M. MRÁZ 1, Z. LACINOVÁ 1, P. KAVÁLKOVÁ 1, D. HALUZÍKOVÁ 1,2, P. TRACHTA 1, J. DRÁPALOVÁ 1, V. HANUŠOVÁ 1, M. HALUZÍK 1 - PDF

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Physiol. Res. 60: , 2011 Serum Concentrations of Fibroblast Growth Factor 19 in Patients With Obesity and Type 2 Diabetes Mellitus: the Influence of Acute Hyperinsulinemia, Very-Low Calorie Diet

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Physiol. Res. 60: , 2011 Serum Concentrations of Fibroblast Growth Factor 19 in Patients With Obesity and Type 2 Diabetes Mellitus: the Influence of Acute Hyperinsulinemia, Very-Low Calorie Diet and PPAR-α Agonist Treatment M. MRÁZ 1, Z. LACINOVÁ 1, P. KAVÁLKOVÁ 1, D. HALUZÍKOVÁ 1,2, P. TRACHTA 1, J. DRÁPALOVÁ 1, V. HANUŠOVÁ 1, M. HALUZÍK 1 1 Third Department of Medicine, General University Hospital and First Medical Faculty, Charles University, Prague, Czech Republic, 2 Department of Sports Medicine, General University Hospital and First Medical Faculty, Charles University, Prague, Czech Republic Received October 12, 2010 Accepted April 7, 2011 On-line May 16, 2011 Summary The aim of our study was to measure serum concentrations of fibroblast growth factor 19 (FGF-19) in patients with obesity (OB), obesity and type 2 diabetes mellitus (T2DM) and healthy subjects (C) at baseline and after selected interventions. We measured serum FGF-19 levels and other biochemical and hormonal parameters in 29 OB and 19 T2DM females and 30 sexand age-matched control subjects. The interventions were acute hyperinsulinemia during isoglycemic-hyperinsulinemic clamp (n=11 for T2DM and 10 for C), very-low calorie diet (VLCD, n=12 for OB) and 3 months treatment with PPAR-α agonist fenofibrate (n=11 for T2DM). Baseline serum FGF-19 levels were significantly lower in OB relative to C group (132.1±12.7 vs ±16.7 pg/ml, p 0.05), while no significant difference was observed between T2DM and OB or control group. Acute hyperinsulinemia tended to decrease FGF-19 levels in both healthy and T2DM subjects. Three weeks of VLCD in OB group had no significant effect on FGF-19, whereas three months of fenofibrate treatment markedly reduced FGF-19 levels in T2DM patients (194.58±26.2 vs ±25.0 pg/ml, p 0.05). We conclude that FGF-19 levels in our study were at least partially dependent upon nutritional status, but were not related to parameters of glucose metabolism or insulin sensitivity. Key words FGF-19 Obesity Type 2 diabetes mellitus Very low calorie diet Fenofibrate Corresponding author M. Haluzík, U nemocnice 1, Prague 2, Czech Republic. Introduction Fibroblast growth factor 19 (FGF-19) is a recently identified endocrine regulator with multiple effects on metabolic processes and energy homeostasis (Nishimura et al. 1999, Xie et al. 1999, Itoh and Ornitz 2004). Produced predominantly in enterocytes of terminal ileum under the regulation of Farnesoid X receptor (FXR), a bile acid nuclear receptor, and acting mainly in liver through fibroblast growth factor receptor 4 (FGFR4), FGF-19 has been demonstrated to play a central role in the negative feedback loop of bile acid synthesis and gallbladder refilling (Yu et al. 2000, Holt et al. 2003, Inagaki et al. 2005, Yu et al. 2005, Choi et al. 2006). Animal studies have also shown antiadipogenic, anti-diabetic and hypolipidemic effects of either exogenous administration or endogenous overexpression of FGF-19 with altered expression of a number of genes involved especially in lipid metabolism (Tomlinson et al. 2002, Fu et al. 2004). In contrast to animals, only limited data on FGF-19 concentrations, regulation and function are available in humans. In the first study conducted in healthy human volunteers, serum FGF-19 levels exerted a PHYSIOLOGICAL RESEARCH ISSN (print) ISSN (online) 2011 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax , 628 Mráz et al. Vol. 60 pronounced diurnal rhythm following the rise in serum bile acids, which was abolished upon fasting. Treatment with bile acid sequestrants decreased FGF-19 concentrations, while administration