Faculty Bibliography

Insulin signaling in the central nervous system (CNS) influences satiety, counterregulation, and peripheral insulin sensitivity. However, the broad distribution of insulin receptors (InsR) within neurons and glia has hampered mapping of specific neuronal sub-populations that mediate specific effects of insulin. Neurons expressing glucose transporter Glut4 influence peripheral insulin sensitivity. Here, we analyzed the effects of InsR signaling in hypothalamic Glut4 neurons on glucose sensing as well as leptin and amino acid signaling. We show that InsR signaling in Glut4 neurons dampens the glucagon response to hypoglycemia and 2-deoxyglucose-induced neuroglycopenia. Using immunohistochemistry in chemically identified hypothalamic Glut4 neurons, we show that InsR signaling promotes Akt signaling in response to insulin, leptin, and amino acids in a cell-autonomous fashion (i.e., in Glut4 neurons), but also generates an inhibitory signal to reduce Akt function in non-Glut4-neurons. We conclude that hypothalamic Glut4 neurons modulate the glucagon counterregulatory response, and that InsR signaling in Glut4 neurons is required to integrate hormonal and nutritional cues for the regulation of glucose metabolism

Receptor for Advanced Glycation End products (RAGE) has been implicated in amyloid β-peptide (Aβ)-induced perturbation relevant to the pathogenesis of Alzheimer's disease (AD). However, whether and how RAGE regulates Aβ metabolism remains largely unknown. Aβ formation arises from aberrant cleavage of amyloid pre-cursor protein (APP) by β- and γ-secretase. To investigate whether RAGE modulates β- and γ-secretase activity potentiating Aβ formation, we generated mAPP mice with genetic deletion of RAGE (mAPP/RO). These mice displayed reduced cerebral amyloid pathology, inhibited aberrant APP-Aβ metabolism by reducing β- and γ-secretases activity, and attenuated impairment of learning and memory compared with mAPP mice. Similarly, RAGE signal transduction deficient mAPP mice (mAPP/DN-RAGE) exhibited the reduction in Aβ40 and Aβ42 production and decreased β-and γ-secretase activity compared with mAPP mice. Furthermore, RAGE-deficient mAPP brain revealed suppression of activation of p38 MAP kinase and glycogen synthase kinase 3β (GSK3β). Finally, RAGE siRNA-mediated gene silencing or DN-RAGE-mediated signaling deficiency in the enriched human APP neuronal cells demonstrated suppression of activation of GSK3β, accompanied with reduction in Aβ levels and decrease in β- and γ-secretases activity. Our findings highlight that RAGE-dependent signaling pathway regulates β- and γ-secretase cleavage of APP to generate Aβ, at least in part through activation of GSK3β and p38 MAP kinase. RAGE is a potential therapeutic target to limit aberrant APP-Aβ metabolism in halting progression of AD.

OBJECTIVE: Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block neoangiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. APPROACH AND RESULTS: Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated neoangiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1(early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, neoangiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. CONCLUSIONS: These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease.

In mammals, changes in the metabolic state, including obesity, fasting, cold challenge and high fat diets activate complex immune responses. In many strains of rodents, high fat diets induce a rapid systemic inflammatory response and lead to obesity. Little is known about the molecular signals required for high fat diet (HFD)-induced phenotypes. Here we studied the function of the receptor for advanced glycation products (RAGE) in the development of phenotypes associated with high fat feeding in mice. RAGE is highly expressed on immune cells, including macrophages. High fat feeding induced expression of RAGE ligand HMGB1 and carboxy methyl lysine (CML)-advanced glycation endproducts (AGE) epitopes in liver and adipose tissue. Genetic deficiency of RAGE prevented the effects of HFD on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. RAGE deficiency had no effect on genetic forms of obesity caused by impaired melanocortin signaling. Hematopoietic deficiency of RAGE or treatment with soluble RAGE partially protected against peripheral HFD-induced inflammation and weight gain. These data argue that high fat feeding induces peripheral inflammation and weight gain in a RAGE-dependent manner, providing a foothold in the pathways that regulate diet-induced obesity and offering the potential for therapeutic intervention.

Sustained increases in glucose flux via the aldose reductase (AR) pathway have been linked to diabetic vascular complications. Previous studies revealed that glucose flux via AR mediates endothelial dysfunction and leads to lesional hemorrhage in diabetic human AR (hAR) expressing mice in an apoE(-/-) background. Our studies revealed sustained activation of Egr-1 with subsequent induction of its downstream target genes tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in diabetic apoE(-/-)hAR mice aortas and in high glucose-treated primary murine aortic endothelial cells expressing hAR. Furthermore, we observed that flux via AR impaired NAD(+) homeostasis and reduced activity of NAD(+)-dependent deacetylase Sirt-1 leading to acetylation and prolonged expression of Egr-1 in hyperglycemic conditions. In conclusion, our data demonstrate a novel mechanism by which glucose flux via AR triggers activation, acetylation, and prolonged expression of Egr-1 leading to proinflammatory and prothrombotic responses in diabetic atherosclerosis.

OBJECTIVE: Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)beta, it was logical to investigate the role of PKCbeta in modulation of atherosclerosis in diabetes mellitus. APPROACH AND RESULTS: ApoE(-/-) and PKCbeta(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCbeta(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCbeta reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCbeta and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1beta in U937 macrophages. CONCLUSIONS: These data link enhanced activation of PKCbeta to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCbeta in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCbeta activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Peripheral neuropathy and insensate limbs and digits cause significant morbidity in diabetic individuals. Previous studies showed that deletion of the receptor for advanced end-glycation products (RAGE) in mice was protective in long-term diabetic neuropathy. Here, we tested the hypothesis that RAGE suppresses effective axonal regeneration in superimposed acute peripheral nerve injury attributable to tissue-damaging inflammatory responses. We report that deletion of RAGE, particularly in diabetic mice, resulted in significantly higher myelinated fiber densities and conduction velocities consequent to acute sciatic nerve crush compared with wild-type control animals. Consistent with key roles for RAGE-dependent inflammation, reconstitution of diabetic wild-type mice with RAGE-null versus wild-type bone marrow resulted in significantly improved axonal regeneration and restoration of function. Diabetic RAGE-null mice displayed higher numbers of invading macrophages in the nerve segments postcrush compared with wild-type animals, and these macrophages in diabetic RAGE-null mice displayed greater M2 polarization. In vitro, treatment of wild-type bone marrow-derived macrophages with advanced glycation end products (AGEs), which accumulate in diabetic nerve tissue, increased M1 and decreased M2 gene expression in a RAGE-dependent manner. Blockade of RAGE may be beneficial in the acute complications of diabetic neuropathy, at least in part, via upregulation of regeneration signals.

The multi-ligand receptor RAGE was discovered on account of its ability to bind and transduce the cell stress-provoking signals of advanced glycation endproducts (AGEs). The finding that RAGE also bound pro-inflammatory molecules set the stage for linking RAGE and inflammation to the pathogenesis of diabetic macro- and microvascular complications. In this review, we focus on the roles of RAGE and its ligands in diabetes complications. We recount the findings from mice, rats, swine and human subjects suggesting that RAGE action potently contributes to vascular, inflammatory and end-organ stress and damage in types 1 and 2 diabetes. We detail the efforts to track ligands and RAGE in human subjects with diabetes to address if this axis may be a biomarker reflective of the state of the diabetic complications. Lastly, we suggest specific strategies to tackle AGE-ligand-RAGE interactions as potential therapeutic targets for diabetes and its complications.

Rationale: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. Objective: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. Methods and Results: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3beta, and consequent smooth muscle cell migration. Conclusions: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.