THE IMPLICATIONS OF CNR1 GENE S POLYMORPHISMS IN THE MODULATION OF THE ENDOCANNABINOID SYSTEM S EFFECTS

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1 UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA THE IMPLICATIONS OF CNR1 GENE S POLYMORPHISMS IN THE MODULATION OF THE ENDOCANNABINOID SYSTEM S EFFECTS PhD thesis summary Scientific Supervisor Prof. Univ. Dr. MIHAI CRUCE PhD Student ILIE-ROBERT DINU CRAIOVA 2011

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3 BACKGROUND The endocannabinoid system (ECS) is a very wide spread system, both at central and peripheral level, that has many important functions. There are proofs that this system has autoprotective roles, of recovery after stress and it remains inactive in repaus physiological conditions. Its implication in metabolic control, as part of the thrifty phenotype, appears to be key for understandaing obesity, type 2 diabetes mellitus, dslipidemia etc. This paper focuses on an issue of cell and molecular biology and genetics and it offers new perspectives in discovering the role of the endocannabinoid system in the pathogeny of obesity, insulin resistance, insulin secretion, focusing on the implications of the CNR1 gene s polymorphisms, the gene that encodes the main receptor of this system. The objectives of this paper are: - The evaluation of the association of CNR1 gene s polymorphisms with obesity, abdominal obesity, type 2 diabetes mellitus or impaired glucose metabolism, dyslipidemia, adipokines levels (adiponectin, leptin), metabolic syndrome. - The identification of the CNR1 gene s polymorphisms associated with the metabolic phenotype in order to find new targets for the treatment of obesity, insulinresistance, diabetes and dyslipidemia - The identification of CNR1 polymorphisms involved in the modulation of the endocannabinoid s role in the pathogeny of insulin secretion. We wanted to realise a cross-sectional study that includes subjects from the south-west part of Romania and to evaluate biological parameters and CNR1 gene s polymorphisms and the association of these polymorphisms with several parameters. Key words: endocannabinoid system, CNR1, CB1, polymorphism, type 2 diabetes mellitus, obesity, adiponectin, leptin, leptin, metabolic syndrome. STATE OF KNOWLEDGE Chapter 1 The endocannabinoid system presents a short history and the structure of this system, composed of the cannabinoids receptors, endogenous ligands (the endocanmabinoids), the enzymes that mediate the biosynthesis and degradation of the endogenous ligands. The CB1 receptor is the most important receptor of the endocannabinoid system and it is located at central and peripheral level. The CNR1 gene encodes this receptor and it is located at 6q14-15 level. This chapter also presents the CB2 receptor and the other targets for the cannabinoids and the endocannabinoids (anandamide and 2-arachydonoil glycerol being the most important ligands) and the cannabinoid receptor agonists ant the main mechanisms of biosynthesis and degradation of the endocannabinoids are described. Chapter 2 The endocannabinoid system as a modulator of some physiological processes describes the implication of this system in some important processes of the human physiology at central and peripheral level. The main function of the ECS is accomplished by the CB1 receptors and it is expressed in humans by the lipid accumulation. For the accomplishment of this function, the ECS acts on three main organs: the brain that controls food ingestion, the liver that is the only organ producing lipids and the adipose tissue that stores lipids. 1

4 The ECS is found in the digestive tube and its role is to facilitate the energy storage through various mechanisms. The liver is an important target of the endocannabinoids and the effect is the increase of fatty acids synthesis (lipogenesis). CB1 receptors, endocannabinoids and biosynthesizing enzymes were found in the adipose tissue. The effects of SEC at the adipose tissue are focused on energy storage achieved by two mechanisms: the increase of fatty acids uptake by the adipocites and stimulations of lipogenesis. Chapter 3 entitled The cannabinoid receptors and their implication in pathology presents the main pathological processes where the ECS is involved. Several studies indicated the implication of ECS in neuroprotection. It appears that the endocannabinoids levels are increased after in vivo brain injury. Several animal models indicated that the activation of the CB1 receptors located in the prefrontal cortex can determine schizophrenia. The administration of cannabinoid agonists had significant improvements of pain and spasticity in subjects with multiple sclerosis. There are some proofs that anandamide can inhibit the development of human breast and prostate cancer cells. Chapter 4 Mutational analysis of the CB1 cannabinoid receptor presents the mutational variants observed at the CB1 receptor. There were identified several polymorphisms of the human receptor. The first polymorphism identified was located in the intron that preceded the exon encoding the receptor. Several authors evaluated the association of this gene s polymorphisms with obesity, abdominal obesity, type 2 diabetes mellitus, and dyslipidemia. OWN CONTRIBUTIONS Chapter 5 Material and methods creates a brief description of the studied group. The inclusion criteria were: >30 years old, unrelated Caucasian subjects, signed consent. The exclusion criteria were: type 1 diabetes mellitus, diseases and drugs that interfere with insulin secretion, insulin resistance, lipid profile, pregnancy, lactation. For the diagnosis of type 2 diabetes mellitus and of impaired glucose metabolism (impaired fasting glucose, impaired glucose tolerance), the oral glucose tolerance test with 75g glucose was performed. The anthropometrical parameters evaluated were body weight, height, waist circumference, body mass index (BMI). Blood samples were collected for the evaluation of plasma glucose, total cholesterol, HDL-cholesterol, triglycerides, insulin, adiponectin and leptin levels. For the genetic analysis, blood samples were collected and DNA was extracted. The stages of genetic analysis were: DNA extraction, the evaluation of its concentration and purity, Taq reaction, statistical analysis. The Hardy-Weinberg is used to evaluate if in the study group there are changes in the genotype frequency. Hardy-Weinberg test was applied for biallelic markers, for all the analyzed polymorphisms. Pearson s Chi square test was used and the inbreeding coefficient was determined. The calculation if odds ratio, confidence interval, the test for Hardy- Weinberg equilibrium and 2 tests were done using DeFinetti s programme. The descriptive analysis and the comparison of the parameters were realized using SPSS 17.o and Excel