of chenodeoxycholic acid had opposite effect (Lundasen et al. 2006). In two other studies circulating FGF-19 was found to be increased in patients with end-stage renal disease and extrahepatic cholestasis caused by tumorous obstruction of biliary ducts (Schaap et al. 2009, Reiche et al. 2010). Chronically malnourished patients with anorexia nervosa did not show any changes in systemic FGF-19 compared to normal-weight subjects (Dostalova et al. 2008b). The only two trials exploring in part the relation of FGF-19 to glucose metabolism disorders could not find significant difference in its circulating levels between patients with type 2 diabetes mellitus or insulin resistance relative to healthy controls (Brufau et al. 2010, Schreuder et al. 2010). To our best knowledge, no targeted information about the influence of obesity on circulating FGF-19 levels is available, nor are any data about the dynamic changes in FGF-19 concentrations after dietary intervention or acute hyperinsulinemia. The main regulator of FGF-19 production FXR is an important link between bile acid and triglyceride metabolism, interacting with a variety of genes and receptors, among others PPAR-α (Smelt 2010). The exact role of FGF-19 in these interactions and the influence of lipid-lowering PPAR-α agonists on FGF-19 levels are largely unknown as well. In the present study we therefore addressed the question whether circulating FGF-19 levels are influenced by increased body weight and the presence of T2DM and whether any changes in its concentrations could contribute to positive metabolic effects of very-low calorie diet in patients with obesity or lipid-lowering therapy with PPAR-α agonists in subjects with obesity and type 2 diabetes. To this end we measured serum FGF-19 concentrations in patients with obesity and T2DM and studied its changes after above mentioned interventions. Methods Study subjects and interventions Overall, 29 women with obesity (defined as BMI 30) without type 2 diabetes mellitus, 19 women with obesity and type 2 diabetes mellitus and 30 age-matched healthy control women were included into the study. The body weight of all study participants remained stable for at least 3 months before the beginning of the study. Very low calorie diet sub-study 12 out of 29 obese subjects without type 2 diabetes mellitus underwent a 3-week very low calorie diet (VLCD) program. They were hospitalized in the Third Department of Medicine, General University Hospital, Prague and were given a very low calorie diet with energy content of 2500 kj/day. Fenofibrate treatment sub-study 11 obese females with type 2 diabetes mellitus and 10 non-obese women were included into fenofibrate intervention study, which was described in detail elsewhere (Haluzik et al. 2009). T2DM patients were treated with diet, metformin alone or combination of metformin and glimepiride before the beginning of the study. Diabetic medication remained unchanged from three months before the start throughout the entire study. Diabetic subjects were treated with PPAR-α agonist fenofibrate (Lipanthyl M 267) for three months. Their insulin sensitivity before and after 3 months of fenofibrate treatment was examined by isoglycemichyperinsulinemic clamp as described previously (Anderlova et al. 2007). Ten lean healthy non-diabetic subjects, who were free of any medication, underwent a single isoglycemic-hyperinsulinemic clamp and served as a control group in fenofibrate intervention study. Written informed consent was signed by all participants before being enrolled into the study. The study was approved by the Human Ethical Review Committee, First Faculty of Medicine and General University Hospital, Prague, Czech Republic and was performed in accordance with the principles of the Declaration of Helsinki as revised in Anthropometric examination, blood and tissue sampling All subjects were measured and weighted and their body mass index (BMI) was calculated. Blood samples were withdrawn between 07:00 h and 08:00 h after overnight fasting. Blood samples were separated by centrifugation for 10 minutes at 1000x g within 30 minutes from blood collection. Serum was subsequently stored in aliquots at 70 C until further analysis. Clinical and hormonal parameters in obese patients on VLCD were measured one day before the beginning of the diet and at the end of the third week of VLCD. Clinical and hormonal parameters in type 2 diabetes patients in the fenofibrate substudy were measured at baseline and after 3 months of treatment with PPAR-α agonist fenofibrate (200 mg, Lipanthyl 267M). 