Rosiglitazone Restores Endothelial Dysfunction in a Rat Model of Metabolic Syndrome through PPARγ- and PPARδ-Dependent Phosphorylation of Akt and eNOS

Vascular endothelial dysfunction has been demonstrated in metabolic syndrome (MS). Chronic administration of rosiglitazone ameliorates endothelial dysfunction through PPARγ-mediated metabolic improvements. Recently, studies suggested that single dose of rosiglitazone also has direct vascular effects, but the mechanisms remain uncertain. Here we established a diet-induced rat model of MS. The impaired vasorelaxation in MS rats was improved by incubating arteries with rosiglitazone for one hour. Importantly, this effect was blocked by either inhibition of PPARγ or PPARδ. In cultured endothelial cells, acute treatment with rosiglitazone increased the phosphorylation of Akt and eNOS and the production of NO. These effects were also abolished by inhibition of PPARγ, PPARδ, or PI3K. In conclusion, rosiglitazone improved endothelial function through both PPARγ- and PPARδ-mediated phosphorylation of Akt and eNOS, which might help to reconsider the complex effects and clinical applications of rosiglitazone

Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performed in a microchemotaxis chamber using chemoattractants formylated peptide Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α). Proteins were identified by immunoblotting and quantitative in-cell Western assay. The effects of TRP channel-inhibitor 2–aminoethoxydiphenylborane (2-APB) and small interfering RNA knockdown of TRPC3 were investigated. We observed an increased fMLP-induced migration of monocytes from hypertensive patients compared with normotensive control subjects (246±14% vs 151±10%). The TNF-α-induced migration of monocytes in patients with essential hypertension was also significantly increased compared to normotensive control subjects (221±20% vs 138±18%). In the presence of 2-APB or after siRNA knockdown of TRPC3 the fMLP-induced monocyte migration was significantly blocked. The fMLP-induced changes of cytosolic calcium were significantly increased in monocytes from hypertensive patients compared to normotensive control subjects. The fMLP-induced monocyte migration was significantly reduced in the presence of inhibitors of tyrosine kinase and phosphoinositide 3-kinase. We conclude that increased monocyte migration in patients with essential hypertension is associated with increased TRPC3 channels

Curcumin suppresses cell proliferation and reduces cholesterol absorption in Caco-2 cells by activating the TRPA1 channel

Abstract Background Curcumin (Cur) is a bioactive dietary polyphenol of turmeric with various biological activities against several cancers. Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Intestinal cholesterol homeostasis is associated with CRC. Chemotherapy for CRC is related to varied adverse effects. Therefore, natural products with anti-cancer properties represent a potential strategy for primary prevention of CRC. Methods The present study used Cur as a therapeutic approach against CRC using the Caco-2 cell line. The cells were treated with different concentrations of Cur for different duration of time and then the proliferation ability of cells was assessed using Cell Counting Kit-8 and 5-Ethynyl-2′-deoxyuridine assays. Oil red O staining and cholesterol assay kit were used to evaluate cellular lipid content and cholesterol outward transportation. Finally, the protein expressions of cholesterol transport-related protein and signal transduction molecules were assessed using Western blot assay. Results Cur inhibited cell proliferation in Caco-2 cells in a dose- and time-dependent manner by activating the transient receptor potential cation channel subfamily A member 1 (TRPA1) channel. Activation of the TRPA1 channel led to increased intracellular calcium, peroxisome proliferator-activated receptor gamma (PPARγ) upregulation, and the subsequent downregulation of the specificity protein-1 (SP-1)/sterol regulatory element-binding protein-2 (SREBP-2)/Niemann-Pick C1-like 1 (NPC1L1) signaling pathway-related proteins, and finally reduced cholesterol absorption in Caco-2 cells. Conclusions Cur inhibits cell proliferation and reduces cholesterol absorption in Caco-2 cells through the Ca2+/PPARγ/SP-1/SREBP-2/NPC1L1 signaling by activating the TRPA1 channel, suggesting that Cur can be used as a dietary supplement for the primary prevention of CRC. Graphical Abstract In Caco-2 cells, Cur first stimulates calcium influx by activating the TRPA1 channel, further upregulates PPARγ and downregulates SP-1/SREBP-2/NPC1L1 signaling pathway, and finally inhibits the absorption of cholesterol. TRPA1, transient receptor potential cation channel subfamily A member 1; NPC1L1, Niemann-Pick C1-like 1; PPARγ, peroxisome proliferator-activated receptor gamma; SP-1, specificity protein-1; SREBP-2, sterol regulatory element-binding protein-2; Cur, curcumin

