Hydrolyzed Chicken Meat Extract and Its Bioactive Cyclopeptides Protect Neural Function by Attenuating Inflammation and Apoptosis via PI3K/AKT and AMPK Pathways.

Cognitive decline is inevitable with age, and due to the lack of well-established pharmacotherapies for neurodegenerative disorders, dietary supplements have become important alternatives to ameliorate brain deterioration. Hydrolyzed chicken meat extract (HCE) and its bioactive components were previously found to improve neuroinflammation and cognitive decline by regulating microglia polarization. However, the effects and mechanisms of these bioactives on neurons remain unclear. Here, the most potent bioactive component on neural function in HCE was screened out, and the detailed mechanism was clarified through in vivo and in vitro experiments. We found that HCE, cyclo(Val-Pro), cyclo(Phe-Phe), cyclo(His-Pro), cyclo(Leu-Lys), and arginine exerted stronger anti-inflammatory and antioxidant effects among the 12 bioactives in amyloid ß (Aß)-treated HT-22 cells. Further transcriptome sequencing and polymerase chain reaction (PCR) array analysis showed that these bioactives participated in different signaling pathways, and cyclo(Val-Pro) was identified as the most potent cyclic dipeptide. In addition, the antiapoptotic and neuroprotective effect of cyclo(Val-Pro) was partly regulated by the activation of PI3K/AKT and AMPK pathways, and the inhibition of these pathways abolished the effect of cyclo(Val-Pro). Moreover, cyclo(Val-Pro) enhanced cognitive function and neurogenesis and alleviated neuroinflammation and oxidative stress in middle-aged mice, with an effect similar to HCE. Hippocampal transcriptome analysis further revealed that HCE and cyclo(Val-Pro) significantly enriched the neuroactive ligand-receptor interaction pathway, verified by enhanced neurotransmitter levels and upregulated neurotransmitter receptor-related gene expression. Therefore, the mechanism of cyclo(Val-Pro) on neural function might be associated with PI3K/AKT and AMPK pathway-mediated antiapoptotic effect and neurogenesis and the activation of the neurotransmitter-receptor pathway.

Morus alba L. (Sangzhi) alkaloids mitigate atherosclerosis by regulating M1/M2 macrophage polarization.

BACKGROUND: Atherosclerosis (AS) is an important cause of cardiovascular disease, posing a substantial health risk. Recognized as a chronic inflammatory disorder, AS hinges on the pivotal involvement of macrophages in arterial inflammation, participating in its formation and progression. Sangzhi alkaloid (SZ-A) is a novel natural alkaloid extracted from the mulberry branches, has extensive pharmacological effects and stable pharmacokinetic characteristics. However, the effects and mechanisms of SZ-A on AS remain unclear. PURPOSE: To explore the effect and underlying mechanisms of SZ-A on inflammation mediated by macrophages and its role in AS development. METHODS: Atherosclerosis was induced in vivo in apolipoprotein E-deficient mice through a high-fat and high-choline diet. We utilized macrophages and vascular endothelial cells to investigate the effects of SZ-A on macrophage polarization and its anti-inflammatory properties on endothelial cells in vitro. The transcriptomic analyses were used to investigate the major molecule that mediates cell-cell interactions and the antiatherogenic mechanisms of SZ-A based on AS, subsequently validated in vivo and in vitro. RESULTS: SZ-A demonstrated a significant inhibition in vascular inflammation and alleviation of AS severity by mitigating macrophage infiltration and modulating M1/M2 macrophage polarization in vitro and in vivo. Moreover, SZ-A effectively reduced the release of the proinflammatory mediator C-X-C motif chemokine ligand (CXCL)-10, predominantly secreted by M1 macrophages. This reduction in CXCL-10 contributed to improved endothelial cell function, reduced recruitment of additional macrophages, and inhibited the inflammatory amplification effect. This ultimately led to the suppression of atherogenesis. CONCLUSION: SZ-A exhibited potent anti-inflammatory effects by inhibiting macrophage-mediated inflammation, providing a new therapeutic avenue against AS. This is the first study demonstrating the efficacy of SZ-A in alleviating AS severity and offers novel insights into its anti-inflammatory mechanism.

CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status.

BACKGROUND AND OBJECTIVES: Chemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated. METHODS AND RESULTS: The expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1-/- bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1-/- mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. CONCLUSION: Pharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

CC chemokine ligand 3 deficiency ameliorates diet-induced steatohepatitis by regulating liver macrophage recruitment and M1/M2 status in mice.

