Early diabetes-induced biochemical changes in the retina: comparison of rat and mouse models.

AIMS/HYPOTHESIS: Recently, various transgenic and knock-out mouse models have become available for studying the pathogenesis of diabetic retinopathy. At the same time, diabetes-induced retinal changes in the wild-type mice remain poorly characterised. The present study compared retinal biochemical changes in rats and mice with similar (6-week) durations of streptozotocin-induced diabetes. MATERIALS AND METHODS: The experiments were performed on Wistar rats and C57Bl6/J mice. Retinal glucose, sorbitol, fructose, lactate, pyruvate, glutamate, alpha-ketoglutarate and ammonia were measured spectrofluorometrically by enzymatic methods. Vascular endothelial growth factor (VEGF) protein was assessed by ELISA, and poly(ADP-ribosyl)ation by immunohistochemistry and western blot analysis. Free mitochondrial and cytosolic NAD(+)/NADH ratios were calculated from the glutamate and lactate dehydrogenase systems. RESULTS: Retinal glucose concentrations were similarly increased in diabetic rats and mice, vs controls. Diabetic rats manifested approximately 26- and 5-fold accumulation of retinal sorbitol and fructose, respectively, whereas elevation of both metabolites in diabetic mice was quite modest. Correspondingly, diabetic rats had (1) increased retinal malondialdehyde plus 4-hydroxyalkenal concentrations, (2) reduced superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase and glutathione transferase activities, (3) slightly increased poly(ADP-ribose) immunoreactivity and poly(ADP-ribosyl)ated protein abundance, and (4) VEGF protein overexpression. Diabetic mice lacked these changes. SOD activity was 21-fold higher in murine than in rat retinas (the difference increased to 54-fold under diabetic conditions), whereas other antioxidative enzyme activities were 3- to 10-fold lower. With the exception of catalase, the key antioxidant defence enzyme activities were increased, rather than reduced, in diabetic mice. Diabetic rats had decreased free mitochondrial and cytosolic NAD(+)/NADH ratios, consistent with retinal hypoxia, whereas both ratios remained in the normal range in diabetic mice. CONCLUSIONS/INTERPRETATION: Mice with short-term streptozotocin-induced diabetes lack many biochemical changes that are clearly manifest in the retina of streptozotocin-diabetic rats. This should be considered when selecting animal models for studying early retinal pathology associated with diabetes.

Gastric distension-induced pyloric relaxation: central nervous system regulation and effects of acute hyperglycaemia in the rat.

1. The pylorus plays an important role in the regulation of gastric emptying. In addition to the autonomic neuropathy associated with long-standing diabetes, acute hyperglycaemia per se has effects on gastric emptying. In this study, the role of the central nervous system in modulating the effects of hyperglycaemia on gastric distension-induced pyloric relaxation was investigated. 2. Gastric distension-induced pyloric relaxation was significantly reduced by subdiaphragmatic vagotomy, hexamethonium (20 mg kg(-1)) and N (G)-nitro-L-arginine methyl ester (L-NAME; 10 mg kg(-1)), a nitric oxide synthase (NOS) biosynthesis inhibitor, in anaesthetized rats. In contrast, neither splanchnectomy nor guanethidine (5 mg kg(-1)) had an effect. 3. An intravenous (I.V.) infusion of D-glucose (20 %) for 30 min, which increased blood glucose concentrations from 5.4 to 12.8 mM, significantly inhibited gastric distension-induced pyloric relaxation. 4. An intracerebroventricular (I.C.V.) injection of D-glucose (3 micromol) also significantly inhibited gastric distension-induced pyloric relaxation without affecting peripheral blood glucose concentrations. 5. I.V. infusion of D-glucose significantly elevated hypothalamic neuropeptide Y (NPY) concentrations. 6. Intracerebroventricular (I.C.V.) administration of NPY (0.03--3 nmol) and a Y1 receptor agonist, [leu(31), pro(34)] NPY (0.03--3 nmol), significantly inhibited gastric distension-induced pyloric relaxation in a dose-dependent manner. 7. I.C.V. administration of a Y1 receptor antagonist, BIBP 3226 (30 nmol), and of a NPY antibody (titre 1:24 000, 3 microl) abolished the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation. 8. Taken together, these findings suggest that gastric distension-induced pyloric relaxation is mediated via a vago-vagal reflex and NO release. Acute hyperglycaemia stimulates hypothalamic NPY release, which, acting through the Y1 receptor, inhibits gastric distension-induced pyloric relaxation in rats exposed to acute elevations in blood glucose concentrations.

