Age-Dependent Changes of Adipokine and Cytokine Secretion From Rat Adipose Tissue by Endogenous and Exogenous Toll-Like Receptor Agonists.

White adipose tissue but recently also brown adipose tissue have emerged as endocrine organs. Age-associated obesity is accompanied by prolonged and elevated lipopolysaccharide (LPS)-induced sickness symptoms and increased cytokine and adipokine levels in the circulation partially originating from adipose tissue. In the present study, ex vivo fat explants were used to investigate how the exogenous pathogen-associated molecular pattern (PAMP) LPS or the endogenous danger-associated molecular patterns (DAMPs) high mobility group box-1 protein (HMGB1) and biglycan modulate the release of cytokines and adipokines/batokines and, thus, could influence systemic and/or local inflammation. The response of adipose tissue (epididymal, retroperitoneal, subcutaneous, and brown) was compared between young lean and old obese rats (2 vs. 24 months old). LPS induced a strong interleukin (IL)-6 and tumor necrosis factor (TNF) alpha release into the supernatant of all adipose tissue types investigated. HMGB1 (subcutaneous) and biglycan (retroperitoneal) led to an increased release of IL-6 and TNFalpha (HMGB1) and decreased visfatin and adiponectin (biglycan) secretion from epididymal adipose tissue (young rats). Visfatin was also decreased by HMGB1 in retroperitoneal adipose tissue of old rats. We found significantly higher leptin (all fat pads) and adiponectin (subcutaneous) levels in supernatants of adipose tissue from old compared to young rats, whereas visfatin secretion showed the opposite. The expression of the biglycan receptor Toll-like receptor (TLR) 2 as well as the LPS and HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE) were reduced with age (TLR4/RAGE) and by stimulation with their ligands (subcutaneous). Overall, we revealed that adipokines/adipose-tissue released cytokines show some modulation of their release caused by mediators of septic (batokines) and sterile inflammation with potential implication for acute and chronic disease. Moreover, aging may increase or decrease the release of fat-derived mediators. These data show that DAMPS and LPS locally modulate cytokine secretion while only DAMPS but not LPS can locally alter adipokine secretion during inflammation.

Advanced glycation end products evoke inflammatory reactions in proximal tubular cells via autocrine production of dipeptidyl peptidase-4.

We have previously shown that albuminuria and renal levels of advanced glycation end products (AGEs), receptor for AGEs (RAGE), and oxidative stress are suppressed in dipeptidyl peptidase-4 (DPP-4)-deficient diabetic rats, thus suggesting the crosstalk between AGE-RAGE axis and DPP-4 in experimental diabetic nephropathy. Therefore, we examined here the role of DPP-4 in AGE-evoked inflammatory reactions in human proximal tubular cells. Proteins were extracted from proximal tubular cells, and conditioned medium was collected, both of which were subjected to western blot analysis using anti-DPP-4 antibody. RAGE-aptamer was prepared using a systemic evolution of ligands by exponential enrichment. NF-κB p65 and monocyte chemoattractant protein-1 (MCP-1) gene expression was analyzed by reverse transcription-polymerase chain reaction. AGEs significantly increased DPP-4 expression and soluble DPP-4 production by tubular cells, the latter of which was attenuated by RAGE-aptamer or an anti-oxidant, N-acetylcysteine. AGEs or DPP-4 up-regulated NF-κB p65 or MCP-1 mRNA levels in tubular cells, which were suppressed by linagliptin, an inhibitor of DPP-4. AGEs stimulated NF-κB p65 gene expression in tubular cells isolated from control rats, but not from DPP-4-deficient rats. Our present results suggest that the AGE-RAGE-mediated oxidative stress could evoke inflammatory reactions in proximal tubular cells via autocrine production of DPP-4.

Uremic Advanced Glycation End Products and Protein-Bound Solutes Induce Endothelial Dysfunction Through Suppression of Krüppel-Like Factor 2.