2011 FGF-19 in Obesity and T2DM: Influence of Diet and Fibrates 629 Table 1. Anthropometric, biochemical and hormonal characteristics of control group of healthy women (Control), obese women without type 2 diabetes mellitus (Obese) and type 2 diabetic women (T2DM). Values are means ± S.E.M. Statistical significance is from Oneway ANOVA or ANOVA on Ranks as appropriate. *p 0.05 vs. control group; p 0.05 T2DM vs. Obese. Group Control Obese T2DM Number of subjects Age (years) 48.4 ± ± ± 1.5 BMI (kg/m 2 ) 23.8 ± ± 1.5* 36.7± 1.2* Blood glucose (mmol/l) 4.30 ± ± 0.33* ± 0.84* Cholesterol (mmol/l) 5.07 ± ± ± 0.21 Triglycerides (mmol/l) 1.16 ± ± 0.15* 2.82 ± 0.26* Insulin (miu/l) ± ± 2.34* 46.60± 4.02* HOMA index 2.50 ± ± 0.66* 18.05± 1.46* Leptin (ng/ml) ± ± 2.19* 28.1± 3.30* Adiponectin (μg/ml) ± ± 1.19* 13.02± 2.11* Hormonal and biochemical assays Serum FGF-19 levels were measured by a sandwich enzyme immunoassay (BioVendor, Brno, Czech Republic), following the manufacturer s instructions. Serum samples for FGF-19 measurement were diluted 1:1 with a dilution buffer prior to the assay. The standard curve range for the assay was ng/ml. Sensitivity was 4.8 pg/ml and the intra- and interassay variability was 3-8 % and %, respectively. Serum insulin concentrations were measured by commercial RIA kit (Cis Bio International, Gif-sur-Yvette Cedex, France). Sensitivity was 2.0 μiu/ml. Serum adiponectin levels were measured by commercial ELISA kit (Linco Research, St. Charles, Missouri, USA). Sensitivity was 0.78 ng/ml. Serum leptin concentrations were measured by commercial ELISA kit (Biovendor, Brno, Czech Republic). Sensitivity was 0.17 ng/ml. The intra- and interassay variability of all kits was less than 5.0 % and 9.0 % respectively. Biochemical parameters were measured in the Department of Biochemistry of General University Hospital, Prague by standard laboratory methods. Homeostasis model assessment (HOMA-R) index was calculated as previously described (Matthews et al. 1985) using the following formula: fasting serum insulin (miu/l) fasting serum glucose (mmol/l)/22.5. Statistical analysis Statistical analysis was performed using SigmaStat software (SPSS Inc., Chicago, IL). The results are expressed as means ± standard error means (S.E.M.). Changes in FGF-19 and hormonal parameters between studied groups were evaluated by One-way ANOVA followed by Holm-Sidak method or ANOVA on Ranks followed by Dunn's test and Paired t-test or Wilcoxon Signed Rank Test as appropriate. Changes of FGF-19 during the clamp were analyzed by One-way RM ANOVA on Ranks followed by Dunn's method. Spearman or Pearson correlation test and multiple regression analysis were used to calculate the relationships between FGF-19 levels and other parameters. Statistical significance was assigned to p 0.05. Results Baseline characteristics of patients with obesity, patients with type 2 diabetes mellitus and control group Anthropometric, biochemical and hormonal characteristics of obese, T2DM, and control group are shown in Table 1. As expected, BMI, blood glucose levels, HOMA index, serum triglyceride, leptin and insulin concentrations were significantly higher in both obese and T2DM group relative to control subjects. On the contrary, adiponectin levels were significantly lower in both of these groups. No differences in serum cholesterol were found between the groups. When comparing T2DM with obese group, diabetic patients had significantly lower BMI and leptin levels, while blood glucose, triglycerides, insulin