low in human adipose tissue as is the activity of the enzyme glycerol kinase -LSB- 18 -RSB- . This enzyme is responsible for the phosphorylation of glycerol into glycerol 3-phosphate , making it available for re-esterification . Abdominal SAT lipolytic response to beta-adrenergic stimulation A blunted isoprenaline-induced increase in total glycerol release per unit abdominal SAT was observed in obese men , indicating that in vivo beta-adrenergic mediated lipolytic response in abdominal SAT of obese participants is blunted . Our data are consistent with evidence of catecholamine resistance in vitro and in situ in obese individuals -LSB- 2 -- 4 -RSB- , in children with obesity -LSB- 5 , 6 -RSB- and also in relatives of obese individuals -LSB- 7 -RSB- . Defects in catecholamine signal transduction have been observed at the β2-adrenoceptor level and further downstream or directly involving hormone-sensitive lipase -LSB- 8 , 19 -- 22 -RSB- . However , from our experiments it is not possible to determine at which level the observed defect is located . Interestingly , catecholamine resistance has been observed in adipose tissue of first-degree relatives of obese participants -LSB- 7 -RSB- and persists after weight reduction -LSB- 4 -RSB- , suggesting that catecholamine resistance may be a primary defect in obesity . Furthermore , plasma insulin concentrations may play an important role in regulating lipolysis -LSB- 23 -RSB- . Therefore , we can not fully rule out the possibility that the blunted lipolytic response per unit adipose tissue mass that we observed is a secondary phenomenon , due to the higher degree of hyperinsulinaemia during beta-adrenergic stimulation in obese compared with lean participants . However , this explanation seems unlikely , since a blunted in situ lipolytic response in abdominal SAT of obese women was still observed when the confounding influence of hyperinsulinaemia had been excluded using a pancreatic hormonal clamp -LSB- 2 -RSB- . In contrast to the present study , two in situ microdialysis studies performed in men found that the increase in interstitial glycerol during isoprenaline administration did not differ between lean and obese individuals -LSB- 24 , 25 -RSB- . A possible explanation for this is that in microdialysis studies interstitial glycerol is used as a measure of lipolysis . Since glycerol is taken up by adipose tissue , interstitial glycerol concentration may not reflect the overall rate of lipolysis . Rather , it may be the net result of TAG and glycerol metabolism , thus reflecting net glycerol turnover -LSB- 26 -RSB- . Whole-body beta-adrenergically mediated lipolytic response Whole-body lipolytic response during isoprenaline infusion tended to be higher in obese participants . This was reflected by a higher increase in circulating NEFA and glycerol concentrations during beta-adrenergic stimulation in obese than in lean participants . Expressed per unit of FM , beta-adrenergically mediated lipolysis -LRB- glycerol Ra -RRB- was significantly lower in obese than in lean men . This suggests that the increased whole-body beta-adrenergically mediated lipolytic response in obese individuals is directly linked to the increased adipose tissue mass , as has been shown before in upper body obese women -LSB- 2 -RSB- . Increased release of NEFA into the circulation increases NEFA delivery to the liver , resulting in increased hepatic VLDL-TAG output and hence increased circulating TAG levels during beta-adrenergic stimulation , as was observed in our obese participants . The control of whole-body lipid metabolism is , to a large extent , dependent on the efficient regulation of lipid metabolism in adipose tissue and the liver . Moreover , hepatic VLDL-TAG is a precursor of TAG stored in adipose tissue -LSB- 27 , 28 -RSB- . Consequently , a greater VLDL-TAG delivery to adipose tissue and greater lipoprotein lipase-mediated hydrolysis might explain the tendency towards increased positive TAG flux across abdominal SAT of obese participants during beta-adrenergic stimulation . Our observation is in agreement with a study by Samra et al. -LSB- 29 -RSB- showing an increased rate of action of lipoprotein lipase during epinephrine infusion . These in vivo findings are in contrast with in vitro studies showing that lipoprotein lipase expression and activity are suppressed by epinephrine -LSB- 30 , 31 -RSB- . Future studies are needed to elucidate whether an increased TAG flux across SAT might contribute to the increased TAG storage in adipose tissue of obese participants . Conclusion The present study demonstrates in vivo that obese men have a blunted beta-adrenergically mediated lipolytic response in abdominal SAT . Therefore , a blunted lipolysis during beta-adrenergic stimulation may be an important factor in the development or maintenance of increased TAG stores and obesity .

O O O O O O O O O B I B O O O O O O B O O O O B O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O B I O O O O O B O O O O O O O B I O O O O O B O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O B I O O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O B O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O B I B O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O B O B I O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O B O O B I O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O B O O O O B I O O B O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O

adipose tissue
catecholamines
glycerol
in vivo
lipolysis
obesity
triacylglycerol

arterio–venous differences
beta-adrenergic stimulation