5 Chapter 6 Results The distribution of the subjects by age and gender indicates that subjects aged between 50 and 70 are the majority both in male and female (Figure 1). Figure 1. The distribution of the subjects by age and gender. The distribution of the subjects by gender is in Figure 2 it can be noticed that women are predominant. Figure 2. The distribution of the subjects by gender. The presence of obesity (BMI 30kg/m 2 ) is considered a marker for IR. It e\was found in 35,7% of the subjects included in the study (Figure 3). 3

6 Figure 3. The distribution of the subjects by BMI Abdominal obesity was found in 80% of the subjects when defined by the IDF criteria and in 57,39% of the subjects when defined by the AHA/NHLBI criteria ( (Figure 4) % 57.39% 42.61% 20.15% Cu obezitate abdominală Fără obezitate abdominală Cu obezitate abdominală Fără obezitate abdominală IDF AHA/NHLBI Figure 4. The distribution of abdominal circumference. Increased HOMA-IR values, indicating the presence of IR were found in 84,83% of the subjects (Figure 5) % 15.17% C <1,4 >1,4 HOMA-IR Figure 5. The distribution of the subjects by HOMA-IR. The rs polymorphism is a single nucleotide polymorphism where guanine (G) is replaced by adenine (A). First, the Hardy-Weinberg equilibrium was tested and p=0,903 indicates that this equilibrium is not violated. 4

7 The genotype frequency was 13,23% for GG genotype, 46,63% for AG genotype and 40,14% for AA genotype. frequency was 0,366 +/- 0,0153 for ancestral G allele and 0,634 +/- 0,0153 for A allele. The ANOVA test indicates that there is a significant between 1h blood glucose during OGTT between the genotypes (Figure 6). Figure 6. Mean 1h glucose levels during OGTT We evaluated the association of this polymorphism with obesity. From the study group, two subgroups were used: one of 179 subjects with obesity (BMI > 30 kg/m 2 ) and one control subgroup with 129 subjects with BMI< 25 kg/m 2. Table 1. The results of the association test of rs polymorphism with obesity OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p

8 As the allele frequency indicates, this polymorphism is not associated with obesity in the study group (Table 1). We evaluated the association of this polymorphism with abdominal obesity. There was no deviation from Hardy-Weinberg equilibrium both in control group control (p=0,650), and in the group with abdominal obesity (p=0,925). The genotype distribution was not significantly different in both groups. Table 2. The results of the association test of rs with abdominal obesity OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency indicates, this polymorphism is not associated with abdominal obesity (Table 2). The evaluation of the association of rs polymorphism with impaired glucose metabolism was performed. There was no deviation from Hardy Weinberg equilibrium both in control group (p=0,988), and in subjects with impaired glucose metabolism (0,887). The genotype distribution was not different between the two groups. Table 3. The results of the association test of rs with impaired glucose metabolism OR %C.I

9 Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency indicates, this polymorphism is not associated with impaired glucose metabolism in the study group (Table 3). The association of rs polymorphism with dyslipidemia was evaluated. There was no deviation from Hardy-Weinberg equilibrium in the control group (p=0,739), and in subjects with dyslipidemia (0,747). Table 4. The results of the association test of rs with dyslipidemia OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p It appears that this polymorphism is not associated with dyslipidemia (Table 4). The association of rs with low adiponectin levels was evaluated. Table 5. The results of the association test of rs with hypoadiponectinemia 7

10 OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with low adiponectin levels (Table 5). The association of rs polymorphism with low leptin levels was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,834), and in the group with hyperleptinemia (0,708). Table 6. The results of the association test of rs with hyperleptinemia OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with high leptin levels (Tabel 6). 8

11 We evaluated the association of rs with metabolic syndrome. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,996), and in the group with metabolic syndrome (0,870). Table 7. The results of the association test of rs with metabolic syndrome. OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with metabolic syndrome (Table 7). The rs polymorphism is a single nucleotid polymorphism where guanine (G) is replaced by adenine (A). First, the Hardy-Weinberg equilibrium was tested and p=0,091 indicates that this equilibrium is not violated. The genotype frequency was 9,98% for GG genotype, 37,88% for AG genotype and 52,14% for AA genotype. frequency was 0,289 +/- 0,0150 for ancestral G allele and 0,711 +/- 0,0150 for A allele. The ANOVA test indicates that there are significant s between total cholesterol, triglycerides and HDL-cholesterol (Figures 7, 8, 9). 9

12 p=0,044 Figure 7. Mean total cholesterol levels 160 rs p=0, TG GG AG AA Figure 8. Mean tryglicerides levels 60 rs p=0,040 HDL GG AG AA Figure 9. Mean HDL-cholesterol levels 10

