Henrik Ortsäter

Differentiating between effects of streptozotocin per se and subsequent hyperglycemia on renal function and metabolism in the streptozotocin-diabetic rat model

The animal model with streptozotocin (STZ)‐induced diabetes mellitus is associated with progressive renal disturbances. The aim of this study was to differentiate between toxic effects of STZ and the effect of hyperglycemia. Previous studies have been limited to investigating the influence of STZ on glomerular filtration rate (GFR), albuminuria and renal morphology. The present study presents a new approach when transplanting β‐cells to cure the STZ‐treated animals and extends the evaluation to include both renal function and oxygen metabolism.

Diazoxide-induced β-cell rest reduces endoplasmic reticulum stress in lipotoxic β-cells

Elevated levels of glucose and lipids are characteristics of individuals with type 2 diabetes mellitus (T2DM). The enhanced nutrient levels have been connected with deterioration of beta-cell function and impaired insulin secretion observed in these individuals. A strategy to improve beta-cell function in individuals with T2DM has been intermittent administration of K(ATP) channel openers. After such treatment, both the magnitude and kinetics of insulin secretion are markedly improved. In an attempt to further delineate mechanisms of how openers of K(ATP) channels improve beta-cell function, the effects of diazoxide on markers of endoplasmic reticulum (ER) stress was determined in beta-cells exposed to the fatty acid palmitate. The eukaryotic translation factor 2-alpha kinase 3 (EIF2AK3; also known as PERK) and endoplasmic reticulum to nucleus signaling 1 (ERN1; also known as IRE1) pathways, but not the activating transcription factor (ATF6) pathway of the unfolded protein response, are activated in such lipotoxic beta-cells. Inclusion of diazoxide during culture attenuated activation of the EIF2AK3 pathway but not the ERN1 pathway. This attenuation was associated with reduced levels of DNA-damage inducible transcript 3 (DDIT3; also known as CHOP) and beta-cell apoptosis was decreased. It is concluded that reduction of ER stress may be a mechanism by which diazoxide improves beta-cell function.

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets.

Aims/hypothesis. The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO2) and insulin release from individual islets of Langerhans.¶Methods. Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and pO2 with a modified Clark-type electrode inserted into the islets.¶Results. In the presence of 3 mmol/l d-glucose, pO2 was 102 ± 9 mmHg and oscillatory (0.26 ± 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 ± 0.02 oscillations/min). When the d-glucose concentration was increased to 11 mmol/l, pO2 decreased by 30 % to 72 ± 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 ± 2 to 131 ± 16 pmol · g–1· s–1 leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO2 varied between islets but was positively correlated to the amount of insulin released (r2 = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO2 was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l d-glucose, neither pO2 nor insulin release of the islets were changed. Temporal analysis of oscillations in pO2 and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release.¶Conclusion/interpretation. The temporal relation between oscillations in pO2 and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release. [Diabetologia (2000) 43: 1313–1318]

Regulation of 11β-hydroxysteroid dehydrogenase type 1 and glucose-stimulated insulin secretion in pancreatic islets of Langerhans

In rodents, the enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) converts inactive 11‐dehydrocorticosterone (DHC) into active corticosterone. The mRNA and activity of 11β‐HSD1 have been shown to be present in batch‐incubated pancreatic islets from the ob/ob mouse. In other tissues, 11β‐HSD1 expression has been demonstrated to be regulated by glucocorticoids. In the present study, the influence of DHC on 11β‐HSD1 levels and glucose‐induced changes in insulin secretion were studied in pancreatic islets isolated from the ob/ob mouse.

Short-term glucocorticoid treatment increases insulin secretion in islets derived from lean mice through multiple pathways and mechanisms.

Chronic exposure to elevated levels of glucocorticoids leads to metabolic dysfunctions with hyperglycemia and insulin resistance. Long-term treatment with glucocorticoids induces severe impairment of glucose-stimulated insulin secretion. We analyzed the effects of short-, and medium-term (2-120h) treatment with 50-200nM glucocorticoids on primary pancreatic islet cultures derived from lean C57BL/6J mice. In contrast to animal models of insulin resistance, beta-cells from lean mice respond with an increased glucose-stimulated insulin secretion, with a peak effect around 18-24h of treatment. Analyses of the insulin secretion response reveal that early and late phase responses are dissociated upon glucocorticoid treatment. Whereas late phase responses return to basal levels after long treatment, early phase responses remain increased over several days. Increased insulin secretion is also obtained by incubation with the inactive glucocorticoid dehydrocorticosterone, pointing to an important role of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 in mediating glucocorticoid effects in beta-cells. Transcript profiling revealed differential regulation of genes involved in mediation of signal transduction, insulin secretion, stress and inflammatory responses. The results show that short- to medium-term glucocorticoid treatment of pancreatic islets derived from lean mice leads to an increased insulin release and may constitute an important parameter in changing towards a pro-diabetic phenotype.

Protein profiling of pancreatic islets.

The insulin-producing β cell in the islet of Langerhans is central in glucose homeostasis. Its dysfunction is part of the pathogenesis of both Type 1 and 2 diabetes mellitus. In both forms of the disease, there is a cytotoxic component either induced by cytokines, as in Type 1 diabetes, or by elevated levels of glucose and fatty acids, as in Type 2 diabetes. To find the mechanisms responsible for the cytotoxic effects of these compounds proteomic approaches with 2D gel electrophoresis and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry have been undertaken. In this article, we describe these methods, and other methodological aspects of protein profiling of pancreatic islets, and summarize the results obtained with these methods.

Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.Mechanical cues are known to influence endothelial cell behavior. Here Frye et al. show that lymphatic endothelial cell progenitors experience varying degrees of matrix stiffness during development, and that matrix stiffness regulates GATA2 expression to drive lymphatic vessel formation.

Smooth muscle cell recruitment to lymphatic vessels requires PDGFB and impacts vessel size but not identity

Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of Pdgfb prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly, Pdgfb overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin. Summary: Pdgfb mutant mice provide insight into the recruitment and function of smooth muscle cells in the lymphatic vasculature, and shed new light on mechanisms of lymph vessel-associated diseases.

Dynamic measurements of oxygen tension in islets of Langerhans.

Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973). With the development of a sensitive assay for insulin it was possible to characterize the dynamics of glucose-induced insulin release from the individual isolated islet of Langerhans (Bergsten and Hellman, 1993). To evaluate the role of the oxidative metabolism for the kinetics of insulin release a method for determining oxidative metabolism with a sensitivity comparable to that available for insulin is crucial. Glucose oxidation and oxygen consumption have been used to monitor glucose-induced metabolic changes (Ashcroft et al., 1973; Hutton and Malaisse, 1980; Longo et al., 1991). These studies indicate that insulin release and cellular metabolism, oxidation in particular, are tightly linked. However, the previously used techniques lack the sensitivity required to monitor the dynamics of metabolism from individual islets. Thus analysis of the coupling or synchronization between metabolic changes and secretion is hampered. To overcome this problem we have developed a new sensitive technique to monitor oxygen tension, which allows time-resolved measurements from individual islets, and characterized the responses in pO2 following exposure to glycolytic and mitochondrial substrates.