Crucial Roles of 5-HT and 5-HT2 Receptor in Diabetes-Related Lipid Accumulation and Pro-Inflammatory Cytokine Generation in Hepatocytes.

BACKGROUND/AIMS: Previously, we confirmed that liver-synthesized 5-HT rather than non-liver 5-HT, acting on the 5-HT2 receptor (5-HT2R), modulates lipid-induced excessive lipid synthesis (ELS). Here, we further revealed the effects of the hepatocellular 5-HT system in diabetes-related disorders. METHODS: Studies were conducted in male ICR mice, human HepG2 cells, and primary mouse hepatocytes (PMHs) under gene or chemical inhibition of the 5-HT system, key lipid metabolism, and inflammation-related factors. Protein and messenger RNA expression and levels of the factors were determined via western blotting, reverse transcription PCR, and quantitative assay kits, respectively. Hepatic steatosis with inflammation and fibrosis, intracellular lipid droplet accumulation (LDA), and reactive oxygen species (ROS) location were determined via hematoxylin and eosin, Masson's trichrome, Oil red O, and fluorescent-specific staining, respectively. RESULTS: Palmitic acid induced the activation of the 5-HT system: the activation of 5-HT2R, primarily 5-HT2AR, in addition to upregulating monoamine oxidase A (MAO-A) expression and 5-HT synthesis, by activating the G protein/ phospholipase C pathway modulated PKCε activation, resulting in ELS with LDA; the activation of NF-κB, which mediates the generation of pro-inflammatory cytokines, was primarily due to ROS generation in the mitochondria induced by MAO-A-catalyzed 5-HT degradation, and secondarily due to the activation of PKCε. These effects of the 5-HT system were also detected in palmitic acid- or high glucose-treated PMHs and regulated multiple inflammatory signaling pathways. In diabetic mice, co-treatment with antagonists of both 5-HT synthesis and 5-HT2R significantly abolished hepatic steatosis, inflammation, and fibrosis as well as hyperglycemia and dyslipidemia. CONCLUSION: Activation of the hepatocellular 5-HT system plays a crucial role in inducing diabetes-related hepatic dysfunction and is a potential therapeutic target.

5-HT2A Receptor and 5-HT Degradation Play a Crucial Role in Atherosclerosis by Modulating Macrophage Foam Cell Formation, Vascular Endothelial Cell Inflammation, and Hepatic Steatosis.

AIM: Previously, we found that diabetes-related liver dysfunction is due to activation of the 5-HT2A receptor (5-HT2AR) and increased synthesis and degradation of 5-HT. Here, we investigated the role of 5-HT in the development of atherosclerosis. METHODS: The study was conducted using high-fat diet-fed male ApoE-/- mice, THP-1 cell-derived macrophages, and HUVECs. Protein expression and biochemical indexes were determined by Western blotting and quantitative analysis kit, respectively. The following staining methods were used: oil red O staining (showing atherosclerotic plaques and intracellular lipid droplets), immunohistochemistry (showing the expression of 5-HT2AR, 5-HT synthase, and CD68 in the aortic wall), and fluorescent probe staining (showing intracellular ROS). RESULTS: In addition to improving hepatic steatosis, insulin resistance, and dyslipidemia, co-treatment with a 5-HT synthesis inhibitor and a 5-HT2AR antagonist significantly suppressed the formation of atherosclerotic plaques and macrophage infiltration in the aorta of ApoE-/- mice in a synergistic manner. Macrophages and HUVECs exposed to oxLDL or palmitic acid in vitro showed that activated 5-HT2AR regulated TG synthesis and oxLDL uptake by activating PKCε, resulting in formation of lipid droplets and even foam cells; ROS production was due to the increase of both intracellular 5-HT synthesis and mitochondrial MAO-A-catalyzed 5-HT degradation, which leads to the activation of NF-κB and the release of the inflammatory cytokines TNF-α and IL-1ß from macrophages and HUVECs as well as MCP-1 release from HUVECs. CONCLUSION: Similar to hepatic steatosis, the pathogenesis of lipid-induced atherosclerosis is associated with activation of intracellular 5-HT2AR, 5-HT synthesis, and 5-HT degradation.