13 The association of rs polymorphism with obesity was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,124), but the equilibrium was violated in subjects with obesity. Table 8. The results of the association test of rs with obesity OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency test indicated, there is no association between this polymorphism and obesity (Table 8). The association of rs polymorphism with abdominal obesity was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,477), but the equilibrium was violated in subjects with obesity (p=0,011). Table 9. The results of the association test of rs with abdominal obesity OR %C.I Chi p [AA]<- [AA]<->[GG] 11

14 OR %C.I Chi p This polymorphism is not associated with abdominal obesity in the study (Table 9). The association of rs polymorphism with impaired glucose metabolism was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,124), and in subjects with impaired glucose metabolism (p=0,793). Table 10. The results of the association test of rs with impaired glucose tolerance OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency indicates, this polymorphism is associated with impaired glucose metabolism. The G allele appears to confer risk for impaired glucose metabolism (OR=2,047, 95%CI=1,281-3,273) and p=0,002. The association of rs polymorphism with impaired lipid metabolism was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,264), and in subjects with impaired lipid metabolism (p=0,165). Table 11. The results of the association test of rs with dyslipidemia 12

15 OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with dyslipidemia The association of rs polymorphism with low adiponectin levels was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,131), but in subjects with hypoadiponectinemia the equilibrium was violated (p=0,002). Table 12. The results of the association test of rs with hypoadiponectinemia OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p

16 As the allele frequency indicates, this polymorphism was associated with low adiponectin levels. The A allele appears to confer protection for low adiponectin levels (Table 12). The association of rs polymorphism with high leptin levels was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group and in subjects with high leptin levels. Table 13. The results of the association test of rs with hyperleptinemia OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with hyperleptinemia (Table 13). The association of rs polymorphism with metabolic syndrome was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group (p=0,996), and in subjects with metabolic syndrome (p=0,870). Table 14. The results of the association test of rs with metabolic syndrome OR %C.I Chi p

17 [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with the metabolic syndrome (Table 14). The rs polymorphism is a single nucleotide polymorphism where adenine (A) is replaced by guanine (G). First, the Hardy-Weinberg equilibrium was tested and p=0,386 indicates that this equilibrium is not violated. The genotype frequency was 81,06% for AA genotype, 19,33% for AG genotype and 0,61% for GG genotype. The ANOVA test indicates that there are significant s between fasting glucose and 1h glucose levels in the OGTT (Figures 11 and 12). 90% 81.06% 60% 30% 18% 0.61% 0% AA AG GG Figure 10. The frequency of the rs genotypes in the study group Glicemia a jeun p=0, GG AG AA Figure 11. Mean fasting glucose 15

18 Glicemia la 1h TTGO p=0, GG AG AA Figure 12. Mean 1h glucose level at OGTT We evaluated the association of rs with obesity. There was no deviation from Hardy-Weinberg equilibrium in control group and in group with obesity. Table 15. The results of the association test of rs with obesity. OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency indicates, this polymorphism is not associated with obesity (Table 15). The association of rs polymorphism with abdominal obesity was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group and in group with abdominal obesity. Table 16. The results of the association test of rs abdominal obesity. 16

19 OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p As the allele frequency indicates, this polymorphism is associated with abdominal circumference. The A allele appears to confer risk for abdominal obesity while the G allele is protective (Table 16). The association of rs polymorphism with impaired glucose metabolism was evaluated. There was no deviation from Hardy-Weinberg equilibrium in control group and in group with impaired glucose metabolism. Table 17. The results of the association test of rs with impaired glucose metabolism OR %C.I Chi p < <0.001 [AA]<- [AA]<->[GG] OR %C.I Chi p < <0.001 <

20 This polymorphism is associated with impaired glucose metabolism and the G allele appears to be protective for impaired glucose metabolism (OR=0,399, 95%CI=0,238-0,670) and p<0,001. The A allele confers risk for impaired glucose metabolism (OR=9,091, 95%CI=1, ,372; p=0,029) (Table 17). The association of rs polymorphism with impaired glucose metabolism was evaluated. Table 18. The results of the association test of rs with dyslipidemia OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with dyslipidemia (Table 18). The association of rs polymorphism with low adiponectin levels was evaluated. Table 19. The results of the association test of rs with hypoadiponectinemia OR %C.I Chi p

21 [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with hypoadiponectinemia (Table 19). The association of rs polymorphism with metabolic syndrome was evaluated. Table 20. The results of the association test of rs with metabolic syndrome OR %C.I Chi p [AA]<- [AA]<->[GG] OR %C.I Chi p This polymorphism is not associated with metabolic syndrome (Table 20). The rs polymorphism is a single nucleotide polymorphism and cytosine is replaced by adenine. The Hardy Weinberg equilibrium was violated when tested in our subjects. The rs polymorphism is a single nucleotide polymorphism and thymine is replaced by cytosine. The Hardy Weinberg equilibrium was violated when tested in our subjects. 19

