Dysregulation of CXCL1 Expression and Neutrophil Recruitment in Insulin Resistance and Diabetes-Related Periodontitis in Male Mice.

Insulin resistance and hyperglycemia are risk factors for periodontitis and poor wound healing in diabetes, which have been associated with selective loss of insulin activation of the PI3K/Akt pathway in the gingiva. This study showed that insulin resistance in the mouse gingiva due to selective deletion of smooth muscle and fibroblast insulin receptor (SMIRKO mice) or systemic metabolic changes induced by a high-fat diet (HFD) in HFD-fed mice exacerbated periodontitis-induced alveolar bone loss, preceded by delayed neutrophil and monocyte recruitment and impaired bacterial clearance compared with their respective controls. The immunocytokines, CXCL1, CXCL2, MCP-1, TNFα, IL-1ß, and IL-17A, exhibited delayed maximal expression in the gingiva of male SMIRKO and HFD-fed mice compared with controls. Targeted overexpression of CXCL1 in the gingiva by adenovirus normalized neutrophil and monocyte recruitment and prevented bone loss in both mouse models of insulin resistance. Mechanistically, insulin enhanced bacterial lipopolysaccharide-induced CXCL1 production in mouse and human gingival fibroblasts (GFs), via Akt pathway and NF-κB activation, which were reduced in GFs from SMIRKO and HFD-fed mice. These results provided the first report that insulin signaling can enhance endotoxin-induced CXCL1 expression to modulate neutrophil recruitment, suggesting CXCL1 as a new therapeutic direction for periodontitis or wound healing in diabetes. ARTICLE HIGHLIGHTS: The mechanism for the increased risks for periodontitis in the gingival tissues due to insulin resistance and diabetes is unclear. We investigated how insulin action in gingival fibroblasts modulates the progression of periodontitis in resistance and diabetes. Insulin upregulated the lipopolysaccharide-induced neutrophil chemoattractant, CXCL1, production in gingival fibroblasts via insulin receptors and Akt activation. Enhancing CXCL1 expression in the gingiva normalized diabetes and insulin resistance-induced delays in neutrophils recruitment and periodontitis. Targeting dysregulation of CXCL1 in fibroblasts is potentially therapeutic for periodontitis and may also improve wound healing in insulin resistance and diabetes.

Endothelial Cells Induced Progenitors Into Brown Fat to Reduce Atherosclerosis.

BACKGROUND: Insulin resistance (IR) can increase atherosclerotic and cardiovascular risk by inducing endothelial dysfunction, decreasing nitric oxide (NO) production, and accelerating arterial inflammation. The aim is to determine the mechanism by which insulin action and NO production in endothelial cells can improve systemic bioenergetics and decrease atherosclerosis via differentiation of perivascular progenitor cells (PPCs) into brown adipocytes (BAT). METHODS: Studies used various endothelial transgenic and deletion mutant ApoE-/- mice of insulin receptors, eNOS (endothelial NO synthase) and ETBR (endothelin receptor type B) receptors for assessments of atherosclerosis. Cells were isolated from perivascular fat and micro-vessels for studies on differentiation and signaling mechanisms in responses to NO, insulin, and lipokines from BAT. RESULTS: Enhancing insulin's actions on endothelial cells and NO production in ECIRS1 transgenic mice reduced body weight and increased systemic energy expenditure and BAT mass and activity by inducing differentiation of PPCs into beige/BAT even with high-fat diet. However, positive changes in bioenergetics, BAT differentiation from PPCs and weight loss were inhibited by N(gamma)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of eNOS, in ECIRS1 mice and eNOSKO mice. The mechanism mediating NO's action on PPC differentiation into BAT was identified as the activation of solubilized guanylate cyclase/PKGIα (cGMP protein-dependent kinase Iα)/GSK3ß (glycogen synthase kinase 3ß) pathways. Plasma lipidomics from ECIRS1 mice with NO-induced increased BAT mass revealed elevated 12,13-diHOME production. Infusion of 12,13-diHOME improved endothelial dysfunction and decreased atherosclerosis, whereas its reduction had opposite effects in ApoE-/-mice. CONCLUSIONS: Activation of eNOS and endothelial cells by insulin enhanced the differentiation of PPC to BAT and its lipokines and improved systemic bioenergetics and atherosclerosis, suggesting that endothelial dysfunction is a major contributor of energy disequilibrium in obesity.

Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy.

Diabetic nephropathy (DN) arises from systemic and local changes in glucose metabolism and hemodynamics. We have reported that many glycolytic and mitochondrial enzymes, such as pyruvate kinase M2 (PKM2), were elevated in renal glomeruli of DN-protected patients with type 1 and type 2 diabetes. Here, mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated to uncover the renal protective function of PPKM2Tg as a potential therapeutic target that prevented elevated albumin/creatinine ratio (ACR), mesangial expansion, basement membrane thickness, and podocyte foot process effacement after 7 months of streptozotocin-induced (STZ-induced) diabetes. Furthermore, diabetes-induced impairments of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes and clinically correlated with estimated glomerular filtration (GFR) - but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocytes. These findings demonstrate that PKM2 structure and enzymatic activation in podocytes can preserve the entire glomerular mitochondrial function against toxicity of hyperglycemia via paracrine factors such as VEGF and prevent DN progression.