Role of 5-HT degradation in acute liver injury induced by carbon tetrachloride.

5-hydroxytryptamine (5-HT) is involved in the pathological processes of several liver diseases. Acute liver injury underlies the development of many liver diseases, but the mechanism remains unclear. We aimed to investigate the role of 5-HT in carbon tetrachloride (CCl4)-induced acute liver injury. Acute liver injury was induced with CCl4 (10 mg/kg) in mice pretreated with the 5-HT2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP). LO2 cells were treated with CCl4, 5-HT or 2,5-dimethoxy-4-idopametamine and pretreated with SH, CDP or the monoamine oxidase A (MAO-A) inhibitor clorgyline. Hematoxylin-eosin staining, immunohistochemistry, Real-time quantitative PCR, western blotting, fluorescent probe and biochemical markers were used to evaluate liver compromise. 5-HT2A receptor, 5-HT synthetase and MAO-A were expressed in hepatocytes; their gene and protein expression were upregulated by CCl4, which led to the degradation of mitochondrial 5-HT and overproduction of reactive oxygen species (ROS). Hepatic injury may be aggravated by ROS, which induce oxidative stress and the phosphorylation of p38 mitogen-activated protein kinase, Jun N-terminal kinase, extracellular regulated protein kinase, signal transducer and activator of transcription 3 and nuclear factor kappa-B. 5-HT2A receptor may contribute to acute liver injury by modulating 5-HT synthase and MAO-A expression. The synergistic action of SH and CDP treatment may inhibit CCl4-induced acute liver injury in a dose-dependent manner. Hence, CCl4-induced acute liver injury is due to an increase in mitochondrial ROS production caused by increased 5-HT degradation and probably involves increases in 5-HT2A receptor expression and 5-HT synthesis.

Renal ischemia/reperfusion injury in rats is probably due to the activation of the 5-HT degradation system in proximal renal tubular epithelial cells.

AIMS: To explore the relationship between renal ischemia/reperfusion injury (RIRI) and the activation of the renal 5-HT degradation system, including 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase-A (MAO-A). MAIN METHODS: Rat RIRI was induced by removing the right kidney, causing ischemia of the left kidney for 45 min and reperfusion for different times. RIRI model (ischemia for 45 min and reperfusion for 24 h) was pretreated with 5-HT2AR antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthase inhibitor carbidopa. In HK-2 cells, cellular damage was induced by hypoxia (24 h)/reoxygenation (12 h) (H/R) and treated with SH, carbidopa or the MAO-A inhibitor clorgyline. Hematoxylin-eosin, immunohistochemistry, TUNEL and fluorescent probe staining, RT-qPCR, western blotting, ELISA, etc. were used in the tests. KEY FINDINGS: The development of RIRI and the emergence of the RIRI peak were consistent with renal 5-HT degradation system activation. The highest expression regions of the 5-HT degradation system overlapped with those of the most severe lesions in the kidney, which were in proximal renal tubules. Rat RIRI and HK-2 cell damage, including oxidative stress, inflammation and apoptosis, could be almost abolished by synergistic inhibition of SH and carbidopa. Clorgyline also abolished the cellular damage induced by H/R. H/R-induced production of mitochondrial ROS in HK-2 cells was due to MAO-A-catalyzed 5-HT degradation, and 5-HT2AR was involved by mediating the expression of 5-HT synthases and MAO-A. SIGNIFICANCE: These findings revealed a close association between RIRI and activation of the renal 5-HT degradation system.

Nephrotoxicity induced by cisplatin is primarily due to the activation of the 5-hydroxytryptamine degradation system in proximal renal tubules.