BACKGROUND AND AIMS: The global prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing. Chemokines and their receptors have potential as therapeutic targets of NAFLD. We investigated the role of CC chemokine ligand 3 (CCL3) in the development of murine and human NAFLD. METHODS: CCL3-knockout mice (CCL3-/-) and littermate CCL3 wild-type control mice (WT) were fed a high-cholesterol and high-fat (CL) diet for 16 weeks to induce NAFLD. We investigated the impact of CCL3 gene deletion in bone marrow cells and leptin-deficient ob/ob mice on CL diet-induced steatohepatitis. We assayed the serum CCL3 levels in 36 patients with biopsy-proven NAFLD and nine healthy control subjects. RESULTS: Compared with normal chow (NC), the CL diet induced steatohepatitis and hepatic fibrosis and elevated the plasma CCL3 level. In the liver, CCL3 protein colocalized with F4/80+ macrophages, especially CD11c+ M1-like macrophages, rather than other cell types. CCL3-/- attenuated CL diet-induced steatohepatitis and fibrosis associated with M2-dominant liver macrophages compared with the WT. The reconstitution of bone marrow (BM) cells from CCL3-/- attenuated steatohepatitis in WT mice fed a CL diet. Furthermore, crossing CCL3-/- onto the ob/ob background prevented CL diet-induced NAFLD in ob/ob mice, which was associated with a lesser inflammatory phenotype of liver macrophages. Also, the serum and hepatic levels of CCL3 were significantly increased in patients with non-alcoholic steatohepatitis (NASH) compared to those with simple fatty liver (NAFL) and healthy subjects. CONCLUSION: Our data indicate that CCL3 facilitates macrophage infiltration into the liver and M1 polarization in the progression of steatohepatitis and highlight the need for further studies to determine the effect of CCL3-CCR1 and -CCR5 signaling blockade on the treatment of NAFLD.

Spermidine improves gut barrier integrity and gut microbiota function in diet-induced obese mice.

Obesity is associated with impaired intestinal barrier function and dysbiosis of the gut microbiota. Spermidine, a polyamine that acts as an autophagy inducer, has important benefits in patients with aging-associated diseases and metabolic dysfunction. However, the mechanism of spermidine on obesity remains unclear. Here, we show that spermidine intake is negatively correlated with obesity in both humans and mice. Spermidine supplementation causes a significant loss of weight and improves insulin resistance in diet-induced obese (DIO) mice. These effects are associated with the alleviation of metabolic endotoxemia and enhancement of intestinal barrier function, which might be mediated through autophagy pathway and TLR4-mediated microbial signaling transduction. Moreover, spermidine causes the significant alteration of microbiota composition and function. Microbiota depletion compromises function, while transplantation of spermidine-altered microbiota confers protection against obesity. These changes might partly be driven by an SCFA-producing bacterium, Lachnospiraceae NK4A136 group, which was decreased in obese subjects and subsequently increased by spermidine. Notably, the change of Lachnospiraceae NK4A136 group is significantly correlated with enhanced gut barrier function induced by spermidine. Our results indicate that spermidine supplementation may serve as a viable therapy for obesity.

The Gut Microbiota and Its Metabolites, Novel Targets for Treating and Preventing Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent metabolic disorders worldwide, along with obesity and type 2 diabetes. NAFLD involves a series of liver abnormalities from simple hepatic steatosis to non-alcoholic steatohepatitis, which can ultimately lead to liver cirrhosis and cancer. The gut-liver axis plays an important role in the development of NAFLD, which depends mainly on regulation of the gut microbiota and its bacterial products. These intestinal bacterial species and their metabolites, including bile acids, tryptophan catabolites, and branched-chain amino acids, regulate adipose tissue and intestinal homeostasis and contribute to the pathogenesis of NAFLD/non-alcoholic steatohepatitis. In this review, the current evidence regarding the key role of the gut microbiota and its metabolites in the pathogenesis and development of NAFLD is highlighted, and the advances in the progression and applied prospects of gut microbiota-targeted dietary and exercise therapies is also discussed.

DPP-4 Inhibition with Anagliptin Reduces Lipotoxicity-Induced Insulin Resistance and Steatohepatitis in Male Mice.

