The effect of glucose variability on the risk of microvascular complications in type 1 diabetes.
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Blood Glucose↗
Biomedical subjects
Michael Brownlee
Publications and source records attributed to Michael Brownlee.
Method for chronological recording of antigen appearance in human head-hair shafts and its use for monitoring glycation products in diabetes.
We describe immunochemical assays of non-enzymatic glycation products in human head-hair protein extracts and hair cross sections using Western blots and a novel "dot-block" methodology. In the latter, groups of approximately 15 hair fibers, clipped at about 1 mm proximal to the scalp-skin were aligned, wound around, and attached to 3 mm diameter araldite screw rods. Up to 40 such rods were next embedded lengthwise in additional araldite polymer creating a solid block and the top surface of the block was sectioned off to the half-diameters of the screw rods thus exposing accurately transected hair cross sections at regular ( approximately 0.5 cm) intervals. Early- and advanced-glycation products (EGAs and AGEs, respectively) were determined in the exposed cross sections in-situ using specific antibodies and ECL densitometry as in conventional Western blots. Both Western blots and this technique demonstrated 3.1 fold EGAs increases in the proximal 2 cm of hair of diabetics as compared to non-diabetics. Dot-blocks, in addition, were less variable and demonstrated exponential EGAs decreases along fibers distally, with calculated intercepts (at the hair roots) of 4.9 fold increases in diabetics as opposed to non-diabetics and half-lives of 6.0, 5.9 and 9.0 months in hair of non-diabetics, gestational diabetics and diabetic patients, respectively. Correlations in amounts of BG vs. HbA1(c), BG vs. EGAs, and HbA1(c) vs. EGAs, using dot-block and clinical lab data were all significant (p<0.05). Acute onset T1D patients, defined as previously unsuspected patients diagnosed upon hospitalization due to diabetic complications, exhibited nearly identical EGAs levels in their proximal 0-9 cm hair as did T1D patients with long-established diabetes, thus supporting the notion of long and insidious T1D etiology. Removal of 1-2 microm layers from dot-block surfaces enabled their re-use for multiple assays. Applied anti-AGEs antibodies demonstrated slight decreases or no significant changes in CML and MGI along hair shafts of normal and diabetic subjects. Fluctuations in EGAs and AGEs along hair shafts, indicating alterations in glycemic control were also observed. We conclude that the dot-block method has a potential for early diagnosis and monitoring of diabetes, and more generally, as a long term "biological record" of various chronic medical conditions.
Adult↗
Glycemic variability: a hemoglobin A1c-independent risk factor for diabetic complications.
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Blood Glucose Self-Monitoring↗
Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation.
Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals previously implicated in hyperglycemia-induced vascular damage and inactivated 2 important antiatherogenic enzymes, prostacyclin synthase and eNOS. In 2 nondiabetic rodent models--insulin-resistant, obese Zucker (fa/fa) rats and high-fat diet-induced insulin-resistant mice--inactivation of prostacyclin synthase and eNOS was prevented by inhibition of FFA release from adipose tissue; by inhibition of the rate-limiting enzyme for fatty acid oxidation in mitochondria, carnitine palmitoyltransferase I; and by reduction of superoxide levels. These studies identify what we believe to be a novel mechanism contributing to the accelerated atherogenesis and increased cardiovascular disease risk occurring in people with insulin resistance.
Animals↗
Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription.
Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.
Amino Acid Sequence↗
Tenilsetam prevents early diabetic retinopathy without correcting pericyte loss.