Long-Term High-Fat Diet Decreases Renal Insulin-Degrading Enzyme Expression and Function by Inhibiting the PPARγ Pathway

SCOPE: Long-term high-fat diet (HFD) causes insulin resistance, which is a primary etiological factor in the development of obesity and type 2 diabetes mellitus. Impaired insulin clearance is not only a consequence but also a cause of insulin resistance. The kidney is a major site of insulin clearance, where the insulin-degrading enzyme (IDE) plays a vital role in the proximal tubule. Thus, the study investigates the role of renal IDE in the regulation of insulin resistance in HFD-induced obese mice. METHODS AND RESULTS: Twenty four-weeks of HFD in C57BL/6 mice causes insulin resistance and impaires insulin clearance, accompanied by a decrease in renal IDE expression and activity. Palmitic acid decreases IDE mRNA and protein expressions in HK-2 cells. RNA-Seq analysis found that the PPAR pathway is involved. 24-weeks of HFD decreases renal PPARγ, but not PPARα or PPARβ/δ mRNA expression. The inhibition of IDE expression by palmitic acid is prevented by the PPARγ agonist rosiglitazone. The amount of PPARγ bound to the promoters of IDE is decreased in palmitic acid-treated cells. Rosiglitazone improves insulin clearance and insulin resistance and increases renal IDE expression in HFD fed-mice. CONCLUSION: Long-term HFD decreases renal IDE expression and activity, and causes insulin resistance, which involves PPARγ

Specific siRNA against TRPC3 blocks the migration in monocytes, but did not affect spontaneous migration of monocytes.

<p><b>A</b>; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. <b>B</b>; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. <b>C</b>; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). <b>D</b>; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. <b>E</b>; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. <b>F</b>; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.</p

Increased fMLP-induced changes of cytosolic calcium in monocytes from patients with essential hypertension.

<p><b>A</b>, <b>B</b>; Representative fluorescence tracings in monocytes from normotensive control subjects (<b>A</b>) and hypertensive patients (<b>B</b>) after administration of fMLP (100 nm/L) in the absence and presence of TRP channel-inhibitor 2-APB. <b>C</b>; Summary data of changes of intracellular calcium concentration in monocytes from normotensive control subjects (NT; open bars) and patients with essential hypertension (HT; filled bars) by several doses of fMLP (10 nmol/L, 50 nmol/L, and 100 nm/L). Each n = 10; *p<0.05 for the comparison HT vs. NT. <b>D</b>, <b>E</b>; fMLP-induced cation influx into monocytes indicated by quenching of fura-2 by manganese (Mn²⁺, 1 µmol/L). Representative raw data, showing mamganese quenching with a stimulus (fMLP, 100 nmol/L) or without stimulus (control, w/o fMLP) in monocytes from normotensive control subjects (<b>D</b>; open black or grey circle; NT) and patients with essential hypertension (<b>E</b>; filled black or grey circle; HT). <b>F</b>; Bar graph shows the summary data of manganese quenching rate obtained from normotensive control subjects (NT, n = 6) and patients with essential hypertension (HT, n = 8) under the control conditions or after administration of fMLP (100 nmol/L). *p<0.05 or **p<0.01 for the comparison with their controls; and #p<0.05 or ## p<0.01 for the comparison HT (filled bars) vs. NT (open bars).</p

Increased monocyte migration associated with tyrosine kinase and phosphoinositide 3-kinase (PI3K) or ERK in essential hypertension.

<p><b>A</b>; Summary data of fMLP-induced monocytes migration was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per 40× fields from duplicate wells. Experiments were performed under control conditions (fMLP, n = 6), in the presence of genistein (Geni, n = 6) or wortmannin (Wort, n = 6) and PD98059 (PD, n = 3). Data are mean ± SEM of three to six independent experiments. **p<0.01 compared to their chemoattractant (fMLP) alone; # p<0.05 for comparison HT (filled bars) vs. NT (open bars).</p