22 Chapter 7 Discussions integrates our study with other studies from the literature. This is the first study that evaluates the association of CNR1 gene s polymorphisms with metabolic parameters in Romanian subjects. Several studies indicated that increased BMI is associated with various CNR1 gene s polymorphisms but the association of increased waist circumference was noticed only with rs and rs polymorphisms. Although our study did not found any association of the CNR1 gene s polymorphisms and metabolic syndrome (MetS) but only of several polymorphisms with some components of MetS, there are some studies that indicated this association. A recent study indicated that subjects with AA and AG genotypes of rs polymorphism have a lower prevalence of MetS. Due to its numerous interactions with other physiological systems, the understanding of the ECS may be the key to decipher the thrifty phenotype. In chapter 8 entitled Conclusions the main ideas of this paper are presented: This paper is the first study that analyzes the association of CNR1 gene s polymorphisms with biological parameters in Romanian subjects. These results indicate that rs polymorphism is associated with impaired glucose metabolism and low adiponectin levels and the A allele of rs polymorphism is associated with abdominal obesity and impaired glucose metabolism. The implication of ECS in obesity, especially in abdominal obesity, is proved by the association of the A allele of rs with increased abdominal circumference This study is a good tool for understanding the physiology and physiopathology of ECS and the pathogeny of obesity and thrifty phenotype An important role of this study is to stimulate the development of new drugs targeting CB1 receptors in order to treat type 2 diabetes mellitus, dyslipidemia, and obesity. 20

23 Bibliography Barsh GS, Schwartz MW. (2002) Genetic approaches to studying energy balance: perception and integration. Nat Rev Genet. 3(8), Benzinou M, Chèvre JC, Ward KJ, et al. (2008) Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations. Hum Mol Genet. 17(13), Bermudez-Silva FJ, Suarez J, Baixeras E, et al. (2008) Presence of functional cannabinoid receptors in human endocrine pancreas. Diabetologia. 51, Black SC. (2004) Cannabinoid receptor antagonists and obesity. Curr Opin Investig Drugs. 5, Cota D, Marsicano G, Tschop M, et al. (2003) The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. J Clin Invest. 112, de Luis DA, González Sagrado M, Aller R, et al. (2011) Relation of G1359A polymorphism of the cannabinoid receptor gene (CB1) with metabolic syndrome by ATP III clasification. Diabetes Metab Res Rev. doi: /dmrr Di Marzo, V. (2008). The endocannabinoid system in obesity and type 2 diabetes. Diabetologia. 51, Di Marzo V, Matias I. (2005) Endocannabinoid control of food intake and energy balance. Nat Neurosci. 8, Di Marzo V, Goparaju SK, Wang L, et al. (2001) Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature. 410, Dinu IR, Popa S, Mota M, et al. (2010b) The association of rs polymorphism of the CNR1 gene with glucose metabolism abnormalities. Ann. Rom. Soc. Cell Biol. 1(15), Dinu IR, Popa SG, Mota M, et al. (2011) The role of the endocannabinoid system in the pathogeny of type 2 diabetes in: Type 2 Diabetes / Book 4, ISBN Intech, Croatia, Howlett AC, Breivogel CS, Childers SR, et al. (2004) Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology. 47, Marsicano G, Goodenough S, Monory K, et al. (2003) CB1 cannabinoid receptors and ondemand defense against excitotoxicity. Science. 302, 84 8 Matias I, Cristino L, Di Marzo V. (2008). Endocannabinoids: Some Like it Fat (and Sweet Too). J Neuroendocrinol. 20, Milewicz A, Tworowska-Bardzińska U, Jędrzejuk D, et al. (2011) Are endocannabinoid type 1 receptor gene (CNR1) polymorphisms associated with obesity and metabolic syndrome in postmenopausal Polish women? Int J Obes. 35(3), Pagotto U, Marsicano G, Cota D, et al. (2006) The emerging role of the endocannabinoid system in endocrine regulation and energz balance. Endocr Rev. 27, Patti ME. (2010). Rehashing endocannabinoid antagonists: can we selectively target the periphery to safely treat obesity and type 2 diabetes? J Clin Invest. 120(8), Peeters A, Beckers S, Mertens I, et al. (2007) The G1422A variant of the cannabinoid receptor gene (CNR1) is associated with abdominal adiposity in obese men, Endocrine. 31, Pertwee R. (2005) Cannabinoids. Springer ISBN X Reggio PH. (2009) The Cannabinoid Receptors. Humana Press. USA. ISBN

24 CURRICULUM VITAE Name Ilie-Robert Dinu Date and place of birth Craiova Adress Strada Dr. Dimitrie Gerota, nr. 36, Bl K1 Ap 14, Craiova, Romania Phone Current status and job: o PhD student in Cell and Molecular Biology with the theme: "The implications of the CNR1 gene s polymorphisms in the modulation of the endocannabinoid system s effects". UMF Craiova, Scientific supervisor: Prof. Dr. Mihai Cruce o Resident doctor, speciality Diabetes mellitus, Nutrition, Metabolic Diseases, Emergency Clinical County Hospital Craiova Grants: 1. Grant IDEI, contract no 234/ : New perspectives in the study of the endocannabinoid system the polymorphisms of the CNR1 gene and the metabolic phenotype. Project Director Prof. Dr. Mota Maria. 2. Grant Nr /2008: Studies regarding the usefulness of some functional foods in preventing and reversing the complications of type 2 diabetes mellitus, technological, genetic, nutritional, biochemical and clinical correlations. Project Director: Prof. Dan Mircea Cheta; Responsable: Prof. Dr Maria Mota. Member of professional associations: o European Association for the Study of Diabetes (EASD) 2008 o Romanian Society of Diabetes, Nutrition and Metabolic Diseases o Romanian Federation of Diabetes, Nutrition and Metabolic Diseases Scientific papers o Books: 1. Type 2 Diabetes/ Book 4. ISBN Coleen Croniger. Croatia. In press 2. The Romanian Treaty of Metabolic Diseases Viorel Șerban. Coauthor of 1 chapter. 3. Nutritional metabolic pathology. Mihaela Dincă, Maria Moţa. ISBN Co-author of 2 chapters 22

