Olga Bloch

Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-producing RINm Cells

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and [3H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Young patients with both type 1 diabetes mellitus and asthma have a unique IL-12 and IL-18 secretory pattern

Rachmiel M, Bloch O, Shaul AA, Ben‐Yehudah G, Bistritzer Z, Weintrob N, Ofan R, Rapoport MJ. Young patients with both type 1 diabetes mellitus and asthma have a unique IL‐12 and IL‐18 secretory pattern.

GLP-1 Receptor Is Expressed in Human Stomach Mucosa Analysis of Its Cellular Association and Distribution within Gastric Glands

The stomach is a target organ of the incretin hormone glucagon-like peptide-1 (GLP-1). However, the cellular expression and glandular distribution of its receptor (GLP-1R) in human gastric mucosa are not known. We determined the expression of GLP-1R in different regions of human stomach mucosa and its specific cellular association and distribution within gastric glands. Tissue samples from stomach body and antrum were obtained from 20 patients during routine esophagogastroduodenoscopy. mRNA encoding GLP-1R protein expression was evaluated by RT-PCR. Determination of cell types bearing GLP-1R, their localization, and their frequency in gastric glands in different gastric regions were estimated by immunohistochemical morphological analysis. Levels of GLP-1R mRNA were similar in body and antrum. GLP-1R immunoreactivity was found throughout the gastric mucosa in various types of glandular cells. The highest frequency of GLP-1R immunoreactive cells was found in the neck area of the principal glands in cells morphologically identified as parietal cells. GLP-1R immunostaining was also found on enteroendocrine-like cells in the pyloric glands. This study provides the first description of GLP-1R expression in human gastric glands and its specific cellular association. Our data suggest that GLP-1 may act directly on the gastric mucosa to modulate its complex functions.

Sensitivity of HaCat keratinocytes to diabetogenic toxins.

Metabolic, genetic and environmental factors very likely play an important role in the development of skin lesions in diabetes mellitus. While these lesions are involved in secondary diabetes complications, various diabetogenic genotoxic agents may induce direct skin damage. In the present study we examined the potential of known diabetogenic agents (streptozotocin (STZ) and alloxan (AL)), with different mechanisms of action, for induction of direct injury in an immortal human keratinocyte HaCat cell line. In contrast to STZ, which induces alkylation of DNA, a genotoxic effect of AL is achieved through reactive oxygen species. We found that HaCat cells are highly sensitive to STZ, but not to AL. At a concentration of 10mM STZ, cell viability decreased to 32 +/-13% of control (P<0.05), as compared to 82 +/-14% with 10mM of AL. Cells treated with 10 and 20mM STZ showed a significant increase in apoptosis (3.9- and 6.7-fold), but not in necrosis, compared to naive cells (P<0.05). In contrast to STZ, no increase in apoptotic and necrotic cell death was observed after AL treatment. Pretreatment with non-metabolizable 3-O-methyl glucose (3-OMG), which can blockade glucose transporter, or with poly(ADP-ribose) polymerase inhibitors (nicotinamide or 3-aminobenzamide), did not protect keratinocytes from STZ injury. Our results show that STZ, but not AL, is highly toxic to the HaCat cell line. Unlike insulin-producing cells, STZ-induced injury of immortal human keratinocyte HaCat cells is independent of the glucose transporters as well as of the activation of poly(ADP-ribose) polymerase.

Reduced GLP-1R Expression in Gastric Glands of Patients With Type 2 Diabetes Mellitus

BACKGROUND The incretin effect is reduced in type 2 diabetes mellitus (T2DM) patients. Whether the impaired function of the enteropancreatic axis in these patients is due to defective GLP-1 receptor (GLP-1R) expression in extrapancreatic target organs is not known. AIMS AND METHODS To compare the GLP-1R expression and distribution in gastric mucosa biopsies of patients with (n =22) and without (n =22) T2DM referred for routine esophagogastroduodenoscopies. GLP-1R mRNA levels were estimated by real-time PCR. The intensity of GLP-1R immunostaining, frequency, and types of glandular cells bearing GLP-1R and their glandular distribution in different stomach mucosa regions were evaluated by immunohistochemical morphological semiquantitative and quantitative analysis. RESULTS Mean mRNA GLP-1R levels were significantly reduced in patients with T2DM compared with nondiabetic patients (P < .02). Immunohistochemical analysis revealed that the reduced GLP-1R expression in T2DM patients was due to a decreased intensity of immunostaining (P < .01). The number of glandular GLP-1R-bearing cells in both body and antrum mucosa was decreased in T2DM patients. Most notably, the frequency of GLP-1R immunoreactive acid-secreting parietal cells was reduced in the neck area of the gastric principal glands of T2DM patients (P < .01). No correlation was found between the reduced GLP-1R expression and clinical parameters including body mass index, age, glycosylated hemoglobin, and disease duration. CONCLUSION This is the first evidence of reduced GLP-1R expression in gastric glands of T2DM patients. These data demonstrate that the defective function of the incretin axis in T2DM may also result from decreased GLP-1R expression in its extrapancreatic target organs.