Excessive hepatic lipid accumulation drives the innate immune system and aggravates insulin resistance, hepatic inflammation, and fibrogenesis, leading to nonalcoholic steatohepatitis (NASH). Dipeptidyl peptidase-4 (DPP-4) regulates glucose metabolism and is expressed in many different cell types, including the cells of the immune system. In addition, DPP-4 may be involved in macrophage-mediated inflammation and insulin resistance. This study investigated the effects of anagliptin (Ana), an inhibitor of DPP-4, on macrophage polarity and phenotype in the livers of mice with steatohepatitis. We investigated the effects of Ana on steatohepatitis induced via a high-cholesterol high-fat (CL) diet or a choline-deficient L-amino acid-defined, high-fat (CDAHF) diet. DPP-4 activity, liver histology, and insulin sensitivity were evaluated, and liver DPP-4+ macrophages were quantified using fluorescence-activated cell sorting (FACS). Liver and plasma DPP-4 activity increased significantly in mice on both diets. FACS revealed that, compared with chow-fed mice, the CL-fed mice exhibited a significant increase in the proportion of DPP-4+ liver macrophages, particularly the M1-type macrophages. Ana decreased hepatic lipid and M1 macrophage accumulation and stimulated M2 macrophage accumulation in the liver, thereby attenuating insulin resistance, steatohepatitis, and fibrosis. Importantly, Ana alleviated hepatic fibrosis and steatohepatitis in mice fed CL diet and CDAHF diet. Using Ana to inhibit DPP-4 reduced lipotoxicity-induced hepatic insulin resistance through regulating the M1/M2 macrophage status.

Xanthine oxidase inhibition attenuates insulin resistance and diet-induced steatohepatitis in mice.

Hyperuricemia drives the development of nonalcoholic fatty liver disease (NAFLD). Pharmacological inhibition of xanthine oxidase (XO), a rate-limiting enzyme for uric acid (UA) production, has been demonstrated to improve hepatic steatosis in diet-induced obese mice. However, it remains unclear whether inhibition of XO improves nonalcoholic steatohepatitis (NASH), a more advanced form of NAFLD, in terms of both liver inflammation and fibrosis. Here, we investigated the effects of febuxostat and allopurinol, two XO inhibitors clinically used for gout, on a mouse model of NASH. Furthermore, we conducted a single-arm, open-label intervention study with febuxostat for NAFLD patients with hyperuricemia. Despite a similar hypouricemic effect of the XO inhibitors on blood UA level, febuxostat, but not allopurinol, significantly decreased hepatic XO activity and UA levels in the NASH model mice. These reductions in hepatic XO activity and UA levels were accompanied by attenuation of insulin resistance, lipid peroxidation, and classically activated M1-like macrophage accumulation in the liver. Furthermore, in NAFLD patients with hyperuricemia, treatment with febuxostat for 24 weeks decreased the serum UA level, accompanied by reductions in the serum levels of liver enzymes, alanine aminotransferase and aspartate aminotransferase. XO may represent a promising therapeutic target in NAFLD/NASH, especially in patients with hyperuricemia.

Adipose Tissue Macrophage Phenotypes and Characteristics: The Key to Insulin Resistance in Obesity and Metabolic Disorders.

Obesity is one of the most serious global health problems, with an incidence that increases yearly and coincides with the development of a variety of associated comorbidities (e.g., type 2 diabetes, nonalcoholic fatty liver disease, some immune-related disorders). Although many studies have investigated the pathogenesis of overweight and obesity, multiple regulatory factors underlying the onset of obesity-related metabolic disorders remain elusive. Macrophages contribute to modulation of obesity-related inflammation and insulin resistance (IR); adipose tissue macrophages are particularly important in this context. Based on newly identified links between the chemokine system and obesity, macrophage polarization has become an essential target of new therapies for obesity-related IR. The findings of multiple studies imply that variations in gut microbiota and its metabolites might contribute to the regulation of obesity and related metabolic disorders. Recently, several novel antidiabetic drugs, applied as treatment for weight loss, were shown to be effective for obesity-induced IR and other comorbidities. The present review will discuss the properties and functions of macrophages in adipose tissue under conditions of obesity from three perspectives: the chemokine system, the gut microbiota, and antidiabetic drug application. It is proposed that macrophages might be a key therapeutic target for obesity-induced complications.

Lycopene Alleviates Obesity-Induced Inflammation and Insulin Resistance by Regulating M1/M2 Status of Macrophages.