Effects of insulin-like growth factor-1 on retinal endothelial cell glucose transport and proliferation.

Insulin-like growth factor-1 (IGF-1) plays important roles in the developing and mature retina and in pathological states characterized by retinal neovascularization, such as diabetic retinopathy. The effects of IGF-1 on glucose transport and proliferation and the signal transduction pathways underlying these effects were studied in a primary bovine retinal endothelial cell (BREC) culture model. IGF-1 stimulated uptake of the glucose analog 2-deoxyglucose in a dose-dependent manner, with a maximal uptake at 25 ng/mL (3.3 nM) after 24 h. Increased transport occurred in the absence of an increase in total cellular GLUT1 transcript or protein. IGF-1 stimulated activity of both protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase), and both pathways were required for IGF-1-mediated BREC glucose transport and thymidine incorporation. Use of a selective inhibitor of the beta isoform of PKC, LY379196, revealed that IGF-1 stimulation of glucose transport was mediated by PKC-beta; however, inhibition of PKC-beta had no effect on BREC proliferation. Taken together, these data suggest that the actions of IGF-1 in retinal endothelial cells couple proliferation with delivery of glucose, an essential metabolic substrate. The present studies extend our general understanding of the effects of IGF-1 on vital cellular activities within the retina in normal physiology and in pathological states such as diabetic retinopathy.

Co-localization of GLUT1 and GLUT4 in the blood-brain barrier of the rat ventromedial hypothalamus.

The ventromedial hypothalamus (VMH) has been proposed to be a glucose sensor within the brain and appears to play a critical role in initiating the counterregulatory response to hypoglycemia. Transport of glucose across the brain capillaries and into neurons in this region is mediated by different isoforms of the sodium-independent glucose transporter gene family. The objective of the present study was to identify the specific glucose transporter isoforms present, as well as their cellular localization, within the VMH. Immunohistochemistry was performed for GLUT1, GLUT2 and GLUT4 in frozen sections of hypothalami from normal rats. GLUT1 was present on the endothelial cells of the blood-brain barrier (BBB) of the VMH. GLUT2 immunoreactivity was seen in the ependymal cells of the third ventricle and in scattered cells in the arcuate and periventricular nuclei. There was no GLUT2 expression in the VMH. The insulin-sensitive GLUT4 isoform was localized to vascular structures within the VMH. Double-labeled immunohistochemistry demonstrated co-localization of GLUT4 with GLUT1 and with the tight junction protein ZO-1 in the VMH and suggested that VMH GLUT4 expression was restricted to the BBB. The role of GLUT4 in the brain and within the VMH is unknown, but given its location on the BBB, it may participate in brain sensing of blood glucose concentrations.

Enhancement of glucose transport by vascular endothelial growth factor in retinal endothelial cells.

PURPOSE: To investigate effects of vascular endothelial growth factor (VEGF) on glucose transport and GLUT1 glucose transporter expression in primary bovine retinal endothelial cell (BREC) cultures. METHODS: Glucose transport in control and VEGF-treated BREC cultures was determined by measurement of [14C]-3-O-methylglucose (3MG) uptake. GLUT1 protein and mRNA was determined by Western and Northern blot analyses, respectively. Protein kinase C (PKC) activity was measured in control and VEGF-treated cultures, and glucose transport was determined with and without prior PKC depletion and PKC inhibition. RESULTS: Dose-dependent increases in 3MG uptake were seen in the VEGF-treated cultures, with an increase of 69% after a 24-hour exposure to 50 ng/ml VEGF (P < 0.001). Total cellular GLUT1 mRNA or protein, however, was unchanged. Western blot analysis of plasma membrane fractions revealed a 75% increase in plasma membrane GLUT1 in VEGF-treated cultures (P = 0.02), suggesting that the VEGF-stimulated increase in glucose transport was due to a translocation of GLUT1 to the cell membrane. VEGF stimulated a 90% increase in PKC activity in membrane fractions from cultures treated with VEGF, and VEGF-stimulated enhancement of glucose transport was abolished by cellular PKC depletion and by general and PKC beta inhibition. CONCLUSIONS: The present study demonstrates VEGF-mediated enhancement of retinal endothelial cell glucose transport and suggests that this increase is due to PKC beta-mediated translocation of cytosolic GLUT1 to the plasma membrane surface. Upregulation of retinal endothelial cell glucose transport by various factors associated with the development of retinopathy may be responsible for the metabolic derangements observed in the diabetic inner blood-retinal barrier in vivo.

