Final Report Summary - HGF-INSR (Hepatocyte Growth Factor and Insulin Resistance)
1. Summary.
Insulin resistance is a hallmark of human diseases, including diabetes and obesity, which lead to abnormal glucose and lipid metabolism as a result of impaired metabolic response to insulin. The mechanism underlying the development of insulin resistance remains incompletely understood.
Excessive production of pro-inflammatory cytokines by the adipose tissue is linked to insulin resistance. Hepatocyte Growth Factor (HGF) is an adipocytokine elevated in obese subjects, which strongly correlated with the metabolic syndrome. However, the role of HGF in the aetiology of insulin resistance remains unknown. HGF is a potent activator of glucose transport and metabolism, and also a strong inhibitor of fatty acid oxidation in rodent myotubes. To gain insights into the role of HGF in the aetiology of insulin resistance, we have generated (i) a conditional transgenic mouse of HGF in the skeletal muscle, and (ii) a conditional knockout mouse of the HGF receptor (c-met) in skeletal muscle. In addition, we have investigated the role of HGF as a pro-inflammatory cytokine in the regulation of glucose and lipid metabolism in preterm neonates and in pregnancies complicated with obesity.
Our main hypothesis is that HGF improves insulin sensitivity in vivo. To test this hypothesis, we initially propose two aims: 1) To investigate the relationship between plasma HGF levels and insulin resistance in humans. 2) To investigate the role of HGF in the insulin resistant skeletal muscle. In addition, we have included a third aim: 3) To investigate the role of HGF as a mechanistic link between maternal obesity and fetal metabolic programing in utero.
Accomplishing this project has two goals: First, to clarify the aetiology and pathophysiology of insulin resistance in humans, and second, to determine its contribution to the fetal origin of adult metabolic diseases. A mechanistic understanding of the role of HGF in the aetiology of insulin resistance and fetal metabolic programing in utero will facilitate the development of therapeutic strategies and the identification of new drug targets in the prevention and treatment of diabetes and obesity.
2. Description of the work performed since the beginning of the project.
The work performed since the beginning of this project is described below:
Specific AIM 1: To investigate the relationship between HGF and insulin resistance in humans.
Performed activities: During these four years we have recruited adult lean (n=50) and obese (n=80) patients at the Endocrinology Clinics of HUPM. Anthropometric measurements and blood samples have been obtained from these subjects. We also have recruited 170 children (male, n=95; female n=75) at the Paediatrics Clinics of HUPM. Anthropometric measurements and blood samples have been obtained from these subjects. In addition, we have recruited 19 preterm (gestational age, 28.04 ± 2.39 weeks) and 24 term (gestational age, 39.37 ± 0.95 weeks) newborns at the Paediatrics Clinics of HUPM. Anthropometric parameters and metabolic indices were evaluated.
Specific AIM 2: To investigate the role of HGF in the insulin-resistant skeletal muscle.
Performed activities: During these four years we have obtained and expanded the DTG-HGF colony mice. The phenotypic characterization of DTG-HGF mice fed regular diet has been performed. Whole body glucose metabolism, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed.
Specific AIM 3: To investigate the role of HGF in the aetiology of adult metabolic diseases (obesity and diabetes).
Performed activities: During the last two years we have recruited pregnant women without complications (n=65), pregnant women with pregnancies complicated with gestational diabetes mellitus (GDM; n=16), and pregnant women with pregnancies complicated with obesity (n=8) at the Endocrinology Clinics of HUPM. Anthropometric measurements, blood samples and placental explants have been obtained from these patients.
3. Description of the main results achieved.
Main results achieved for specific AIM 1:
Cross-sectional analyses of data from adult lean (n=50) and obese (n=80) patients at the Endocrinology Clinics of HUPM was performed. Informed consent was obtained from all subjects in accordance with ethics committee guidelines of the HUPM and University of Cádiz. Anthropometric measurements and blood samples have been obtained from these subjects. All blood samples have been obtained after pharmacological treatment withdrawn. At this point, we are analyzing our data to determine whether or not in our study cohort we have the following four groups: Group 1, lean subjects without insulin resistance (BMI ≤24.9; HOMA<3, n=25); Group 2, obese patients without insulin resistance (BMI ≥35; HOMA<3, n=25); Group 3, obese patients with moderate resistance (BMI ≥35; 35, n=25). A preliminary analysis of our study group has demonstrated that our population of study is matched by age and sex. We have determined plasma glucose, insulin, triglycerides, cholesterol, AST, ALT, creatinine, pro-HGF, active HGF, HGFA and PAI-1. We calculated the HOMA index using biochemical parameters. Finally, we are performing statistical analyses using SPSS software. For comparisons between groups will be performed using two-tailed t-test; and between multiple groups using ANOVA. Univariate analysis will be used to determine an association between serum HGF levels and various parameters (BMI, HOMA, biochemical data, HGFA, PAI-1, etc). Multivariate linear regression analysis will be used to determine correlation coefficients between serum HGF levels and various parameters. Statistical significance will be defined as p<0.05.