BACKGROUND: Cardiovascular disease is the leading cause of morbidity and mortality in patients with end-stage renal disease. The accumulation of uremic solutes in this patient population is associated with endothelial dysfunction and accelerated cardiovascular disease. In this study, we examined the impact of the uremic milieu on the endothelial transcription factor, Krüppel-like factor 2 (KLF2), a key regulator of endothelial function and activation. METHODS AND RESULTS: Using serum from uremic pigs with chronic renal insufficiency, our results show that KLF2 expression is suppressed by the uremic milieu and individual uremic solutes in vitro. Specifically, KLF2 expression is significantly decreased in human umbilical vein endothelial cells after treatment with uremic porcine serum or carboxymethyllysine-modified albumin, an advanced glycation end product (AGE) known to induce endothelial dysfunction. AGE-mediated suppression of KLF2 is dependent on activation of the receptor for AGE, as measured by small interfering RNA knockdown of the receptor for AGE. Furthermore, KLF2 suppression promotes endothelial dysfunction, because adenoviral overexpression of KLF2 inhibits reactive oxygen species production and leukocyte adhesion in human umbilical vein endothelial cells. In addition, the application of hemodynamic shear stress, prolonged serum dialysis, or treatment with the receptor for AGE antagonist azeliragon (TTP488) is sufficient to prevent KLF2 suppression in vitro. To decipher the mechanism by which uremic AGEs suppress KLF2 expression, we assessed the role of the receptor for AGE in activation of nuclear factor-κB signaling, a hallmark of endothelial cell activation. Using a constitutively active form of IκBα, we show that translocation of p65 to the nucleus is necessary for KLF2 suppression after treatment with uremic AGEs. CONCLUSIONS: These data identify KLF2 suppression as a consequence of the uremic milieu, which may exacerbate endothelial dysfunction and resultant cardiovascular disease.

Zerumbone, a Phytochemical of Subtropical Ginger, Protects against Hyperglycemia-Induced Retinal Damage in Experimental Diabetic Rats.

Diabetic retinopathy (DR), the most ordinary and specific microvascular complication of diabetes, is a disease of the retina. Zerumbone (ZER) is a monocyclic sesquiterpene compound, and based on reports, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The aim of the current study is to evaluate the protective effect of zerumbone against DR in streptozotocin (STZ)-induced diabetic rats. STZ-diabetic rats were treated with ZER (40 mg/kg) once a day orally for 8 weeks. ZER administration significantly (p < 0.05) lowered the levels of plasma glucose (32.5% ± 5.7% lower) and glycosylated hemoglobin (29.2% ± 3.4% lower) in STZ-diabetic rats. Retinal histopathological observations indicated that disarrangement and reduction in thickness of retinal layers were reversed in ZER-treated diabetic rats. ZER downregulated both the elevated levels of advanced glycosylated end products (AGEs) and the higher levels of the receptors for AGEs (RAGE) in retinas of diabetic rats. What's more, ZER significantly (p < 0.05) ameliorated diabetes-induced upregulation of tumor necrosis factor-α, interleukin (IL)-1 and IL-6. ZER also attenuated overexpression of vascular endothelial growth factor and intercellular adhesion molecule-1, and suppressed activation of nuclear factor (NF)-κB and apoptosis in the retinas of STZ-diabetic rats. Our results suggest ZER possesses retinal protective effects, which might be associated with the blockade of the AGEs/RAGE/NF-κB pathway and its anti-inflammatory activity.

Short-term effects of dietary advanced glycation end products in rats.

Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.

Advanced Glycation End Products (AGE) Potently Induce Autophagy through Activation of RAF Protein Kinase and Nuclear Factor κB (NF-κB).

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people because of high amounts of three- or four-carbon sugars derived from glucose, thereby causing multiple consequences, including inflammation, apoptosis, obesity, and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to the activation of autophagy (self-eating) that might result in obesity. We detected AGE as one of the potent inducers of autophagy compared with doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3K and potentiated by the autophagosome maturation blocker bafilomycin. It increases autophagy in different cell types, and that correlates with the expression of its receptor, receptor for AGE. LC3B, the marker for autophagosomes, is shown to increase upon AGE stimulation. AGE-mediated autophagy is partially suppressed by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases sterol regulatory element binding protein activity, which leads to an increase in lipogenesis. Although AGE-mediated lipogenesis is affected by autophagy inhibitors, AGE-mediated autophagy is not influenced by lipogenesis inhibitors, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we provide data showing that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, that are involved in autophagy and simultaneously help with the accumulation of lipid droplets that are not cleared effectively by autophagy, therefore causing obesity.

Nε-(carboxymethyl) lysine-induced mitochondrial fission and mitophagy cause decreased insulin secretion from ß-cells.

Nε-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic ß-cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in the cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, the effects of α-lipoic acid (ALA) on mitochondria in AGE-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 wk increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxide production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells. Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 protein suggest that fragmented mitochondria were associated with increased mitophagic activity, and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic ß-cells. The findings from this study suggest that increased concentration of AGEs may damage ß-cells and reduce insulin secretion.

RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator-activated receptor-γ (PPARG)-responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68(+) macrophages from atherosclerotic plaques of Ldlr(-/-) mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr(-/-) mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.