Modeling of Environmental Effects in Genome-Wide Association Studies Identifies SLC2A2 and HP as Novel Loci Influencing Serum Cholesterol Levels

Genome-wide association studies (GWAS) have identified 38 larger genetic regions affecting classical blood lipid levels without adjusting for important environmental influences. We modeled diet and physical activity in a GWAS in order to identify novel loci affecting total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels. The Swedish (SE) EUROSPAN cohort (NSE = 656) was screened for candidate genes and the non-Swedish (NS) EUROSPAN cohorts (NNS = 3,282) were used for replication. In total, 3 SNPs were associated in the Swedish sample and were replicated in the non-Swedish cohorts. While SNP rs1532624 was a replication of the previously published association between CETP and HDL cholesterol, the other two were novel findings. For the latter SNPs, the p-value for association was substantially improved by inclusion of environmental covariates: SNP rs5400 (pSE,unadjusted = 3.6×10−5, pSE,adjusted = 2.2×10−6, pNS,unadjusted = 0.047) in the SLC2A2 (Glucose transporter type 2) and rs2000999 (pSE,unadjusted = 1.1×10−3, pSE,adjusted = 3.8×10−4, pNS,unadjusted = 0.035) in the HP gene (Haptoglobin-related protein precursor). Both showed evidence of association with total cholesterol. These results demonstrate that inclusion of important environmental factors in the analysis model can reveal new genetic susceptibility loci

A Journey to Understand Glucose Homeostasis: Starting from Rat Glucose Transporter Type 2 Promoter Cloning to Hyperglycemia

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.ope

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells

Glucose concentrations of normal human airway surface liquid are ~12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of d-glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5–10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway

A novel therapeutic strategy for pancreatic neoplasia using a novel RNAi platform targeting PDX-1

Bi-functional shRNA (bi-shRNA), a novel RNA interference (RNAi) effector platform targeting PDX-1 utilizing a systemic DOTAP-Cholesterol delivery vehicle, was studied in three mouse models of progressive pancreatic neoplasia. Species-specific bi-functional PDX-1 shRNA (bi-shRNAPDX-1) lipoplexes inhibited insulin expression and secretion while also substantially inhibiting proliferation of mouse and human cell lines via disruption of cell cycle proteins in vitro. Three cycles of either bi-shRNA<sup>mousePDX-1</sup> or shRNA<sup>mousePDX-1</sup> lipoplexes administered intravenously prevented death from hyperinsulinemia and hypoglycemia in a lethal insulinoma mouse model. Three cycles of shRNA<sup>mousePDX-1</sup> lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of pancreatic neoplasia. Moreover, three cycles of the bi-shRNA<sup>humanPDX-1</sup> lipoplexes resulted in near complete ablation of tumor volume and considerably improved survival in a human PANC-1 implanted SCID-mouse model. Human pancreatic neoplasia specimens also stained strongly for PDX-1 expression. Together, these data support the clinical development of a novel therapeutic strategy using systemic bi-shRNA<sup>PDX-1</sup> lipoplexes against pancreatic neoplasia

Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior

Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic β-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.Fil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad de Concepción; ChileFil: Millán, Carola. Universidad de Concepción; Chile. Universidad Adolfo Ibañez; ChileFil: Barahona, María José. Universidad de Concepción; ChileFil: Recabal, Antonia. Universidad de Concepción; ChileFil: Salgado, Magdiel. Universidad de Concepción; ChileFil: Martinez, Fernando. Universidad de Concepción; ChileFil: Ordenes, Patricio. Universidad de Concepción; ChileFil: Elizondo Vega, Roberto. Universidad de Concepción; ChileFil: Sepúlveda, Fernando. Universidad de Concepción; ChileFil: Uribe, Elena. Universidad de Concepción; ChileFil: García Robles, María de los Ángeles. Universidad de Concepción; Chil

Hyperglycemia Induction in HepG2 Cell Line

Background: Hyperglycemia is one of the important featurs of diabetes. In cell culture studies different methods are used to mimic the hyperglycemia condition. In this study we investigate response of human liver cancer cell line (HepG2) to high insulin, high glucose, and high insulin/ high glucose medium exposure.Methods: HepG2 cells were settled in DMEM+0.1% FBS or DMEM free-serum medium with high concentrations of d-glucose (30 mm) and/or insulin (1μM) for 24h after an overnight starving in serum- free medium. The level of hyperglycemia was estimated in the supernatants via GOD-POD method.Results: Serum-free madium with high insulin/ high glucose consentration made the higher level of hypreglycemia in HepG2 cells.Conclusions: Our study interduced high insulin/ high glucose treatment as the best way to induction hyperglycemia.

Hyperglycemia Induction in HepG2 Cell Line

Background: Hyperglycemia is one of the important featurs of diabetes. In cell culture studies different methods are used to mimic the hyperglycemia condition. In this study we investigate response of human liver cancer cell line (HepG2) to high insulin, high glucose, and high insulin/ high glucose medium exposure.Methods: HepG2 cells were settled in DMEM+0.1% FBS or DMEM free-serum medium with high concentrations of d-glucose (30 mm) and/or insulin (1μM) for 24h after an overnight starving in serum- free medium. The level of hyperglycemia was estimated in the supernatants via GOD-POD method.Results: Serum-free madium with high insulin/ high glucose consentration made the higher level of hypreglycemia in HepG2 cells.Conclusions: Our study interduced high insulin/ high glucose treatment as the best way to induction hyperglycemia.

Inhibitors of the sodium-glucose transporter type 2 and new possibilities for managing vascular age in patients with type 2 diabetes mellitus

The article discusses the pathophysiological mechanisms of the development of vascular aging as a combination of the influence on the  body of  genetic, environmental, regulatory, metabolic and other factors causing biochemical, enzymatic and cellular changes in the arterial vascular bed. The concept of “early vascular aging” and “healthy vascular aging” is defined depending on the ratio of the biological and chronological age of the vessels. The role of diabetes mellitus in increasing vascular stiffness, early vascular aging, as well as the  progression of  atherosclerotic cardiovascular diseases and their complications is considered in detail. Approaches to multifactorial management of vascular age in patients with type 2 diabetes (lifestyle modification with strategy of aggressive treatment of modifiers of atherosclerosis, rejection of bad habits, adherence to dietary recommendations and the use of modern organo- and vasoprotective antidiabetic drugs) are revealed. The mechanism of realization of vasoprotective effects of inhibitors of sodium-glucose transporter-2 (iNGLT-2) is described in detail. The results of completed large random ized trials EMPA-REG Outcome and EMPA-REG BP of the most studied representative of the IGLT-2 group, empagliflozin, are presented. It has been shown that due to their glucose and natriuretic effects, the ability to reduce body weight and blood pressure, improve myocardial metabolism and bioenergetics, decrease the activity of the sympathetic nervous system, as well as positive effects on vascular stiffness, NGLT-2 inhibitors are the drugs of choice in patients with type 2 diabetes mellitus (T2DM) and cardiovascular diseases. This makes it possible to widely use this group of drugs for managing the vascular age of patients and represents a new opportunity in the prevention of vascular aging in T2DM