Cross-sectional analyses of data from 170 Spanish 10- and 12-year-olds participating were included in our study. The present analyses included 49 controls, 98 obese and 23 obese with criteria for metabolic syndrome. Inflammatory markers included C-reactive protein (CRP), total-HGF and activated-HGF total hepatocyte growth factor (t-HGF), activated-HGF (a-HGF) and activated-hepatocyte growth factor activator (a-HGFA). The CVD risk factors included blood pressure, glucose, insulin, triglycerides. Low-density lipoprotein cholesterol (LDL-c), and high density lipoprotein cholesterol (HDL-c). We found that the ratio a-HGFA/t-HGF is lower in obese children than in lean children. The plasma levels of inactivated-HGF were associated with metabolic syndrome. Finally, lower plasma a-HGF levels were associated with reduced insulin sensitivity in obese children. Our study demonstrates a relationship between plasma HGF levels and insulin resistance in obese children.
Cross-sectional analyses of data from 43 Spanish newborns were included in our study. We found that immediately after birth, preterms exhibited higher a-HGF levels than term neonates, despite similar t-HGF levels. Additionally, a-HGF inversely correlated with gestational age and birth weight. Furthermore, preterm newborns showed a time-dependent upregulation of t-HGF and a-HGF serum levels in the early postnatal period. We demonstrate that regulation of HGF activity and circulating HGF levels differ between term and preterm neonates along fetal development. In conclusion, our study underscores a time-dependent regulation of t-HGF and a-HGF levels along fetal development, suggesting that HGF is a marker of neonatal maturity.
Main results achieved for specific AIM 2:
Phenotypic characterization of DTG-HGF mice was performed in feeding conditions of regular diet at two doses (low, 0.5 mg/mL; and high, 1 mg/mL) of DOX for 1 month. Body weight, analyses of motor activity, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed in control and transgenic mice. As a result of these studies, we did not find metabolic differences between control and transgenic mice. For this reason, we performed the phenotypic characterization of DTG-HGF mice fed high fat diet (HFD). Phenotypic characterization of DTG-HGF mice was performed in feeding conditions of HFD (60% fat) at high dose (1 mg/mL) of DOX for 4 months. Body weight, analyses of motor activity, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed in control and transgenic mice. As a result of these studies, we found that DT-HGF exhibited reduced plasma insulin levels and improved glucose tolerance, despite similar body weight and plasma glucose levels. In conclusion, our results suggest that overexpression of HGF in skeletal muscle of obese mice improves total body glucose metabolism and insulin sensitivity.
Main results achieved for specific AIM 3:
We aimed to further understand the mechanistic link between maternal obesity, through its associated inflammatory uteroplacental environment, and the regulation of placental metabolism. To this end, we tested the hypothesis that the pro-inflammatory cytokine HGF alters placental glucose and lipid metabolism leading to accumulation of placental triglycerides. In obese women, total- and -activated-HGF levels in amniotic fluid were elevated ~24%, and placental HGF levels were 2-fold higher than in lean women. At a similar dose to that present in amniotic fluid of obese women, HGF (30 ng/mL) increased glucose uptake in placental explants. In parallel, HFG decreased fatty acid oxidation, whereas esterification and de novo fatty acid synthesis increased, leading to 2-fold triglyceride accumulation in placental explants. In conclusion, HGF is a mechanistic link between the intrauterine pro-inflammatory milieu of pregnancies complicated with obesity and its associated placental lipotoxicity. Our results support the notion that HGF, through modifying the availability of nutrient supply to the fetus, may play an indirect role on fetal macrosomia and increase the risk to suffer metabolic diseases such as obesity and diabetes later in life.
4. Expected final results and socio-economic impact.
The epidemic of obesity represents one of the most public health challenges of the 21st century for Europe. The prevalence of obesity has tripled in many European countries in the last two decades and the numbers of those affected continue to rise at an alarming rate, particularly among children. Half of all adults and one in five children in Europe are overweight. The trend is particularly alarming in children and adolescents, thus passing the epidemic into adulthood and creating a growing health burden for the next generation.
In view of these health issues, the main goal of this proposal is translational research. Accomplishing this project will enhance our knowledge about the factors involved in the aetiology and pathophysiology of two devastating diseases such as of obesity and type 2 diabetes (T2D), in order to facilitate its prevention, diagnosis and treatment.