Hyperglycemia-induced mitochondrial overproduction of reactive oxygen species leads to the activation of different biochemical pathways involved in endothelial damage of the diabetic retina. Tenilsetam [(+/-)-3-(2-thienyl)-2-piperazinone] is a dicarbonyl scavenger in the millimolar range and a transition metal ion chelator in the micromolar range. We tested its effect on experimental diabetic retinopathy, and on endothelial cell characteristics in vitro. Streptozotocin diabetic male Wistar rats (60 mg/kg BW) received 50 mg/kg BW tenilsetam (D-T) for 36 weeks, or no treatment (D). The impact of tenilsetam (0-30 mM) on endothelial proliferation, apoptosis, sprouting, cytokine-induced leucocyte-endothelial interaction, and VEGF expression was tested in vitro. Tenilsetam did not affect glycemic control or body weight in diabetic animals. The 3.7 fold increase in acellular capillaries in diabetic rats [p < 0.001 vs. non-diabetic controls (N)] was reduced by 70% (p < 0.001) through treatment, but pericyte loss (D vs. N -33%; p < 0.001) remained unaffected. In vitro, tenilsetam inhibited endothelial proliferation at lower doses, while inducing apoptosis at high doses. Leucocyte adhesion was only inhibited at high doses. Sprouting angiogenesis of bovine retinal endothelial cells was promoted at lower doses (< or = 10 mM). At micromolar concentrations, endothelial VEGF expression was upregulated by 100%. Long-term treatment with the AGE-inhibitor and iron-chelating compound tenilsetam inhibits the formation of acellular capillaries without correcting pericyte loss. The compound has dose-dependent effects on endothelial cell function. These data suggest that, independent of known properties, tenilsetam shows important rescue functions on endothelial cells which could be useful for the treatment of early diabetic retinopathy.
Animals↗
Glycation of mitochondrial proteins from diabetic rat kidney is associated with excess superoxide formation.
Chronic hyperglycemia causes structural alterations of proteins through the Maillard reaction. In diabetes, methylglyoxal (MGO)-induced hydroimidazolones are the predominant modification. In contrast to acute hyperglycemia, mitochondrial respiration is depressed in chronic diabetes. To determine whether MGO-derived protein modifications result in abnormalities in mitochondrial bioenergetics and superoxide formation, proteomics and functional studies were performed in renal cortical mitochondria isolated from rats with 2, 6, and 12 mo of streptozotocin-induced diabetes. MGO-modified proteins belonged to the following two pathways: 1) oxidative phosphorylation and 2) fatty acid beta-oxidation. Two of these proteins were identified as components of respiratory complex III, the major site of superoxide production in health and disease. Mitochondria from rats with diabetes exhibited a diminution of oxidative phosphorylation. A decrease in the respiratory complex III activity was significantly correlated with the quantity of MGO-derived hydroimidazolone present on mitochondrial proteins in both diabetic and control animals. In diabetes, isolated renal mitochondria produced significantly increased quantities of superoxide and showed evidence of oxidative damage. Administration of aminoguanidine improved mitochondrial respiration and complex III activity and decreased oxidative damage to mitochondrial proteins. Therefore, posttranslational modifications of mitochondrial proteins by MGO may represent pathogenic events leading to mitochondria-induced oxidative stress in the kidney in chronic diabetes.
2,4-Dinitrophenol↗
Paradoxical effects of green tea (Camellia sinensis) and antioxidant vitamins in diabetic rats: improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and cross-linking.
We tested the hypothesis that green tea prevents diabetes-related tissue dysfunctions attributable to oxidation. Diabetic rats were treated daily with tap water, vitamins C and E, or fresh Japanese green tea extract. After 12 months, body weights were decreased, whereas glycated lysine in aorta, tendon, and plasma were increased by diabetes (P < 0.001) but unaffected by treatment. Erythrocyte glutathione and plasma hydroperoxides were improved by the vitamins (P < 0.05) and green tea (P < 0.001). Retinal superoxide production, acellular capillaries, and pericyte ghosts were increased by diabetes (P < 0.001) and improved by green tea and the vitamins (P variable). Lens crystallin fluorescence at 370/440 nm was ameliorated by green tea (P < 0.05) but not the vitamins. Marginal effects on nephropathy parameters were noted. However, suppressed renal mitochondrial NADH-linked ADP-dependent and dinitrophenol-dependent respiration and complex III activity were improved by green tea (P variable). Green tea also suppressed the methylglyoxal hydroimidazolone immunostaining of a 28-kDa mitochondrial protein. Surprising, glycoxidation in tendon, aorta, and plasma was either worsened or not significantly improved by the vitamins and green tea. Glucosepane cross-links were increased by diabetes (P < 0.001), and green tea worsened total cross-linking. In conclusion, green tea and antioxidant vitamins improved several diabetes-related cellular dysfunctions but worsened matrix glycoxidation in selected tissues, suggesting that antioxidant treatment tilts the balance from oxidative to carbonyl stress in the extracellular compartment.
