Dissertation committee:
Παρασκευή Μουτσάτσου, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ, Επιβλέπουσα
Αθανάσιος Παπαβασιλείου, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευανθία Κασσή, Αναπληρώτρια καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Δημητριάδης, Ομότιμος καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Γρηγόριος Καλτσάς, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Βάϊα Λαμπαδιάρη, Αναπληρώτρια καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Χρήστος Κρούπης, Αναπληρωτής καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Type 2 Diabetes Mellitus (T2DM) is a chronic disease characterized by disturbed carbohydrate (hyperglycemia), fat and protein metabolism as well as by decreased secretion and/or effect of insulin, resulting in an absolute or relative insulin-deficiency. T2DM is one of the leading causes of increased morbidity and early mortality worldwide and has seen a dramatic increase in adults, children and adolescents. It is therefore imperative to broaden the scientific knowledge on the mechanisms that are disrupted in T2DM and find new therapeutic strategies.
Many studies support the significant impact of stress in T2DM. Moreover, decreased negative feedback and hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis characterizes T2DM. The glucocorticoid receptor (GR) is a key mediator of HPA axis negative feedback, however its role in linking hypercortisolemia and T2DM-associated hyperglycemia, hyperlipidemia and inflammation is not known. Moreover, it is completely unknown whether the potential abnormalities in the GR-signaling pathway are related to the metabolic disturbances and inflammation that characterize T2DM. Main purpose of this thesis is to investigate the possible role of GR as the “biological link” between hypercortisolemia and the metabolic disturbances as well as the inflammation observed in T2DM. Consequently, we investigated the hypothesis that an impaired GR function (GC resistance) may be associated with HPA axis hyperactivity and increased cortisol levels which in turn lead to abnormalities in peripheral metabolic tissues (such as hyperglycemia, hyperlipidemia) and increased inflammation in immune cells in T2DM patients. Due to limited access in brain GRs, we carried out our investigation in PBMCs since data support that GRs are similarly regulated in the brain and immune system and GR changes in brain parallel those in immune cells. Moreover, PBMCs are considered today as a model system to study the pathophysiology of diabetes and its complications. In 31 T2DM patients and 24 healthy controls a) we assessed various GR signaling parameters in PBMCs, b) we determined salivary cortisol parameters (as a measure of HPA axis activity) and c) we investigated the possible associations between GR- and cortisol parameters as well as with measures of glycaemia, lipidemia, inflammation, and energy metabolism.
Regarding the parameters of the GR-signaling pathway we determined: the total GR protein levels, the phosphorylated bioactive GR protein levels (pGR-S211), the gene expression ratio GRα/GRβ and the glucocorticoid (GC) sensitivity. GC sensitivity was assessed by determining the gene expression levels of dexamethasone (DEX)-induced GILZ (glucocorticoid-induced leucine zipper) and FKBP5 (FK506 binding-protein) as well as basal protein levels of interleukin-1β (IL-1). Regarding the diurnal salivary cortisol curve, it was constructed after determining cortisol levels in four saliva samples and we calculated various cortisol parameters such as the cortisol awaking response (CAR), the reactivity slope (slopeT0-T40), the overall slope (slopeT0-T23), the diurnal cortisol secretion (total area under the curve, AUCtotal) and the cortisol secretion during awaking (area under the curve with respect to increase, AUCi). Furthermore, we measured biochemical markers [fasting glucose, insulin, triglycerides, glycosylated hemoglobin (HbA1), HDL- and LDL-cholesterol], inflammatory markers (intracellular IL-1β levels), energy metabolism markers [AMP-activated protein kinase phosphorylated at Thr172 (pAMPKα-Thr172) protein levels], plasma antiponectin levels, pyruvate dehydrogenase kinase 4 (PDK4) gene expression levels and apoptosis markers. Finally, both GR signaling parameters and salivary cortisol parameters were correlated with indices of glycemia, lipidemia, inflammation, and energy metabolism.