The expected socio-economic impact of this proposal will contribute to improve the health care and well-being of the European citizens, increasing economic growth and enhancing European competitiveness. Our research will improve the diagnosis of the metabolic syndrome and T2D and might facilitate the development of new therapeutic targets in the treatment and cure of obesity, cardiovascular diseases and T2D.
Insulin resistance is a hallmark of human diseases, including diabetes and obesity, which lead to abnormal glucose and lipid metabolism as a result of impaired metabolic response to insulin. The mechanism underlying the development of insulin resistance remains incompletely understood.
Excessive production of pro-inflammatory cytokines by the adipose tissue is linked to insulin resistance. Hepatocyte Growth Factor (HGF) is an adipocytokine elevated in obese subjects, which strongly correlated with the metabolic syndrome. However, the role of HGF in the aetiology of insulin resistance remains unknown. HGF is a potent activator of glucose transport and metabolism, and also a strong inhibitor of fatty acid oxidation in rodent myotubes. To gain insights into the role of HGF in the aetiology of insulin resistance, we have generated (i) a conditional transgenic mouse of HGF in the skeletal muscle, and (ii) a conditional knockout mouse of the HGF receptor (c-met) in skeletal muscle. In addition, we have investigated the role of HGF as a pro-inflammatory cytokine in the regulation of glucose and lipid metabolism in preterm neonates and in pregnancies complicated with obesity.
Our main hypothesis is that HGF improves insulin sensitivity in vivo. To test this hypothesis, we initially propose two aims: 1) To investigate the relationship between plasma HGF levels and insulin resistance in humans. 2) To investigate the role of HGF in the insulin resistant skeletal muscle. In addition, we have included a third aim: 3) To investigate the role of HGF as a mechanistic link between maternal obesity and fetal metabolic programing in utero.
Accomplishing this project has two goals: First, to clarify the aetiology and pathophysiology of insulin resistance in humans, and second, to determine its contribution to the fetal origin of adult metabolic diseases. A mechanistic understanding of the role of HGF in the aetiology of insulin resistance and fetal metabolic programing in utero will facilitate the development of therapeutic strategies and the identification of new drug targets in the prevention and treatment of diabetes and obesity.
2. Description of the work performed since the beginning of the project.
The work performed since the beginning of this project is described below:
Specific AIM 1: To investigate the relationship between HGF and insulin resistance in humans.
Performed activities: During these four years we have recruited adult lean (n=50) and obese (n=80) patients at the Endocrinology Clinics of HUPM. Anthropometric measurements and blood samples have been obtained from these subjects. We also have recruited 170 children (male, n=95; female n=75) at the Paediatrics Clinics of HUPM. Anthropometric measurements and blood samples have been obtained from these subjects. In addition, we have recruited 19 preterm (gestational age, 28.04 ± 2.39 weeks) and 24 term (gestational age, 39.37 ± 0.95 weeks) newborns at the Paediatrics Clinics of HUPM. Anthropometric parameters and metabolic indices were evaluated.
Specific AIM 2: To investigate the role of HGF in the insulin-resistant skeletal muscle.
Performed activities: During these four years we have obtained and expanded the DTG-HGF colony mice. The phenotypic characterization of DTG-HGF mice fed regular diet has been performed. Whole body glucose metabolism, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed.
Specific AIM 3: To investigate the role of HGF in the aetiology of adult metabolic diseases (obesity and diabetes).
Performed activities: During the last two years we have recruited pregnant women without complications (n=65), pregnant women with pregnancies complicated with gestational diabetes mellitus (GDM; n=16), and pregnant women with pregnancies complicated with obesity (n=8) at the Endocrinology Clinics of HUPM. Anthropometric measurements, blood samples and placental explants have been obtained from these patients.
3. Description of the main results achieved.
Main results achieved for specific AIM 1:
Cross-sectional analyses of data from adult lean (n=50) and obese (n=80) patients at the Endocrinology Clinics of HUPM was performed. Informed consent was obtained from all subjects in accordance with ethics committee guidelines of the HUPM and University of Cádiz. Anthropometric measurements and blood samples have been obtained from these subjects. All blood samples have been obtained after pharmacological treatment withdrawn. At this point, we are analyzing our data to determine whether or not in our study cohort we have the following four groups: Group 1, lean subjects without insulin resistance (BMI ≤24.9; HOMA<3, n=25); Group 2, obese patients without insulin resistance (BMI ≥35; HOMA<3, n=25); Group 3, obese patients with moderate resistance (BMI ≥35; 3
Cross-sectional analyses of data from 170 Spanish 10- and 12-year-olds participating were included in our study. The present analyses included 49 controls, 98 obese and 23 obese with criteria for metabolic syndrome. Inflammatory markers included C-reactive protein (CRP), total-HGF and activated-HGF total hepatocyte growth factor (t-HGF), activated-HGF (a-HGF) and activated-hepatocyte growth factor activator (a-HGFA). The CVD risk factors included blood pressure, glucose, insulin, triglycerides. Low-density lipoprotein cholesterol (LDL-c), and high density lipoprotein cholesterol (HDL-c). We found that the ratio a-HGFA/t-HGF is lower in obese children than in lean children. The plasma levels of inactivated-HGF were associated with metabolic syndrome. Finally, lower plasma a-HGF levels were associated with reduced insulin sensitivity in obese children. Our study demonstrates a relationship between plasma HGF levels and insulin resistance in obese children.