Animals↗
The pathobiology of diabetic complications: a unifying mechanism.
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Animals↗
The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species.
Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Here, we analyze adipocytes exposed to hyperglycemia both in vitro as well as in vivo. Comparing cells differentiated at 4 mm to cells differentiated at 25 mm glucose (the standard differentiation protocol) reveals severe insulin resistance in cells exposed to 25 mm glucose. A global assessment of transcriptional changes shows an up-regulation of a number of mitochondrial proteins. Exposure to hyperglycemia is associated with a significant induction of reactive oxygen species (ROS), both in vitro as well as in vivo in adipocytes isolated from streptozotocin-treated hyperglycemic mice. Furthermore, hyperglycemia for a few hours in a clamped setting will trigger the induction of a pro-inflammatory response in adipose tissue from rats that can effectively be reduced by co-infusion of N-acetylcysteine (NAC). ROS levels in 3T3-L1 adipocytes can be reduced significantly with pharmacological agents that lower the mitochondrial membrane potential, or by overexpression of uncoupling protein 1 or superoxide dismutase. In parallel with ROS, interleukin-6 secretion from adipocytes is significantly reduced. On the other hand, treatments that lead to a hyperpolarization of the mitochondrial membrane, such as overexpression of the mitochondrial dicarboxylate carrier result in increased ROS formation and decreased insulin sensitivity, even under normoglycemic conditions. Combined, these results highlight the importance ROS production in adipocytes and the associated insulin resistance and inflammatory response.
3T3-L1 Cells↗
Susceptibility to apoptosis in insulin-like growth factor-I receptor-deficient brown adipocytes.
Fetal brown adipocytes are insulin-like growth factor-I (IGF-I) target cells. To assess the importance of the IGF-I receptor (IGF-IR) in brown adipocytes during fetal life, we have generated immortalized brown adipocyte cell lines from the IGF-IR(-/-) mice. Using this experimental model, we demonstrate that the lack of IGF-IR in fetal brown adipocytes increased the susceptibility to apoptosis induced by serum withdrawal. Culture of cells in the absence of serum and growth factors produced rapid DNA fragmentation (4 h) in IGF-IR(-/-) brown adipocytes, compared with the wild type (16 h). Consequently, cell viability was decreased more rapidly in fetal brown adipocytes in the absence of IGF-IR. Furthermore, caspase-3 activity was induced much earlier in cells lacking IGF-IR. At the molecular level, IGF-IR deficiency in fetal brown adipocytes altered the balance of the expression of several proapoptotic (Bcl-xS and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family. This imbalance was irreversible even though in IGF-IR-reconstituted cells. Likewise, cytosolic cytochrome c levels increased rapidly in IGF-IR-deficient cells compared with the wild type. A rapid entry of Foxo1 into the nucleus accompanied by a rapid exit from the cytosol and an earlier activation of caspase-8 were observed in brown adipocytes lacking IGF-IR upon serum deprivation. Activation of caspase-8 was inhibited by 50% in both cell types by neutralizing anti-Fas-ligand antibody. Adenoviral infection of wild-type brown adipocytes with constitutively active Foxol (ADA) increased the expression of antiapoptotic genes, decreased Bcl-xL and induced caspase-8 and -3 activities, with the final outcome of DNA fragmentation. Up-regulation of uncoupling protein-1 (UCP-1) expression in IGF-IR-deficient cells by transduction with PGC-1alpha or UCP-1 ameliorated caspase-3 activation, thereby retarding apoptosis. Finally, insulin treatment prevented apoptosis in both cell types. However, the survival effect of insulin on IGF-IR(-/-) brown adipocytes was elicited even in the absence of phosphatidylinositol 3-kinase/Akt signaling. Thus, our results demonstrate for the first time the unique role of IGF-IR in maintaining the balance of death and survival in fetal brown adipocytes.