25 4. The guide of the educator for the therapeutical education of patients with type 2 diabetes mellitus. Maria Moţa. ILEX Publishing House. ISBN Coauthor of 1 chapter. 5. Pregnancy and Diabetes. In press. Coauthor of 2 chapters. 6. Monography: Obesity induced insulinorezistence and chronic hepatitis C; Moţa M., Popa S., Moţa E., Dinu I.R., Silosi I., Comănescu V., Comănescu M., Foarfa C., Mitruţ P., Zaharie S.I., Ioana M., Rogoz S., Panus C., Panduru M., Bicu M.L., Gheorghiţoiu I., Vladu M.; Editors: Moţa Maria, Popa Simona. Medicala Universitara Publishing House Craiova, ISBN ; Coauthor of 2 chapters. o Scientific papers published in specialty magazines: 1. The epidemiology of diabetic neuropathy. IR Dinu, Simona Popa, E Mota, Maria Mota. Romanian Journal of Diabetes, Nutrition, Metabolic Diseases. 2011; 2(18). ISSN (in press) 2. The association of the rs polymorphism of the CNR1 gene with hypoadiponectinemia. Robert Dinu, Simona Popa, Maria Mota, Eugen Mota, Camelia Stanciulescu, Mihai Ioana, Mihai Cruce. Romanian Journal of Morphology and Embriology. 2(52) [ISI] in press The use of the incretinic effect for a good glycemic control in a patient with type 2 Diabetes Mellitus case presentation. IR Dinu, Simona Georgiana Popa, Maria Mota, E Mota. Romanian Journal of Diabetes, Nutrition, Metabolic Diseases. 2010; 4(17). ISSN The association of rs polymorphism of the CNR1 gene with glucose metabolism abnormalities. IR Dinu, Simona Popa, Maria Mota, E Mota, M Ioana, M Cruce. Annals of the Romanian Society for Cell Biology. 2010; 1(15) [CNCSIS, EBSCO Academic Search Complete, Index Copernicus Journal Master List, Elsevier] 5. The Metabolic Syndrome New and Old Data. Pircalaboiu Lucretia, Simona G. Popa, Ileana Puiu, R.I. Dinu, Maria Moţa. Rom J Intern Med. 2010; 2(48): [PubMed - indexed for MEDLINE] 6. The diagnosis and causes of secondary arterial hypertension. Amelia Genunche, Daniela Badea, M. Badea, P. Mitrut, R. Dinu. Medicina Modernă. 2010; 5(17): Adiponectin, leptin levels and adiponectin-leptin ratio in obese subjects: correlations with insulin resistance. Maria Mota, Simona Popa, Robert Dinu, Camelia Stanciulescu, Eugen Mota. Diabetes. 2009; 58(1), A350, ISSN [ISI]. 8. The implication of CNR1 gene s polymorphisms in the modulation of endocannabinoid system effects. I. R. Dinu, Simona Popa, Mihaela Bîcu, Eugen Mota, Maria Moţa. Romanian Journal of Internal Medicine. 2009; 1(47):9-18. ISSN [PubMed- MEDLINE] 9. The benefits of thiamine (benfotiamine) use in diabetes mellitus. Mihaela L. Bîcu, D. Bîcu, Simona G. Popa, R.I. Dinu, E. Moţa, Maria Moţa. Romanian Journal of Diabetes, Nutrition, Metabolic Diseases. 2009; 2(16). ISSN The role of adult height, perinatal and familial factors in determining the risk of diabetic kidney disease. Panduru MN, Moţa M, Moţa E, Popa SG, Dinu R. Infomedica, 2009, 1(147):