and HOMA index were significantly higher in T2DM group relative to obese subjects. Anthropometric and clinical characteristics of patients included into VLCD study The influence of VLCD on anthropometric, 630 Mráz et al. Vol. 60 Table 2. Anthropometric, biochemical and hormonal characteristics of control group of healthy women (Control) and obese female subjects before (Obese Baseline) and after (Obese after VLCD) three weeks of VLCD. Values are means ± S.E.M. Statistical significance is from One-way ANOVA or ANOVA on Ranks (Obese Baseline or Obese after VLCD vs. Controls) and from Paired t-test or Wilcoxon Signed Rank Test (Obese Baseline vs. Obese after VLCD) as appropriate. *p 0.05 vs. control group; p 0.05 Obese Baseline vs. Obese after VLCD. Group Control Baseline Obese After VLCD Number of subjects BMI (kg/m 2 ) 23.8 ± ± 2.7* 47.1 ± 2.5* Blood glucose (mmol/l) 4.30 ± ± 0.47* 5.26 ± 0.26* Cholesterol (mmol/l) 5.07 ± ± ± 0.22 Triglycerides (mmol/l) 1.16 ± ± ± 0.10 Insulin (miu/l) ± ± 3.56* ± 4.11* HOMA index 2.50 ± ± 1.09* 6.57 ± 0.88* Leptin (ng/ml) ± ± 3.89* ± 4.77* Adiponectin (μg/ml) ± ± 1.45* ± 1.52* Table 3. Anthropometric, biochemical and hormonal characteristics of control group of healthy females (Control) and obese type 2 diabetic women before (T2DM baseline) and after three months of treatment with PPAR-α agonist fenofibrate (T2DM fenofibrate). Values are means ± S.E.M. Statistical significance is from One-way ANOVA or ANOVA on Ranks as appropriate (T2DM/Baseline or T2DM/Fenofibrate vs. Controls) and from Paired t-test or Wilcoxon Signed Rank Test (T2DM/Baseline vs. T2DM/Fenofibrate) *p 0.05 vs. control group; p 0.05 T2DM/Fenofibrate vs. T2DM/baseline. Group Control Baseline T2DM Fenofibrate Number of subjects BMI (kg/m 2 ) 23.7 ± ± 2.9* 36.5±2.7* Blood glucose (mmol/l) 4.97 ± ± 0.68* 9.43±0.84* Glycated Hemoglobin (%) 3.81 ± ± 0.56* 6.10 ± 0.58* Cholesterol (mmol/l) 5.39 ± ± ±0.25 HDL-cholesterol (mmol/l) 1.55 ± ± 0.11* 1.20 ± 0.11* Triglycerides (mmol/l) 0.98 ± ± 0.39* 2.17±0.24* Insulin (miu/l) 23.0 ± ± 7.1* 47.5±8.0* HOMA index 5.08 ± ± 0.21* ± 0.30* Adiponectin (μg/ml) ± ± ± 2.56 Atherogenic index 1.88 ± ± 0.37* 3.02 ± 0.75* biochemical and hormonal parameters is summarized in Table 2. At baseline, patients with obesity had significantly higher BMI, blood glucose, serum insulin, HOMA index and leptin and lower serum adiponectin relative to control subjects. The groups did not significantly differ in serum triglyceride and cholesterol levels. VLCD induced a significant decrease in BMI and HOMA index. Insulin, glucose and leptin concentrations tended to decrease after VLCD, but the difference did not reach the statistical significance. No significant influence of VLCD on serum cholesterol, triglyceride and adiponectin levels was found. Anthropometric and clinical characteristics of patients included into fenofibrate treatment study Anthropometric, biochemical and hormonal characteristics of the study sub-group have been described in detail elsewhere (Haluzik et al. 2009) and are briefly shown in Table 3. At baseline, BMI, blood glucose, serum triglyceride, insulin, glycated hemoglobin and atherogenic index were significantly higher, while serum HDL cholesterol was significantly lower in T2DM group relative to control subjects. Three months of treatment with fenofibrate led to a significant decrease of 2011 FGF-19 in Obesity and T2DM: Influence of Diet and Fibrates 631 Fig. 1. Serum FGF-19 concentrations in control group (black bar, n=30), obese nondiabetic group (light grey bar, n=29) and type 2 DM group (dark grey bar, n=19). Values are means ± S.E.M. Statistical significance is from One-way ANOVA or ANOVA on Ranks as appropriate. *p 0.05 vs. control group. serum triglyceride concentrations, while blood glucose, HOMA index and glycated hemoglobin slightly but significantly increased. Other parameters including BMI were not affected by fenofibrate treatment. Insulin sensitivity of T2DM group was significantly lower relative to control group and was not affected