A strategy for the engineering of insulin producing cells with a broad spectrum of defense properties

Insulin-producing pancreatic beta cells are known to be extremely susceptible to the oxidative stress and hypoxia generated following islet transplantation in diabetic patients. We hereby present a novel in vivo selection strategy based on the isolation of insulin-producing cells with enhanced protection after repeated rounds of encapsulation and xenotransplantation. Rat insulinoma INS-1 cells were encapsulated in alginate macrobeads and transplanted in the peritoneal cavity of mice. After 2 days the beads were retrieved and cells were recovered from alginate and propagated in vitro until submitted to a second round of encapsulation and transplantation. Three days later, the surviving cells, named INS-1m2, were isolated from the alginate beads and their protection and functional activity examined. Compared to parental INS-1 cells, the selected INS-1m2 cells were more resistant to hydrogen peroxide, nitric oxide, alloxan and hypoxia. This enhanced protection of the selected cells correlated with the increased level of catalase and poly (ADP-ribose) polymerase expression. Although selected cells expressed more insulin than parental cells, no change in their insulin response to glucose was observed. We conclude that the in vivo selection strategy is a powerful tool for the engineering of insulin producing cells with a broad spectrum of defense properties.

Nutrient Induced Type 2 and Chemical Induced Type 1 Experimental Diabetes Differently Modulate Gastric GLP-1 Receptor Expression

T2DM patients demonstrate reduced GLP-1 receptor (GLP-1R) expression in their gastric glands. Whether induced T2DM and T1DM differently affect the gastric GLP-1R expression is not known. This study assessed extrapancreatic GLP-1R system in glandular stomach of rodents with different types of experimental diabetes. T2DM and T1DM were induced in Psammomys obesus (PO) by high-energy (HE) diet and by streptozotocin (STZ) in Sprague Dawly (SD) rats, respectively. GLP-1R expression was determined in glandular stomach by RT PCR and immunohistomorphological analysis. The mRNA expression and cellular association of the GLP-1R in principal glands were similar in control PO and SD rats. However, nutrient and chemical induced diabetes resulted in opposite alterations of glandular GLP-1R expression. Diabetic PO demonstrated increased GLP-1R mRNA expression, intensity of cellular GLP-1R immunostaining, and frequency of GLP-1R positive cells in the neck area of principal glands compared with controls. In contrast, SD diabetic rats demonstrated decreased GLP-1 mRNA, cellular GLP-1R immunoreactivity, and frequency of GLP-1R immunoreactive cells in the neck area compared with controls. In conclusion, nutrient and chemical induced experimental diabetes result in distinct opposite alterations of GLP-1R expression in glandular stomach. These results suggest that induced T1DM and T2DM may differently modulate GLP-1R system in enteropancreatic axis.

Sepsis-induced activation of endogenous GLP-1 system is enhanced in type 2 diabetes

High levels of circulating GLP‐1 are associated with severity of sepsis in critically ill nondiabetic patients. Whether patients with type 2 diabetes (T2D) display different activation of the endogenous GLP‐1 system during sepsis and whether it is affected by diabetes‐related metabolic parameters are not known.

Incretin hormone glucagon-like peptide-1 is increased in patients with acute-phase ST-elevation myocardial infarction treated with a primary percutaneous coronary intervention: a pilot study

ObjectivesThe incretin hormone glucagon-like peptide 1 (GLP-1) is assumed to have a cardioprotective effect. It is not known whether GLP-1 levels are increased in patients with acute myocardial infarction. We investigated the GLP-1 levels in patients presenting with ST-segment elevation myocardial infarction (STEMI). Patients and methodsGLP-1 serum level samples were obtained in 12 consecutive patients presenting with acute STEMI before and 24, 72 h, and 90 days after a percutaneous coronary intervention (PCI). ResultsThe mean GLP-1 levels increased significantly within 24 h after PCI from 27±7.1 to 39.5±11.4 (P<0.04) and reverted to preadmission levels after 3 months. No correlation was found between GLP-1 levels and any of the clinical and laboratory parameters or indicators of myocardial infarction severity. However, both hypertension and smoking history (former and current) were associated with significantly lower GLP-1 levels as compared with normotensive and nonsmoker patients (P<0.01 and P<0.04, respectively). ConclusionA transient and significant increase in GLP-1 levels occurs in patients after STEMI treated with primary PCI. These pilot data may suggest a role for GLP-1 in the physiologic response to acute ischemic heart disease.

Endogenous glucagon-like peptide-1 system response is impaired during ST-elevation myocardial infarction in type 2 diabetes patients

We previously demonstrated increased glucagon‐like peptide‐1 (GLP‐1) secretion during acute ST elevation myocardial infarction (STEMI) in non‐diabetic (ND) patients. Whether the endogenous GLP‐1 system response is different in patients with type 2 diabetes (T2D) during STEMI is unknown. Patients with STEMI (20 ND, 13 T2D) and 3 control groups (non‐STEMI [14 ND, 13 T2D], stable angina pectoris [SAP] [8 ND, 10 T2D] patients and healthy subjects) (n = 25) were studied. Plasma levels of total and active GLP‐1 and soluble dipeptidyl peptidase‐4 (sDPP4) were estimated by enzyme‐linked immunosorbent assay on admission and at 24 and 48 hours after percutaneous coronary intervention in all patients. Sharply elevated levels of total and active GLP‐1 were found in ND STEMI patients at 24 h (P < 0.05 and P < 0.005, respectively), but not in T2D STEMI patients. All patients demonstrated decreased sDPP4 levels compared with healthy controls (P < 0.0005) accompanied by increased active/total GLP‐1 ratio regardless of their ischemic state. These data demonstrate that T2D patients fail to further upregulate their endogenous GLP‐1 system during STEMI. This may underlie their worse cardiovascular outcome.