26 11. Types of carbohidrates and their role in maintaining the glycemic control. Maria Moţa, ML Bîcu, SG Popa, RI Dinu, Sigina R Gîrgavu. Romanian Journal of Diabetes, Nutrition, Metabolic Diseases. 2009; 1(16): ISSN The correlation of the hepatic expression of SOCS3 with the response to PegInterferon in patients with chronic hepatitis C and obesity induced insulin resistance. Maria Moţa, Ilie-Robert Dinu, Simona Popa, Iulian Diaconescu, Mihaela Vladu, Mihaela Bîcu, Eugen Moţa. MÆDICA a Journal of Clinical Medicine. 2008; 3(2):98-104, ISSN , [OmniMedicalSearch]. 13. Leptin is correlated with clinical and biological parameters in subjects with metabolic syndrome. IR Dinu, Simona Popa, Camelia Stanciulescu, E Mota, Maria Mota, Amelia Barca, M Cruce. Annals of the Romanian Society for Cell Biology. 2009; 2(14) [CNCSIS, EBSCO Academic Search Complete, Index Copernicus Journal Master List, Elsevier] 14. The study of the mechanisms which induce a low response to the antiviral therapy in obese patients with insulin resistance in chronic viral hepatitis C. Maria Moţa, Mihaela Bîcu, Simona Popa, Robert Dinu, M Ioana, Mihaela Vladu, P Mitruţ, E Moţa. Romanian Journal of Diabetes, Nutrition and Metabolic Disease. 2008;3(14):5-18. ISSN [CNCSIS]. 15. Types of carbohydrates and their role in maintaining the glycemic control. Maria Moţa, Mihaela L. Bîcu, Simona G. Popa, R. I. Dinu, Sigina R. Gîrgavu. Romanian Journal of Diabetes, Nutrition and Metabolic Diseases. 2009; 1(16):17-28 ISSN [PubMed- MEDLINE] 16. Evaluation of the role of hiperinsulenimia and andipokines in the proliferation of hepatic stellate cells and fibrosis extension in chronic hepatitis C. M. Mota, M. Vladu, S. Popa, IR Dinu, V. Comănescu, I Diaconescu, M. Bîcu, C. Pănuş, E Moţa. Craiova Medicala, 2008;1(10):5-11. ISSN [CNCSIS]. o Papers published at international and national congresses: o Papers published at international congresses: 1. The association of the rs polymorphism of the CNR1 gene with hyperleptinemia in subjects with abdominal obesity. Robert Dinu, Simona Popa, Maria Mota, Eugen Mota, Camelia Stanciulescu, Mihai Ioana. Oral presentation. 25 th Symposium of the Federation of the International Danube Symposia on Diabetes Mellitus Insulin resistance, adipokines and the expression of metabolic syndrome in obese individuals with elevated waist circumference. Simona Popa, Robert Dinu, Maria Mota, Eugen Mota, Camelia Stanciulescu. Poster. 25 th Symposium of the Federation of the International Danube Symposia on Diabetes Mellitus The relation between adipokines, adiponectin-leptin ratio and the metabolic syndrome. Dinu IR, Popa SG, Stanciulescu C, Mota E, Mota M. XV International Symposium on Atherosclerosis - Boston, Adiponectin, leptin levels and adiponectin-leptin ratio in obese subjects: correlations with insulin resistance. M Mota, SG Popa, RI Dinu, C Stanciulescu, E Mota. American Diabetes Association 69th Scientific Sessions. New Orleans, Correlation between adiponectin-leptin ratio, adipokines and insulin resistance in patients with type 2 diabetes and prediabetes. Popa SG, Dinu IR, Stanciulescu C, Bicu M, Mota E, Mota M. XV International Symposium on Atherosclerosis. Boston,

27 6. The role of adult heigh, perinatal and familial factors in determining the risk of diabetic kidney disease. MN Panduru, M Mota, E Mota, SG Popa, R Dinu. European Society of Human Genetics ESHG, The correlation of host insulin resistance with hepatic stellate cell apoptosis and proliferation in chronic hepatitis C. Dinu IR, Popa SG, Bicu ML, Panduru MN, Mota M. 6 th Annual World Congress on the Insulin Resistance Syndrome. Los Angeles, The correlation between adipocitokines, hepatic fibrosis and hepatic stellate cell activation in chronic hepatitis C.Popa SG, Dinu IR, Bicu ML, Comanescu V, Mota M. 6 th Annual World Congress on the Insulin Resistance Syndrome. Los Angeles, The study of vascular manifestations to a proup of workers exposed to sonorous pollution. Dinu Ilie-Robert, Gusti Simona. 11th International Scientific Students Conference for Students and Young Doctors. Poland, Gdansk, 2003 o Papers published at national congresses: 1. The usefulness of anthropometric indices to explain the link of adipokines with insulin resistance and metabolic syndrome in subjects with impaired glucose metabolism. Maria Mota, Robert Dinu, Simona Popa, Eugen Mota, Camelia Stanciulescu. Poster. The 8th Congress of the Romanian Federation of Diabetes, Nutrition, Metabolic Diseases, Cluj, CNR1 gene rs polymorphism is not associated with alterations of carbohydrate metabolism. Robert Dinu, Simona Popa, Maria Moţa, Eugen Moţa, Mihai Ioana. Oral presentation. The 36 th National Congress of the Romanian Society of Diabetes, Nutrition and Metabolic Diseases, Sibiu, Impact of type 1 endocanabinoid receptor gene (CNR1) polymorphisms on the metabolic phenotype. Maria Moţa, Simona Popa, Robert Dinu, Mihai Ioana. Oral presentation. The 36 th National Congress of the Romanian Society of Diabetes, Nutrition and Metabolic Diseases, Sibiu, Involvement of endocanabinoid receptor gene (CNR1) polymorphism rs in the modulation of obesity associated phenotype. Simona Popa, Robert Dinu, Maria Moţa, Eugen Moţa, Mihai Ioana. Oral presentation. The 36 th National Congress of the Romanian Society of Diabetes, Nutrition and Metabolic Diseases, Sibiu, The correlation of the IR markers with the elements of the metabolic syndrome. Dinu FC, Popa SG, Dinu RI, Moga EM, Bîcu ML, Popescu D, Bornagel B, Moţa M. The 36 th National Congress of the Romanian Society of Diabetes, Nutrition and Metabolic Diseases, Sibiu, România. ISSN The utility of CGMS in the evaluation of the metabolic profile in patients with type 2 diabetes mellitus and chronic kidney disease. Popa Simona, Văduva Cristina, Dinu Robert, Protasiewicz Diana, Bîcu Mihaela, Moţa Maria. Oral presentation. UMF Days, Craiova ISSN The cardiovascular risk in patients with type 1 diabetes mellitus and diabetic nephropathy. Clenciu Diana, Vladu Mihaela, Gîrgavu Sigina, Popa Simona, Bîcu Mihaela, Panduru NM, Dinu R, Moţa Maria. Oral presentation. UMF Days Craiova ISSN Molecular mechanisms in metabolic syndrome: the contribution of adipocytokines. Maria Moţa, Simona Popa, Robert Dinu. Conference. National Congress of the Romanian Academic Association. - Bucharest, Correlation of adipokines with insulinresistance markers in subjects with abdominal obesity. Robert Dinu, Simona Popa, Mihaela Bicu, Diana Protasiewicz, Isabela Silosi, 25