SCOPE: Adipose tissue macrophage (ATM) recruitment and polarization are pivotal in the development of insulin resistance. However, treatment modalities targeting ATMs remain limited. The effects of lycopene, an antioxidant carotenoid compound, on adipose tissue inflammation and insulin resistance in high fat (HF)-diet-induced obese mice are examined. METHODS AND RESULTS: C57BL/6J mice are fed an HF diet or an HF diet containing lycopene (HF+LY) for 8 weeks. Lycopene attenuates HF-diet-induced glucose intolerance and hyperinsulinemia. Compared with HF mice, HF+LY mice exhibit attenuated adipocyte hypertrophy and macrophage infiltration in epididymal white adipose tissue (eWAT) and hepatic steatosis and inflammation. Flow cytometry analysis of ATMs demonstrates that lycopene attenuated the increased number of ATMs in HF diet-fed mice. In addition, HF+LY mice have 23% fewer M1-polarized ATMs and 60% more M2-polarized ATMs than HF mice, resulting in the predominance of M2 over M1 in the ATM population. M2-dominant polarization is also seen in hepatic macrophages in HF+LY mice. Moreover, lycopene promotes IL-4-induced M2 polarization by increasing the phosphorylation levels of STAT6 and Akt in Raw 264.7 macrophages. CONCLUSIONS: Lycopene facilitates M2-dominant polarization in ATM, thereby attenuating HF diet-induced inflammation and insulin resistance in eWAT and the liver.

Pirfenidone prevents and reverses hepatic insulin resistance and steatohepatitis by polarizing M2 macrophages.

Nonalcoholic steatohepatitis (NASH) is associated with lipotoxic liver injury, leading to insulin resistance, inflammation, and fibrosis. Despite its increased global incidence, very few promising treatments for NASH are available. Pirfenidone is an antifibrotic agent used to treat pulmonary fibrosis; it suppresses the pulmonary influx of T cells and macrophages. Here, we investigated the effect of pirfenidone in a mouse model of lipotoxicity-induced NASH via a high-cholesterol and high-fat diet. After 12 weeks of feeding, pirfenidone administration attenuated excessive hepatic lipid accumulation and peroxidation by reducing the expression of genes related to lipogenesis and fatty acid synthesis and enhancing the expression of those related to fatty acid oxidation. Flow cytometry indicated that pirfenidone reduced the number of total hepatic macrophages, particularly CD11c+CD206-(M1)-type macrophages, increased the number of CD11c-CD206+(M2)-type macrophages, and subsequently reduced T-cell numbers, which helped improve insulin resistance and steatohepatitis. Moreover, pirfenidone downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 marker genes and upregulated IL-4-induced M2 marker genes in a dose-dependent manner in RAW264.7 macrophages. Importantly, pirfenidone reversed insulin resistance, hepatic inflammation, and fibrosis in mice with pre-existing NASH. These findings suggest that pirfenidone is a potential candidate for the treatment of NASH.

SGLT2 Inhibition by Empagliflozin Promotes Fat Utilization and Browning and Attenuates Inflammation and Insulin Resistance by Polarizing M2 Macrophages in Diet-induced Obese Mice.

Sodium-glucose cotransporter (SGLT) 2 inhibitors increase urinary glucose excretion (UGE), leading to blood glucose reductions and weight loss. However, the impacts of SGLT2 inhibition on energy homeostasis and obesity-induced insulin resistance are less well known. Here, we show that empagliflozin, a SGLT2 inhibitor, enhanced energy expenditure and attenuated inflammation and insulin resistance in high-fat-diet-induced obese (DIO) mice. C57BL/6J mice were pair-fed a high-fat diet (HFD) or a HFD with empagliflozin for 16weeks. Empagliflozin administration increased UGE in the DIO mice, whereas it suppressed HFD-induced weight gain, insulin resistance, and hepatic steatosis. Moreover, empagliflozin shifted energy metabolism towards fat utilization, elevated AMP-activated protein kinase and acetyl-CoA carbolxylase phosphorylation in skeletal muscle, and increased hepatic and plasma fibroblast growth factor 21 levels. Importantly, empagliflozin increased energy expenditure, heat production, and the expression of uncoupling protein 1 in brown fat and in inguinal and epididymal white adipose tissue (WAT). Furthermore, empagliflozin reduced M1-polarized macrophage accumulation while inducing the anti-inflammatory M2 phenotype of macrophages within WAT and liver, lowering plasma TNFα levels and attenuating obesity-related chronic inflammation. Thus, empagliflozin suppressed weight gain by enhancing fat utilization and browning and attenuated obesity-induced inflammation and insulin resistance by polarizing M2 macrophages in WAT and liver.