Glucose transport in brain and retina: implications in the management and complications of diabetes.

Neural tissue is entirely dependent on glucose for normal metabolic activity. Since glucose stores in the brain and retina are negligible compared to glucose demand, metabolism in these tissues is dependent upon adequate glucose delivery from the systemic circulation. In the brain, the critical interface for glucose transport is at the brain capillary endothelial cells which comprise the blood-brain barrier (BBB). In the retina, transport occurs across the retinal capillary endothelial cells of the inner blood-retinal barrier (BRB) and the retinal pigment epithelium of the outer BRB. Because glucose transport across these barriers is mediated exclusively by the sodium-independent glucose transporter GLUT1, changes in endothelial glucose transport and GLUT1 abundance in the barriers of the brain and retina may have profound consequences on glucose delivery to these tissues and major implications in the development of two major diabetic complications, namely insulin-induced hypoglycemia and diabetic retinopathy. This review discusses the regulation of brain and retinal glucose transport and glucose transporter expression and considers the role of changes in glucose transporter expression in the development of two of the most devastating complications of long-standing diabetes mellitus and its management.

Pathological upregulation of inner blood-retinal barrier Glut1 glucose transporter expression in diabetes mellitus.

The pathological changes in the retinal microvasculature characteristic of diabetic retinopathy (DR) are the result of chronic exposure to elevated blood glucose. Since glucose entry into the microvascular endothelial cells comprising the inner blood-retinal barrier (BRB) is mediated by the GLUT1 glucose transporter, changes in GLUT1 expression on the inner BRB in long-standing diabetes mellitus may have a direct impact on the subsequent development of retinopathic changes. In the present study, quantitative immunogold electron microscopy for GLUT1 was employed on ultrathin cross-sections of postmortem retina specimens from 3 individuals with long-standing diabetes and minimal or no clinical retinopathy and from 2 non-diabetic individuals without ocular disorders. In the non-diabetic retinal capillaries, GLUT1 immunogold was distributed asymmetrically between the lumenal and ablumenal membranes with a lumenal-to-ablumenal ratio of 1 to 1.7. In the diabetic microvessels, a bimodal distribution pattern of GLUT1 immunoreactivity was observed. In 17 of 40 of the diabetic microvessels examined, the density and distribution of GLUT1 was no different from that of the non-diabetic vessels; however, in a subpopulation of the diabetic microvessels (23 of 40), a dramatic increase in GLUT1 immunoreactivity on the lumenal and albumenal membrane and in the cytoplasm was noted. On the lumenal membrane, the increased expression of immunoreactive GLUT1 was more than 18 times that of the non-diabetic microvessels. These findings demonstrate that localized upregulation of GLUT1 expression at the inner BRB occurs in long-standing diabetes mellitus with minimal or no clinical retinopathy and suggest that this upregulation may serve to amplify the deleterious effects of chronic hyperglycemia on the retinal microvasculature.

Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia.

An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.

Differential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexus.

The sodium-independent GLUT1 glucose transporter is expressed in high density in human erythrocytes and in tissues which serve a barrier function. In the polarized endothelial cells of the brain capillaries, which comprise the blood-brain barrier (BBB), GLUT1 is expressed on both apical and basolateral membranes; however, in the epithelium of the choroid plexus, GLUT1 expression is restricted to the basolateral surface. The present study examined whether these differences in subcellular localization of GLUT1 at the BBB and choroid plexus could be correlated with differential N-linked or O-linked glycosylation of the protein. Western blot analysis of solubilized brain capillaries (BC) and choroid plexus (CP) revealed that while the BC GLUT1 had an average molecular mass identical to that of the purified human erythrocyte transporter (54 kDa), the CP GLUT1 was of lower molecular mass (47 kDa). Treatment of brain capillaries and choroid plexus with N-glycanase resulted in a shift in the mobility of the GLUT1 of both samples to a lower molecular mass of 42 kDa; however, in contrast, treatment with O-glycanase produced no change in the mobility patterns of GLUT1, but did result in O-linked deglycosylation of another BBB marker, gamma-glutamyl transpeptidase. In conclusion, BBB and choroid plexus GLUT1 are subject to differential N-linked glycosylation with the protein having an N-linked carbohydrate side chain of higher molecular mass at the BBB in comparison to the choroid plexus.

GLUT1 glucose transporter expression in the diabetic and nondiabetic human eye.