28 Eugen Mota, Maria Mota. Poster for the poster session, Annual Congress of the Romanian Medical Association, Bucharest, The evaluation of the glycemic variability using CGMS clinical utility. Protasiewicz D.C., Dinu F., Popa S.G., Dinu R.I., Moţa M. Acta Diabetologica Romana no. 35/2009. Poster. The 36 th National Congress of the Romanian Society of Diabetes, Nutrition and Metabolic Diseases. May 2009 Sinaia 11. The correlation between adipocytokines and insulinresistance markers in subjects with abdominal obesity. Robert Dinu, Simona Popa, Diana Protasiewicz, Isabela Siloşi, Eugen Moţa, Maria Moţa, Amelia Bârcă, Mihai Cruce. Oral presentation. UMF Days, Craiova ISSN: Obesity predictive factor for the severity of the histological lesions at the patients with hepatitis C virus. Popa Simona, Bîcu Mihaela, Moţa Maria, Comănescu Violeta, Dinu Robert, Mitroi Nicoleta, Pănus Camelia, Diaconescu Iulian, Moţa Eugen; Journal of Experimental Medical Surgical Research, Year XV, Suppl. 1-2/2008. ISSN Endocannabinoid system and metabolic phenoptype. Simona Popa, Mihaela Bîcu, Robert Dinu, M. Ioana, Camelia Pănuş, Maria Moţa. III rd Prize at the Poster Session. Annual Congress of the Romanian Medical Association, Bucharest, Biomarkers of obesity evaluating the risk for complications. Robert Dinu, M. Ioana, Simona Popa, Maria Moţa, M. Cruce; poster for poster sessions, Annual Congress of the Romanian Medical Association, Bucharest, Correlation of HOMA-IR and A/L ratio with hepatic stellate cells phenotype in patients with chronic hepatitis C. Simona Popa, Robert Dinu, Mihaela Bicu, Diana Protasiewicz, Violeta Comanescu, Eugen Mota, Maria Mota. Prize at the Poster Session. Annual Congress of the Romanian Medical Association, Bucharest, Accu Check Smart Pix system-utility in analysis of therapeutic decision at 6 children from Dolj County, who use Spirit pumps. Mota Maria, Popescu Daniela, Panus Camelia, Popa Simona, Braicu Daniela, Dinu Robert; Journal of Experimental Medical Surgical Research, Year XV, Suppl. 1-2/2008. ISSN Oral presentation; The 34th National Congress of the Romanian Society of Diabetes, Sinaia May 25-28, The study of the lower limbs amputation made on the chirurgical clinic in 5 years period. Mitroi Nicoleta, Popa Simona, Bicu Mihaela, Dinu Robert, Mota Maria; Journal of Experimental Medical Surgical Research, Year XV, Suppl. 1-2/2008. ISSN The 34th National Congress of the Romanian Society of Diabetes, Sinaia May 25-28, The correlation between waist circumference and TG/HDL>3 ratio as insulinresistance markers with the disturbances of glycemic metabolism. Mihaela L. Bicu, Simona G. Popa, R.I. Dinu, Camelia Panus, Maria Mota. The 7th Congress of the Romanian Federation of Diabetes, Nutrition, Metabolic Diseases, Sibiu, The effects of obesity on VHC, response to antiviral treatment and the evolution of chronic hepatc lesions. Robert Dinu, Simona Popa, Maria Moţa, Mihai Cruce; National Congress of Morphopathology and Embryology, Craiova, The diagnosis and prognosis of the complications of obesity. Dinu IR, Ioana M, Popa Simona, Moţa Maria. Oral presentation. UMF Craiova Days June 6th-7th, ISSN: Observations regarding the echographic-electrocardiographic correlations in the early hypertensive cardiopathy. Dinu Ilie-Robert, Enache Andreea Lorena, Vasile Ileana Manuela, Lungana Florin Mihai, Vasile Ileana, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, Predictive value of the carotidian vascular injury to estimate the severity of the coronarian ischemic injuries. Enache Andreea Lorena, Dinu Ilie-Robert, Lungana 26