Characterization of Glycolytic Enzymes and Pyruvate Kinase M2 in Type 1 and 2 Diabetic Nephropathy.

OBJECTIVE: Elevated glycolytic enzymes in renal glomeruli correlated with preservation of renal function in the Medalist Study, individuals with ≥50 years of type 1 diabetes. Specifically, pyruvate kinase M2 (PKM2) activation protected insulin-deficient diabetic mice from hyperglycemia-induced glomerular pathology. This study aims to extend these findings in a separate cohort of individuals with type 1 and type 2 diabetes and discover new circulatory biomarkers for renal protection through proteomics and metabolomics of Medalists' plasma. We hypothesize that increased glycolytic flux and improved mitochondrial biogenesis will halt the progression of diabetic nephropathy. RESEARCH DESIGN AND METHODS: Immunoblots analyzed selected glycolytic and mitochondrial enzymes in postmortem glomeruli of non-Medalists with type 1 diabetes (n = 15), type 2 diabetes (n = 19), and no diabetes (n = 5). Plasma proteomic (SOMAscan) (n = 180) and metabolomic screens (n = 214) of Medalists with and without stage 3b chronic kidney disease (CKD) were conducted and significant markers validated by ELISA. RESULTS: Glycolytic (PKM1, PKM2, and ENO1) and mitochondrial (MTCO2) enzymes were significantly elevated in glomeruli of CKD- versus CKD+ individuals with type 2 diabetes. Medalists' plasma PKM2 correlated with estimated glomerular filtration rate (r 2 = 0.077; P = 0.0002). Several glucose and mitochondrial enzymes in circulation were upregulated with corresponding downregulation of toxic metabolites in CKD-protected Medalists. Amyloid precursor protein was also significantly upregulated, tumor necrosis factor receptors downregulated, and both confirmed by ELISA. CONCLUSIONS: Elevation of enzymes involved in the metabolism of intracellular free glucose and its metabolites in renal glomeruli is connected to preserving kidney function in both type 1 and type 2 diabetes. The renal profile of elevated glycolytic enzymes and reduced toxic glucose metabolites is reflected in the circulation, supporting their use as biomarkers for endogenous renal protective factors in people with diabetes.

Exogenous Insulin Infusion Can Decrease Atherosclerosis in Diabetic Rodents by Improving Lipids, Inflammation, and Endothelial Function.

OBJECTIVE: The objective of this study is to evaluate whether exogenously induced hyperinsulinemia may increase the development of atherosclerosis. APPROACH AND RESULTS: Hyperinsulinemia, induced by exogenous insulin implantation in high-fat fed (60% fat HFD) apolipoprotein E-deficient mice (ApoE-/-) mice, exhibited insulin resistance, hyperglycemia, and hyperinsulinemia. Atherosclerosis was measured by the accumulation of fat, macrophage, and extracellular matrix in the aorta. After 8 weeks on HFD, ApoE-/- mice were subcutaneously implanted with control (sham) or insulin pellet, and phlorizin, a sodium glucose cotransporters inhibitor (1/2)inhibitor, for additional 8 weeks. Intraperitoneal glucose tolerance test showed that plasma glucose levels were lower and insulin and IGF-1 (insulin-like growth factor-1) levels were 5.3- and 3.3-fold higher, respectively, in insulin-implanted compared with sham-treated ApoE-/- mice. Plasma triglyceride, cholesterol, and lipoprotein levels were decreased in mice with insulin implant, in parallel with increased lipoprotein lipase activities. Atherosclerotic plaque by en face and complexity staining showed significant reductions of fat deposits and expressions of vascular adhesion molecule-1, tumor necrosis factor-α, interleukin 6, and macrophages in arterial wall while exhibiting increased activation of pAKT and endothelial nitric oxide synthase (P<0.05) comparing insulin-implanted versus sham HFD ApoE-/- mice. No differences were observed in atherosclerotic plaques between phlorizin-treated and sham HFD ApoE-/- mice, except phlorizin significantly lowered plasma glucose and glycated hemoglobin levels while increased glucosuria. Endothelial function was improved only by insulin treatment through endothelial nitric oxide synthase/nitric oxide activations and reduced proinflammatory (M1) and increased anti-inflammatory (M2) macrophages, which were inhibited by endothelial nitric oxide synthase inhibitor. CONCLUSIONS: Exogenous insulin decreased atherosclerosis by lowering inflammatory cytokines, macrophages, and plasma lipids in HFD-induced hyperlipidemia, insulin resistant and mildly diabetic ApoE-/- mice.