As a widely used anticancer drug in the clinic, cisplatin has obvious side effects, especially nephrotoxicity. Previous studies have suggested that the accumulation of intracellular reactive oxygen species (ROS) is a hallmark of cisplatin-induced acute kidney injury. This study aimed to investigate the relationship between ROS accumulation induced by cisplatin and 5-HT degradation. In vivo, by HE and TUNEL staining, we found that cisplatin-induced renal lesions and apoptotic regions, which were located in proximal tubular epithelial cells, were also the regions in which tryptophan hydroxylase 1 (Tph1), aromatic l-amino acid decarboxylase (AADC), 5-HT2A receptor (5-HT2AR) and monoamine oxidase A (MAO-A) were overexpressed, as determined by immunohistochemistry. Notably, the 5-HT2AR antagonist sarpogrelate hydrochloride (SH) and the AADC inhibitor carbidopa (CDP) significantly attenuated cisplatin-induced increases in serum creatinine and blood urea nitrogen levels, renal ROS levels, oxidative stress (SOD activity and MDA), proinflammatory cytokine levels (NF-κB, TNF-α and IL-1ß), proapoptotic factor levels (Bax, Bcl-2, C-caspase 3 and C-caspase 9) and the phosphorylation of p38 and STAT3, as well as renal lesions and apoptosis. The combination of SH and CDP could almost abolish the effects of cisplatin challenge. In vitro, the effects of cisplatin challenge and the inhibitory effects of SH and CDP were also observed in HK-2 cells. Additionally, similar to the combination of SH and CDP, the MAO-A inhibitor clorgyline could also abolish the effects of cisplatin challenge. More importantly, by western blotting, we detected that the upregulation of Tph1, AADC and MAO-A expression induced by cisplatin both in vivo and in vitro could be obviously suppressed by SH to decrease 5-HT synthesis and mitochondrial 5-HT degradation. Altogether, these findings suggested that cisplatin-induced nephrotoxicity is due to the activation of the 5-HT degradation system in proximal tubular epithelial cells, including 5-HT2AR and 5-HT synthesis and degradation. 5-HT2AR plays a role by mediating the expression of MAO-A and the 5-HT synthases Tph1 and AADC.

5-HT 2 receptor mediates high-fat diet-induced hepatic steatosis and very low density lipoprotein overproduction in rats.

BACKGROUND: 5-HT has been shown to mediate abnormality of hepatic lipid metabolism through activation of mammalian target of rapamycin (mTOR). However, it is unclear whether 5-HT is directly involved in high-fat diet (HFD)-induced hepatic steatosis. MATERIALS AND METHODS: Male rats were allocated into seven groups with control, either HFD feeding, 5-HT treatment, or HFD feeding and 5-HT treatment with or without sarpogrelate treatment, all of which were executed for 4 weeks. HepG2 cells were exposed to 5-HT or palmitic acid (PA) with or without rapamycin or Sar treatment. RESULTS: Rats fed with HFD or exposed to 5-HT led to abnormalities with activated hepatic mTOR-S6K pathway, overproduction of hepatic triglycerides and VLDL with steatosis, and hyperlipidemia, which were exacerbated by a combination of HFD and 5-HT. Sarpogrelate significantly inhibited above abnormalities induced by HFD and 5-HT, alone or in a combination. Additionally, HFD caused up-regulation of 5-HT2 receptors (5-HT2R), including 5-HT2AR and 5-HT2BR, and 5-HT synthesis in the liver, without obvious influence on other 5-HT receptors gene expression. In HepG2 cells, both PA and 5-HT induced overproduction of triglycerides and VLDL with lipid droplets, and PA up-regulated 5-HT2AR and 5-HT2BR expression and 5-HT synthesis as well. Rapamycin fully abolished PA or 5-HT-induced mTOR activation, which was more effective than sarpogrelate. However, the inhibitory effects of rapamycin on PA or 5-HT-induced overproduction of triglycerides and VLDL were less than sarpogrelate. CONCLUSIONS: Up-regulation of hepatic 5-HT2R and 5-HT synthesis by HFD is crucial for HFD-induced overproduction of hepatic triglycerides and VLDL with hyperlipidemia.

New Developments in the Treatment of Neuroendocrine Tumours - RADIANT-4, NETTER-1 and Telotristat Etiprate.