Our results show that patients with T2DM have elevated body mass index (BMI), abdominal fat, fasting glucose, insulin, triglycerides and HbA1 levels as well as decreased LDL- and HDL-cholesterol levels and PDK4 gene expression compared to healthy population. These data are consistent with the presence of metabolic disturbances in T2DM. Diabetic patients have elevated total GR protein and GRβ gene expression levels as well as decreased bioactive pGR-S211 protein levels compared to healthy controls. GC resistance in T2DM patients is confirmed by reduced mRNA expression levels of GILZ and FKBP5 as well as by increased basal IL-1β protein levels. Moreover, T2DM patients have decreased basal GILZ and FKBP5 mRNA levels and flattened DEX-induced GILZ and FKBP5 response curves. These results show the existence of GC resistance in patients with T2DM. All of the above findings support an impaired GR-signaling in immune cells of T2DM patients reflecting possibly, an impaired GR-signaling in HPA axis tissues. Decreased salivary cortisol secretion profile is evidenced by flattened diurnal salivary cortisol curves, reduced CAR, slopeT0-T30 and AUCi values as well as by increased AUCtotal scores compared to healthy controls. These data support the reduced function of the HPA axis. Moreover, diabetic patients have elevated IL-1β and decreased pAMPKα-Thr172 protein levels, findidings which are consistent with the presence of increased inflammation and reduced energy metabolism in T2DM. Finally T2DM patients also show lower rate of apoptosis in PBMCs. An important finding of the present study is the statistically significant correlations of GR signaling parameters with inflammation and metabolism parameters. These correlations suggest that the GR dysfunction in PBMCs (which possibly reflects the GR dysfunction in HPA axis in brain) may lead to hyperglycemia, hyperlipidemia and increased inflammation in T2DM. The statistically significant correlations between the diurnal saliva cortisol parameters and measures of metabolism and inflammation support that the HPA axis hyperactivity leads to metabolic disturbances and increased inflammation in T2DM. All of the above correlations seem to confirm the original hypothesis of the study that in T2DM the presence of an impaired GR function and GC resistance in immune cells, are possibly linked to HPA axis GC resistance, HPA axis hyperactivity and hypercortisolemia, leads in turn, to abnormal GC-function in metabolic tissues (hyperglycemia, hyperlipidemia). On the other hand, GC resistance in PBMCs may result in increased inflammation in immune cells. In conclusion, our study supports that the GR dysfunction is the key biological link between hypercortisolism and metabolic disorders and inflammation in T2DM.
Regarding the relationship between diet and T2DM, the guidelines for clinical practice underlie the important role of nutrition in T2DM prevention and treatment. Consequently, patients are often encouraged to use alternative sweeteners, such as steviol glycosides. Although the consumption of steviol glycosides is considered safe, recent data suggest that stevioside (a steviol glycoside) exhibits dexamethasone-like action in macrophages acting through glucocorticoid response elements (GREs). These possible effects of steviol glycosides would be detrimental for metabolism. Hence, another important aim of the present study was to evaluate the impact of steviol, steviol glycosides, and a Greek-derived stevia extract on a number of key steps of GR signaling cascade in PBMCs from 4 healthy individuals. In particular, we measured the mRNA levels of the genes GILZ and FKBP5, the total GR protein levels, and the subcellular compartmentalization of GR. We also assessed the effect of the aforementioned natural sweeteners on GILZ and FKBP5 expression levels in whole blood from healthy volunteers following an oral consumption of a mixture of steviol glycosides. Our results revealed that none of the tested compounds (steviol, steviol glycosides, and Greek-derived stevia extract) altered the expression of the primary GR-target genes GILZ and FKPB5, GR protein levels or GR subcellular localization in PBMCs. These findings support that steviol, steviol glycosides, and a Greek-derived stevia extract do not exert GR-mediated effects in PBMCs and therefore it is unlikely to adversely affect metabolism.