High density lipoprotein modulates osteocalcin expression in circulating monocytes: a potential protective mechanism for cardiovascular disease in type 1 diabetes.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of mortality in type 1 diabetes (T1D). A pro-calcific drift of circulating monocytes has been linked to vascular calcification and is marked by the surface expression of osteocalcin (OCN). We studied OCN+ monocytes in a unique population with ≥50 years of T1D, the 50-Year Joslin Medalists (J50M). METHODS: CD45 bright/CD14+/OCN+ cells in the circulating mononuclear blood cell fraction were quantified by flow cytometry and reported as percentage of CD45 bright cells. Mechanisms were studied by inducing OCN expression in human monocytes in vitro. RESULTS: Subjects without history of CVD (n = 16) showed lower levels of OCN+ monocytes than subjects with CVD (n = 14) (13.1 ± 8.4% vs 19.9 ± 6.4%, p = 0.02). OCN+ monocytes level was inversely related to total high density lipoprotein (HDL) cholesterol levels (r = -0.424, p = 0.02), large (r = -0.413, p = 0.02) and intermediate (r = -0.445, p = 0.01) HDL sub-fractions, but not to small HDL. In vitro, incubation with OxLDL significantly increased the number of OCN+ monocytes (p < 0.01). This action of OxLDL was significantly reduced by the addition of HDL in a concentration dependent manner (p < 0.001). Inhibition of the scavenger receptor B1 reduced the effects of both OxLDL and HDL (p < 0.05). CONCLUSIONS: Low OCN+ monocytes levels are associated with lack of CVD in people with long duration T1D. A possible mechanism for the increased OCN+ monocytes could be the elevated levels of oxidized lipids due to diabetes which may be inhibited by HDL. These findings suggest that circulating OCN+ monocytes could be a marker for vascular disease in diabetic patients and possibly modified by HDL elevation.

Inflammatory cytokines and reactive oxygen species as mediators of chronic kidney disease-related vascular calcification.

BACKGROUND: Vascular calcification, a regulated process in chronic kidney disease (CKD), requires vascular smooth muscle cell (VSMC) differentiation into osteoblast-like cells. This phenomenon can be enhanced by inflammatory cytokines and production of reactive oxygen species (ROS). In CKD rats with vascular calcification, we investigated whether inflammatory cytokines, ROS generation, and downstream signaling events are associated with CKD-related vascular calcification. METHODS: CKD was induced in male Wistar rats by renal mass ablation and vascular calcification was induced with a high calcium-phosphate diet and vitamin D supplementation (Ca/P/VitD). At week 3-6, hemodynamic parameters were determined and thoracic aorta was harvested for assessment of vascular calcification, macrophage infiltration, cytokines expression, VSMC differentiation, ROS generation, and related signaling pathway activation. RESULTS: CKD rats treated with Ca/P/VitD developed medial calcification of thoracic aorta and increased pulse pressure and aortic pulse wave velocity. VSMC differentiation was confirmed by increased bone morphogenetic protein-2 and osteocalcin expression and reduced α-smooth muscle actin expression. The expression of interleukin-1ß, interleukin-6, and tumor necrosis factor were also increased. The expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) were increased, whereas the expression of antioxidant enzymes (SOD1, SOD2, Gpx1, and Prdx1) was reduced in CKD + Ca/P/VitD rats. Oxidized peroxiredoxin, a sensor of ROS generation, was significantly increased and ROS-sensitive signaling pathways were activated in the aorta from CKD + Ca/P/VitD rats. CONCLUSION: This study demonstrates a relationship between inflammation/ROS and arterial calcification in CKD and contributes to understanding of the complex pathways that mediate arterial calcification in CKD patients.

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus.

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline-NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.

Reactive oxygen species are required for the hypothalamic osmoregulatory response.

Free radicals, or reactive oxygen species (ROS), are highly reactive byproducts of oxygen degradation. They are well known for their cellular toxicity, but few studies have analyzed their potential role in homeostatic processes. We investigated ROS production and function during the arginine vasopressin (AVP) hypothalamic response to hyperosmolarity. Six-week-old male C3H/HeJ mice were subjected to salt loading for 2 or 8 d. The osmotic axis was progressively activated and reached a new steady-state status at 8 d as demonstrated by monitoring of plasmatic osmolality and c-Fos and AVP expression in the supraoptic nucleus (SON). Free radicals, visualized by dihydroethidine staining and measured by 2'-7'dichlorofluorescein diacetate assays, were detected after 2 d of salt loading. The activity and expression of superoxide dismutase 2 and catalase were concomitantly up-regulated in the SON, suggesting that free radicals are detoxified by endogenous antioxidant systems, thereby avoiding their deleterious effects. The early phase of the osmoregulatory response has been investigated using an acute hyperosmotic model; free radicals were produced 45 min after an ip injection of 1.5 m NaCl. This was followed by an increase in c-Fos and AVP expression and an increase in superoxide dismutase 2 and catalase activities. α-Lipoic acid, a ROS scavenger, administrated during the 3 d before the hypertonic ip injection, abolished the increase of AVP. These findings establish that hyperosmolarity causes ROS production in the SON, which is essential for AVP increase. This demonstrates the importance of free radicals as physiological signaling molecules in the regulation of body-fluid balance.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines.

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100ß expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.