Cross-sectional analyses of data from 43 Spanish newborns were included in our study. We found that immediately after birth, preterms exhibited higher a-HGF levels than term neonates, despite similar t-HGF levels. Additionally, a-HGF inversely correlated with gestational age and birth weight. Furthermore, preterm newborns showed a time-dependent upregulation of t-HGF and a-HGF serum levels in the early postnatal period. We demonstrate that regulation of HGF activity and circulating HGF levels differ between term and preterm neonates along fetal development. In conclusion, our study underscores a time-dependent regulation of t-HGF and a-HGF levels along fetal development, suggesting that HGF is a marker of neonatal maturity.
Main results achieved for specific AIM 2:
Phenotypic characterization of DTG-HGF mice was performed in feeding conditions of regular diet at two doses (low, 0.5 mg/mL; and high, 1 mg/mL) of DOX for 1 month. Body weight, analyses of motor activity, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed in control and transgenic mice. As a result of these studies, we did not find metabolic differences between control and transgenic mice. For this reason, we performed the phenotypic characterization of DTG-HGF mice fed high fat diet (HFD). Phenotypic characterization of DTG-HGF mice was performed in feeding conditions of HFD (60% fat) at high dose (1 mg/mL) of DOX for 4 months. Body weight, analyses of motor activity, glucose tolerance, insulin sensitivity, and plasma levels of glucose, triglycerides, cholesterol and insulin have been performed in control and transgenic mice. As a result of these studies, we found that DT-HGF exhibited reduced plasma insulin levels and improved glucose tolerance, despite similar body weight and plasma glucose levels. In conclusion, our results suggest that overexpression of HGF in skeletal muscle of obese mice improves total body glucose metabolism and insulin sensitivity.
Main results achieved for specific AIM 3:
We aimed to further understand the mechanistic link between maternal obesity, through its associated inflammatory uteroplacental environment, and the regulation of placental metabolism. To this end, we tested the hypothesis that the pro-inflammatory cytokine HGF alters placental glucose and lipid metabolism leading to accumulation of placental triglycerides. In obese women, total- and -activated-HGF levels in amniotic fluid were elevated ~24%, and placental HGF levels were 2-fold higher than in lean women. At a similar dose to that present in amniotic fluid of obese women, HGF (30 ng/mL) increased glucose uptake in placental explants. In parallel, HFG decreased fatty acid oxidation, whereas esterification and de novo fatty acid synthesis increased, leading to 2-fold triglyceride accumulation in placental explants. In conclusion, HGF is a mechanistic link between the intrauterine pro-inflammatory milieu of pregnancies complicated with obesity and its associated placental lipotoxicity. Our results support the notion that HGF, through modifying the availability of nutrient supply to the fetus, may play an indirect role on fetal macrosomia and increase the risk to suffer metabolic diseases such as obesity and diabetes later in life.
4. Expected final results and socio-economic impact.
The epidemic of obesity represents one of the most public health challenges of the 21st century for Europe. The prevalence of obesity has tripled in many European countries in the last two decades and the numbers of those affected continue to rise at an alarming rate, particularly among children. Half of all adults and one in five children in Europe are overweight. The trend is particularly alarming in children and adolescents, thus passing the epidemic into adulthood and creating a growing health burden for the next generation.
In view of these health issues, the main goal of this proposal is translational research. Accomplishing this project will enhance our knowledge about the factors involved in the aetiology and pathophysiology of two devastating diseases such as of obesity and type 2 diabetes (T2D), in order to facilitate its prevention, diagnosis and treatment.
The expected socio-economic impact of this proposal will contribute to improve the health care and well-being of the European citizens, increasing economic growth and enhancing European competitiveness. Our research will improve the diagnosis of the metabolic syndrome and T2D and might facilitate the development of new therapeutic targets in the treatment and cure of obesity, cardiovascular diseases and T2D.