Neuroendocrine tumours (NETs) are a heterogeneous group of neoplasms whose incidence has increased significantly in recent years, and whose optimal management remains controversial. We report the latest innovations in their management, in particular the results of three trials concerning the use of the mammalian target of rapamycin (mTOR) inhibitor, everolimus, in non-functional NETs of lung/ gastrointestinal (GI) origin, the first randomised trial of radiolabelled 177Lu-DOTATATE in patients with mid-gut NETs, and the use of the 5-HT synthesis inhibitor, telotristat etiprate, in patients with the carcinoid syndrome.

Relationship between fatigue caused by type 2 diabetes mellitus and 5-HT degradation in skeletal muscle / 药学学报

Fatigue is a common complication of type 2 diabetes mellitus (T2DM). We examined the relationship between T2DM fatigue and the skeletal muscle 5-hydroxytryptamine (5-HT) system. In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin. The mice were treated with the 5-HT2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP) (separately and in combination). In cell culture experiments, C2C12 cells were stimulated with D-glucose, palmitic acid or 5-HT. 5-HT2AR, 5-HT synthesis and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor. The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University. The results showed that 5-HT2AR, 5-HT synthase and MAO-A were expressed in mouse skeletal muscle and C2C12 cells. The expression of these proteins was significantly up-regulated in T2DM mice or when C2C12 cells were exposed to palmitic acid and D-glucose; palmitic acid was a stronger stimulant of their expression than D-glucose. Rotating rod experiments and biochemical index tests have shown that T2DM fatigue is associated with an increase in skeletal muscle 5-HT2AR, 5-HT synthesis and 5-HT degradation. 5-HT2AR mediates the expression of MAO-A and the synthesis of 5-HT, which indirectly regulates the degradation of 5-HT. MAO-A regulates cell inflammation, mitochondrial ROS production and membrane potential depolarization by mediating 5-HT degradation. MAO-A also inhibits the expression of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1), carnitine palmitoyltransferase-1 (CPT1) and ATP synthase-6 (ATP6), thus inhibiting mitochondrial functions such as fatty acid β oxidation and ATP synthesis. SH and CDP can effectively treat T2DM fatigue, and can also reduce blood glucose and blood lipid, and the combination of SH and CDP has a clear synergistic effect.

The role of renal 5-hydroxytryptamine synthesis and degradation in hyperglycemia-induced kidney injury / 药学学报

Hyperglycemic kidney injury (HKI) is a common complication of diabetic patients. We examined the relationship between HKI and the abnormal expression of 5-hydroxytryptamine (5-HT) system induced by hyperglycemia in type 2 diabetes mellitus (T2DM). In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin. The mice were treated with the 5-HT2A receptor (5-HT2AR) antagonist sarpogrelate hydrochloride (SH) and 5-HT synthesis inhibitor carbidopa (CDP) (respectively or in combination). In cell culture experiments, human glomerular mesangial cells (HMC) were stimulated with D-glucose (D-Glu), and 5-HT2AR, 5-HT synthesis, and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor clorgyline. Periodic acid-Schiff (PAS) staining and Masson staining, immunohistochemistry and Western blot, fluorescent probe, and enzyme linked immunosorbent assay (ELISA) and enzyme reagent were respectively used to detect histopathology, protein expression, intracellular reactive oxygen species (ROS), and biochemical indexes. The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University. The results showed that 5-HT2AR, 5-HT synthases, and MAO-A were expressed in glomerular basement membrane and kidney tubular epithelial cells of mouse kidney and HMC. The expression of these proteins was significantly up-regulated in T2DM mice or when HMC cells were exposed to high concentration of D-Glu. HKI, characterized by abnormal renal function, glomerular swelling, and glomerular basement membrane thickening and fibrosis, is closely associated with an increase in kidney 5-HT2AR, 5-HT synthesis, and 5-HT degradation. Among them, 5-HT2AR can mediate the expression of 5-HT synthases and MAO-A; MAO-A can catalyze the degradation of 5-HT to increase the production of mitochondrial ROS, leading to the phosphorylation of nuclear factor kappa B (NF-κB) with the production of inflammatory cytokines, and the up-regulation of matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) with the production of collagens. SH and CDP can effectively treat HKI, and the combination of SH and CDP has a clear synergistic effect.