I-Hsien Wu

Loss of Insulin Signaling in Vascular Endothelial Cells Accelerates Atherosclerosis in Apolipoprotein E Null Mice

To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls but reduced to below control values by a VCAM-1-blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.

Inhibition of Insulin Signaling in Endothelial Cells by Protein Kinase C-induced Phosphorylation of p85 Subunit of Phosphatidylinositol 3-Kinase (PI3K)

Background: Insulin actions are decreased in endothelial cells causing vascular dysfunction in diabetic and insulin-resistant states. Results: IRS2 and p85 subunit of PI3K are targets of PKC and angiotensin activation inhibiting insulin signaling. Conclusion: PKC and angiotensin activation inhibit selective insulin activation of Akt/eNOS in endothelial cells. Significance: We provide a biochemical mechanism by which PKC activation inhibits insulin signaling and protective actions in endothelial cells. The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulins activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (ł50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Characterization of Circulating and Endothelial Progenitor Cells in Patients With Extreme-Duration Type 1 Diabetes

OBJECTIVE We characterized and correlated endothelial progenitor cells (EPCs) and circulating progenitor cells (CPCs) with lack of vascular complications in the Joslin Medalist Study in patients with type 1 diabetes for 50 years or longer. RESEARCH DESIGN AND METHODS EPC and CPC levels were ascertained by flow cytometry and compared among Medalists (n = 172) with or without diabetic retinopathy (DR; n = 84 of 162), neuropathy (n = 94 of 165), diabetic nephropathy (DN; n = 18 of 172), cardiovascular disease (CVD; n = 63 of 168), age-matched controls (n = 83), type 2 diabetic patients (n = 36), and younger type 1 diabetic patients (n = 31). Mitogens, inflammatory cytokines, and oxidative markers were measured in blood or urine. Migration of cultured peripheral blood mononuclear cells (PBMCs) from Medalists and age-matched controls were compared. RESULTS Medalists’ EPC and CPC levels equaled those of their nondiabetic age-matched controls, were 10% higher than those in younger type 1 diabetic patients, and were 20% higher than those in age-matched type 2 diabetic patients. CPC levels were 15% higher in Medalists without CVD and nephropathy than in those affected, whereas EPC levels were significantly higher in those without peripheral vascular disease (PVD) than those with PVD. Stromal-derived factor 1 (SDF-1) levels were higher in Medalists with CVD, DN, and DR than in those not affected and their controls. IGF-I levels were lower in Medalists and correlated inversely with CPC levels. Additionally, cultured PBMCs from Medalists migrated more than those from nondiabetic controls. CONCLUSIONS Normal levels of EPC and CPC in the Medalists, unlike other groups with diabetes, especially those without CVD, support the idea that endogenous factors exist to neutralize the adverse effects of metabolic abnormalities of diabetes on vascular tissues.

Beta 2-adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications

Macrophage activation is increased in diabetes and correlated with the onset and progression of vascular complications. To identify drugs that could inhibit macrophage activation, we developed a cell-based assay and screened a 1,040 compound library for anti-inflammatory effects. Beta2-adrenergic receptor (β2AR) agonists were identified as the most potent inhibitors of phorbol myristate acetate-induced tumor necrosis factor-α production in rat bone marrow macrophages. In peripheral blood mononuclear cells isolated from streptozotocin-induced diabetic rats, β2AR agonists inhibited diabetes-induced tumor necrosis factor-α production, which was prevented by co-treatment with a selective β2AR blocker. To clarify the underlying mechanisms, THP-1 cells and bone marrow macrophages were exposed to high glucose. High glucose reduced β-arrestin2, a negative regulator of NF-κB activation, and its interaction with IκBα. This subsequently enhanced phosphorylation of IκBα and activation of NF-κB. The β2AR agonists enhanced β-arrestin2 and its interaction with IκBα, leading to downregulation of NF-κB. A siRNA specific for β-arrestin2 reversed β2AR agonist-mediated inhibition of NF-κB activation and inflammatory cytokine production. Treatment of Zucker diabetic fatty rats with a β2AR agonist for 12 weeks attenuated monocyte activation as well as pro-inflammatory and pro-fibrotic responses in the kidneys and heart. Thus, β2AR agonists might have protective effects against diabetic renal and cardiovascular complications.

Protection from Severe Periodontal Disease among People with Type 1 Diabetes with Duration of 50 Years or Longer

Periodontal disease (PD) is more common and severe in people with diabetes than the general population. The presence of severe PD is correlated with decreased survival, since it can potentially affect glycemic control and severity of complications in people with diabetes. We have reported that Medalists (people with T1DM of 50 years or longer duration) may have endogenous protective factors for the development of diabetic nephropathy (DN) and retinopathy (DR). This study assessed the prevalence of PD in the Medalist cohort and correlated it to the known risk factors of PD and diabetic complications. Severity of PD was defined in a subset (n=170) of Medalists with comparable characteristics of the whole cohort, according to the AAP criteria. The prevalence of severe PD was dramatically decreased in Medalists (13.5%) compared with other studies of people with diabetes of similar age, approximately 33%. Clinical parameters, such as male gender, chronological age, age at diagnosis, and total insulin dose were correlated positively with severity of PD (p=0.04, 0.01, 0.03, and 0.02, respectively), while duration of disease, hemoglobin A1c, BMI, and lipid profiles did not exhibit any correlation. Interestingly, detectible plasma C-peptide levels correlated inversely with severity of PD (p=0.04). Systemic inflammatory markers, such as plasma IL-6, clearly correlated positively with the severity of PD (p=0.01). Serum antibody titer against Porphyromonas gingivalis (Pg), a known pathogen of PD, trended with severity of PD (p=0.08). Amongst the various complications, only the prevalence of CVD correlated positively with severity of PD (p=0.02). These results suggest that Medalists are protected from severe PD even with hyperglycemia. The endogenous protective factors for PD could be similar to those for CVD, possibly related to endogenously produced insulin to neutralize the chronic inflammation caused by residual infection with Pg in the gingival tissues. Disclosure T. Shinjo: None. A. Ishikado: Employee; Self; Sunstar Inc.. Employee; Spouse/Partner; Sunstar Inc.. H. Hasturk: None. L.J. Tinsley: None. D.M. Pober: None. I. Wu: None. T.E. Van Dyke: None. R.J. Genco: None. G.L. King: Research Support; Self; Sanofi-Aventis.