PURPOSE: The GLUT1 glucose transporter is expressed in endothelial and epithelial barriers, including the retinal capillary endothelium and the retinal pigment epithelium (RPE) of the eye. The present studies were undertaken to determine whether GLUT1 is expressed in additional cell types within the human eye and whether retinal endothelial GLUT1 is aberrantly expressed in diabetic proliferative retinopathy in humans. METHODS: Immunohistochemical staining of sections of human eyes obtained at surgery or autopsy from patients with and without diabetes was performed with polyclonal antisera directed against the human GLUT1 glucose transporter. RESULTS: In the course of this study, an unexpected multicellular localization of GLUT1 in different cellular barriers of the human eye was observed. In the nondiabetic eye, specific staining for GLUT1 was seen in the nerve fiber layer, the ganglion and photoreceptor cell bodies, the capillaries and the RPE of the retina, the basal infoldings of the pigmented and nonpigmented layers of the ciliary body, the capillary endothelium and posterior epithelium of the iris, the corneal epithelium and endothelium, and the endothelium lining of the canal of Schlemm. Müller cells, a type of retinal glial cell identified by morphology and by parallel staining for glial fibrillary acidic protein, also stained intensely positive for GLUT1. The pattern of GLUT1 immunoreactivity in the diabetic eyes was virtually identical to that in the nondiabetic specimens, with the notable exception that the neovascular endothelium of proliferative retinopathy did not stain for GLUT1. CONCLUSIONS: These studies describe the heretofore unrecognized expression of immunoreactive GLUT1 in the ganglion cell layer of the retina, the endothelium lining the canal of Schlemm, the corneal endothelium, and the basal cells of the corneal epithelium of the human eye. The present study also provides evidence for immunoreactive GLUT1 in glial cells of the central nervous system. Because the expression of GLUT1 is characteristic of tissues that possess a barrier function, the absence of GLUT1 immunoreactivity in the neovascular tissue of proliferative diabetic retinopathy suggests that the loss of selective permeability is associated with an absence of facilitated glucose transport in this disorder.

Absorptive-mediated endocytosis of cationized albumin and a beta-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport.

Cationized albumin (pI greater than 8), unlike native albumin (pI approximately 4), enters cerebrospinal fluid (CSF) rapidly from blood. This suggests that a specific uptake mechanism for cationized albumin may exist at the brain capillary wall, i.e. the blood-brain barrier. Isolated bovine brain capillaries rapidly bound cationized [3H]albumin and approximately 70% of the bound radioactivity was resistant to mild acid wash, which is assumed to represent internalized peptide. Binding was saturable and a Scatchard plot gave a maximal binding capacity (Ro) = 5.5 +/- 0.7 micrograms/mgp (79 +/- 10 pmol/mgp), and a half-saturation constant (KD) = 55 +/- 8 micrograms/ml (0.8 +/- 0.1 microM). The binding of cationized [3H]albumin (pI = 8.5-9) was inhibited by protamine, protamine sulfate, and polylysine (molecular weight = 70,000) with a Ki of approximately 3 micrograms/ml for all three proteins. The use of cationized albumin in directed delivery of peptides through the blood-brain barrier was examined by coupling [3H]beta-endorphin to unlabeled cationized albumin (pI = 8.5-9) using the bifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)proprionate. The [3H]beta-endorphin-cationized albumin chimeric peptide was rapidly bound and endocytosed by isolated bovine brain capillaries, and this was inhibited by unlabeled cationized albumin but not by unconjugated beta-endorphin or native bovine albumin. Cationized albumin provides a new tool for studying absorptive-mediated endocytosis at the brain capillary and may also provide a vehicle for directed drug delivery through the blood-brain barrier.

Chimeric peptides as a vehicle for peptide pharmaceutical delivery through the blood-brain barrier.

A new strategy for peptide delivery through the brain capillary wall, i.e., the blood-brain barrier (BBB), is the synthesis of chimeric peptides which are formed by the covalent coupling of a non-transportable peptide (e.g., beta-endorphin) to a transportable peptide that undergoes receptor- or absorptive-mediated transcytosis at the BBB. beta-endorphin was covalently coupled via disulfide linkage to cationized albumin (pI greater than or equal to 9) which, owing to it's highly basic charge, undergoes rapid absorptive-mediated transport into brain from blood. The [3H]labeled beta-endorphin-cationized albumin chimera was rapidly taken up by isolated brain capillaries in vitro and by rat brain in vivo; conversely, the BBB uptake of native [3H]beta-endorphin was negligible. The synthesis of chimeric peptides is a new strategy for solving the problem of peptide delivery through the BBB.