29 Florin Mihai, Vasile Ileana Manuela, Lungana Ana Maria Cristina, Vasile Ileana, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, Children s effort inducted astma. Dinu Ilie-Robert, Enache Andreea Lorena, Lungana Florin Mihai, Lungana Ana Maria Cristina, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, Therapeutically options in the diabetic leg s injuries. Lungana Florin Mihai, Lungana Ana Maria Cristina, Enache Andreea Lorena, Dinu Ilie-Robert, Siloşi Cristian Adrian, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, Profilaxis of tromboembolic events a desideratum in general surgery. Enache Andreea Lorena, Dinu Ilie-Robert, Lungana Florin Mihai, Lungana Ana Maria Cristina, Păun Mariana, Teodorescu Mădălin, Mogoş Dan, Păun Ion, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, The value of some serum imonumarkers in acute pancreatic diagnostic severity. Lungana Florin Mihai, Lungana Ana Maria Cristina, Enache Andreea Lorena, Dinu Ilie- Robert, Siloşi Cristian Adrian, National Congress for Students and Young Physicians, XI-th edition, Bucharest, December 6-9, Risk factors involved in trombotic complications for essential thrombocythemia. Dinu Ilie-Robert, Enache Andreea Lorena, Găman Amelia, Craiova International Medical Students Conference November The role of Doppler ultrasound for diagnosis of the severity of vascular diseases. Enache Andreea Lorena, Dinu Ilie-Robert, Vasile Ioana Manuela, Vasile Ileana, Craiova International Medical Students Conference November Profilaxis of tromboembolic events a target in general surgery. Enache Andreea Lorena, Dinu Ilie-Robert, Păun Mariana, Teodorescu Mădălin, Mogoş Dan, Păun Ion, Craiova International Medical Students Conference November The value of serum immunomarkers in diagnosis of the acute pancreatitis. Lungana Florin Mihai, Lungana Ana Maria Cristina, Enache Andreea Lorena, Dinu Ilie-Robert, Silosi Cristian Adrian, Silosi Isabela, Craiova International Medical Students Conference November Therapeutically options at the diabetic leg. Lungana Florin Mihai, Lungana Ana Maria Cristina, Enache Andreea Lorena, Dinu Ilie-Robert, Silosi Cristian Adrian, Craiova International Medical Students Conference November The role of immunohistochemistry in anatomopathological diagnosis in peritonitis of neopasic ethiology. Lungana Florin Mihai, Lungana Ana Maria Cristina, Enache Andreea Lorena, Dinu Ilie-Robert, Silosi Cristian Adrian, Silosi Isabela, Violeta Comănescu, Craiova International Medical Students Conference November Clinical and hemodynamic study of ACE inhibitors. Enache Andreea Lorena, Dinu Ilie-Robert, Vasile Ioana Manuela, Vasile Ileana, Craiova International Medical Students Conference November The incidence and spectrum of the neurological symptoms in an internal medicine clinic. Dinu Ilie-Robert, Enache Andreea-Lorena, Vasile Ileana, National Congress for Students and Young Physicians, Craiova, March 15-18, Ovary cancer between diagnostic dilemmas and therapeutically feed-back whim. Dinu Ilie-Robert, Enache Andreea-Lorena, Păun Ion, Mogoş Dan, National Congress for Students and Young Physicians, Craiova, March 15-18, First injury mirage (vesicular lithiasis) and laparoscopic colecistectomy may delay colic cancer diagnosis. Enache Andreea-Lorena, Dinu Ilie-Robert, Paun Ion, 27

30 Mogoş Dan, National Congress for Students and Young Physicians, X-th edition, Bucharest, November 23-26, Rare cause of urethral obstruction endoscopical approach. Enache Andreea-Lorena, Dinu Ilie-Robert, Mitroi George, National Congress for Students and Young Physicians, X-th edition, Bucharest, November 23-26, The 3-D model of coronarian capillaries at patients sufferinf of dilatative, ischemic or inflammatory cardiomiopathy. Enache Andreea-Lorena, Dinu Ilie-Robert, Forţofoiu Mircea Cătălin, National Congress for Students and Young Physicians, X-th edition, Bucharest, November 23-26, Left ventricular hypertrophy, prediction factor for atrial fibrillation. Enache Andreea-Lorena, Dinu Ilie-Robert, Forţofoiu Mircea Cătălin, National Congress for Students and Young Physicians, X-th edition, Bucharest, November 23-26, Study on morbidity through viral hepatitis. Enache Andreea-Lorena, Dinu Ilie- Robert, Roşu Lucica, National Congress for Students and Young Physicians, X-th edition, Bucharest, November 23-26, The importance of GADD45A in vascular cerebral stroke. Dinu Ilie-Robert, Cruce Mihai, Popa-Wagner Aurel, National Congress for Students and Young Physicians, Craiova, April 14-16, Ethiopatogenic and therapeutical evaluation in early sexualisation. Dinu Ilie-Robert, Cergă Areta, Bistriceanu Marian, National Congress for Students and Young Physicians, Craiova, April 14-16, Diagnosis and therapeuthical attitude in foreign traheobronchic formations. Dinu Ilie-Robert, Deciu Cristian, Anghelina Florin, Ionita Elena, National Congress for Students and Young Physicians, Craiova, April 14-16, Central vascular aspects observed at a batch of cervical spondilosis patients. Dinu Ilie-Robert, Gusti Simona, National Congress for Students and Young Physicians, Craiova, April 15-17, Doppler vascular parameters of measuring cerebral irrigation in physiological and pathological conditions. Dinu Ilie-Robert, Gusti Simona, National Congress for Students and Young Physicians, Craiova, April 15-17, Aspects of vascular behaviour observed at a batch of workers exposed to sound pollution. Dinu Ilie-Robert, Gusti Simona, Univerisity of Medicine and Pharmacy Iuliu Haţeganu Cluj Napoca, December 2-5, Aspects of vascular behaviour observed at a batch of workers exposed to sound pollution. Dinu Ilie-Robert, Gusti Simona, Sesiunea National Congress for Students and Young Physicians, Craiova, June 6-8, Correlations between Doppler fluxmetry and coronarian irrigation observed in a batch of menopause women. Dinu Ilie-Robert, Gusti Simona, National Congress for Students and Young Physicians, Craiova, May 24-26,