by fenofibrate treatment (data not shown here, previously described in (Haluzik et al. 2009). The influence of obesity and obesity/type 2 diabetes mellitus on FGF-19 levels and the relationship of FGF-19 levels with anthropometric and metabolic parameters Circulating concentrations of FGF-19 reached the highest levels in lean control subjects (202.2±16.7 pg/ml), with a non-significant tendency towards lower levels in T2DM group (146.4±15.9 pg/ml) and a further, statistically significant, decrease in the obese non-diabetic group (132.1±12.7 pg/ml, p 0.05). No significant difference in FGF-19 levels was observed between T2DM and obese group (Fig. 1). In a combined population of all three groups as well as in the subpopulation of control subjects combined with obese non-diabetic patients FGF-19 concentrations significantly negatively correlated with BMI (r= , p 0.05 for the whole population and r= , p 0.05 for Control + Obese subgroup) and leptin (r= , p 0.05 for the whole population and r= , p=0.005 Fig. 2. Serum FGF-19 concentrations in control group (black bar, n=30), obese patients before (light grey bar, n=12) and after (dashed bar, n=12) three weeks of very low calorie diet (VLCD). Values are means ± S.E.M. Statistical significance is from Oneway ANOVA or ANOVA on Ranks and from paired t-test or Wilcoxon Signed Rank Test as appropriate. *p 0.05 vs. control group. for Control + Obese subgroup) and significantly positively with adiponectin (r=0.289, p 0.05 for the whole population and r=0.289, p 0.05 for Control + Obese subgroup). In contrast, no such relationship could be seen in the T2DM subgroup. FGF-19 levels were not significantly related to age, serum blood glucose, triglycerides, insulin and cholesterol or HOMA index in any of the studied groups. The influence of VLCD, fenofibrate treatment and hyperinsulinemia during the clamp on serum FGF-19 levels Three weeks of VLCD did not significantly influence serum FGF-19 levels in obese group (140.2±19.7 vs ±27.1 pg/ml, p=0.369) (Fig. 2), while 3 months of treatment with fenofibrate significantly decreased FGF-19 concentrations in T2DM patients (194.6±26.2 vs ±25.0 pg/ml, p=0.007) (Fig. 3). Three hours of acute hyperinsulinemia during isoglycemic-hyperinsulinemic clamp tended to decrease FGF-19 levels in both healthy and T2DM subjects, but the difference did not reach statistical significance (214.4±15.0 vs ±19.9 pg/ml, p=0.130 for control and 198.6±18.0 vs.147.2±24.8 pg/ml, p=0.519 for diabetic group). Nevertheless, there was a significant drop in FGF-19 concentrations in T2DM group after 632 Mráz et al. Vol. 60 Fig. 3. Serum FGF-19 concentrations in control group (black bar, n=10), type 2 DM group before (dark grey bar, n=11) and after three months of fenofibrate treatment (dashed bar, n=11). Values are mean± S.E.M. Statistical significance is from One-way ANOVA or ANOVA on Ranks and from paired t-test or Wilcoxon Signed Rank Test as appropriate. *p 0.05 vs. control group, p 0.05 vs. T2DM group before treatment. Fig. 4. Changes of FGF19 concentrations during isoglycemichyperinsulinemic clamp in control group of healthy females (filled circles, n=10) and obese females with type 2 DM before (open circles, n=11) and after three months of treatment with PPAR-α agonist fenofibrate (filled triangles, n=11). Statistical significance is from One-way RM ANOVA on Ranks. * p 0.05 vs. baseline value of the respective group. 90 minutes of the clamp (198.6±18.0 vs ±21.0 pg/ml, p 0.001), while no such change could be seen in the control group (214.4±15.0 vs ±41.1 pg/ml, p=0.483) (Fig. 4). In T2DM group three months of fenofibrate treatment resulted in markedly decreased initial FGF-19 concentrations with no further change in its levels in the course of the isoglycemichyperinsulinemic clamp (95.7±16.5 vs ±18.0 vs ±25.8 pg/ml, p=0.264) (Fig. 4). Discussion FGF-19 is a novel endocrine and paracrine regulator which plays central role in the suppression of bile acid synthesis and secretion. As bi
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