Adenoviridae↗
Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species.
Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-alpha that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.
AMP-Activated Protein Kinases↗
15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB.
Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.
Animals↗
A novel action of minocycline: inhibition of human immunodeficiency virus type 1 infection in microglia.
Human immunodeficiency virus type 1 (HIV-1) infection of the brain produces a characteristic disease called acquired immunodeficiency syndrome (AIDS) dementia in which productive infection and inflammatory activation of microglia and macrophages play a central role. In this report, the authors demonstrate that minocycline (MC), a second-generation tetracycline with proven safety and penetration to the central nervous system, potently inhibited viral production from microglia. Inhibition of viral release was sustained through the entire course of infection and even when the drug exposure was limited to the first day of infection. Minocycline was effective even at low viral doses, and against R5- and X4R5-HIV, as well as in single-cycle reporter virus assays. Electrophoretic mobility shift analysis showed that minocycline inhibited nuclear factor (NF)-kappaB activation in microglia. HIV-1 long terminal repeat (LTR)-promoter activity in U38 cells was also inhibited. These results, combined with recently demonstrated in vivo anti-inflammatory effects of MC on microglia, suggest a potential utility for MC as an effective adjunct therapy for AIDS dementia.
Cell Culture Techniques↗
Uncoupling proteins prevent glucose-induced neuronal oxidative stress and programmed cell death.
The central role of mitochondria in most pathways leading to programmed cell death (PCD) has focused our investigations into the mechanisms of glucose-induced neuronal degeneration. It has been postulated that hyperglycemic neuronal injury results from mitochondria membrane hyperpolarization and reactive oxygen species formation. The present study not only provides further evidence to support our model of glucose-induced PCD but also demonstrates a potent ability for uncoupling proteins (UCPs) to prevent this process. Dorsal root ganglion (DRG) neurons were screened for UCP expression by Western blotting and immunocytochemistry. The abilities of individual UCPs to prevent hyperglycemic PCD were assessed by adenovirus-mediated overexpression of UCP1 and UCP3. Interestingly, UCP3 is expressed not only in muscle, but also in DRG neurons under control conditions. UCP3 expression is rapidly downregulated by hyperglycemia in diabetic rats and by high glucose in cultured neurons. Overexpression of UCPs prevents glucose-induced transient mitochondrial membrane hyperpolarization, reactive oxygen species formation, and induction of PCD. The loss of UCP3 in DRG neurons may represent a significant contributing factor in glucose-induced injury. Furthermore, the ability to prevent UCP3 downregulation or to reproduce the uncoupling response in DRG neurons constitutes promising novel approaches to avert diabetic complications such as neuropathy.
Animals↗
Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy.
Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.
Angiopoietin-1↗
Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy.
Three of the major biochemical pathways implicated in the pathogenesis of hyperglycemia induced vascular damage (the hexosamine pathway, the advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG)-protein kinase C (PKC) pathway) are activated by increased availability of the glycolytic metabolites glyceraldehyde-3-phosphate and fructose-6-phosphate. We have discovered that the lipid-soluble thiamine derivative benfotiamine can inhibit these three pathways, as well as hyperglycemia-associated NF-kappaB activation, by activating the pentose phosphate pathway enzyme transketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars. In retinas of diabetic animals, benfotiamine treatment inhibited these three pathways and NF-kappaB activation by activating transketolase, and also prevented experimental diabetic retinopathy. The ability of benfotiamine to inhibit three major pathways simultaneously might be clinically useful in preventing the development and progression of diabetic complications.
Animals↗
Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells.
Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.
Animals↗