Glucoraphanin Ameliorates Obesity and Insulin Resistance Through Adipose Tissue Browning and Reduction of Metabolic Endotoxemia in Mice.

Low-grade sustained inflammation links obesity to insulin resistance and nonalcoholic fatty liver disease (NAFLD). However, therapeutic approaches to improve systemic energy balance and chronic inflammation in obesity are limited. Pharmacological activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) alleviates obesity and insulin resistance in mice; however, Nrf2 inducers are not clinically available owing to safety concerns. Thus, we examined whether dietary glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, ameliorates systemic energy balance, chronic inflammation, insulin resistance, and NAFLD in high-fat diet (HFD)-fed mice. Glucoraphanin supplementation attenuated weight gain, decreased hepatic steatosis, and improved glucose tolerance and insulin sensitivity in HFD-fed wild-type mice but not in HFD-fed Nrf2 knockout mice. Compared with vehicle-treated controls, glucoraphanin-treated HFD-fed mice had lower plasma lipopolysaccharide levels and decreased relative abundance of the gram-negative bacteria family Desulfovibrionaceae in their gut microbiomes. In HFD-fed mice, glucoraphanin increased energy expenditure and the protein expression of uncoupling protein 1 (Ucp1) in inguinal and epididymal adipose depots. Additionally, in this group, glucoraphanin attenuated hepatic lipogenic gene expression, lipid peroxidation, classically activated M1-like macrophage accumulation, and inflammatory signaling pathways. By promoting fat browning, limiting metabolic endotoxemia-related chronic inflammation, and modulating redox stress, glucoraphanin may mitigate obesity, insulin resistance, and NAFLD.

DPP-4 Inhibition by Linagliptin Attenuates Obesity-Related Inflammation and Insulin Resistance by Regulating M1/M2 Macrophage Polarization.

Dipeptidyl peptidase 4 (DPP-4) cleaves a large number of chemokine and peptide hormones involved in the regulation of the immune system. Additionally, DPP-4 may also be involved in macrophage-mediated inflammation and insulin resistance. Thus, the current study investigated the effect of linagliptin, an inhibitor of DPP-4, on macrophage migration and polarization in white adipose tissue (WAT) and liver of high-fat diet-induced obese (DIO) mice. DPP-4(+) macrophages in lean and obese mice were quantified by fluorescence-activated cell sorting (FACS) analysis. DPP-4 was predominantly expressed in F4/80(+) macrophages in crown-like structures compared with adipocytes in WAT of DIO mice. FACS analysis also revealed that, compared with chow-fed mice, DIO mice exhibited a significant increase in DPP-4(+) expression in cells within adipose tissue macrophages (ATMs), particularly M1 ATMs. Linagliptin showed a greater DPP-4 inhibition and antioxidative capacity than sitagliptin and reduced M1-polarized macrophage migration while inducing an M2-dominant shift of macrophages within WAT and liver, thereby attenuating obesity-induced inflammation and insulin resistance. Loss of macrophage inflammatory protein-1α, a chemokine and DPP-4 substrate, in DIO mice abrogated M2 macrophage-polarizing and insulin-sensitizing effects of linagliptin. Therefore, the inhibition of DPP-4 by linagliptin reduced obesity-related insulin resistance and inflammation by regulating M1/M2 macrophage status.

Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E.

Hepatic insulin resistance and nonalcoholic steatohepatitis (NASH) could be caused by excessive hepatic lipid accumulation and peroxidation. Vitamin E has become a standard treatment for NASH. However, astaxanthin, an antioxidant carotenoid, inhibits lipid peroxidation more potently than vitamin E. Here, we compared the effects of astaxanthin and vitamin E in NASH. We first demonstrated that astaxanthin ameliorated hepatic steatosis in both genetically (ob/ob) and high-fat-diet-induced obese mice. In a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat diet, astaxanthin alleviated excessive hepatic lipid accumulation and peroxidation, increased the proportion of M1-type macrophages/Kupffer cells, and activated stellate cells to improve hepatic inflammation and fibrosis. Moreover, astaxanthin caused an M2-dominant shift in macrophages/Kupffer cells and a subsequent reduction in CD4(+) and CD8(+) T cell recruitment in the liver, which contributed to improved insulin resistance and hepatic inflammation. Importantly, astaxanthin reversed insulin resistance, as well as hepatic inflammation and fibrosis, in pre-existing NASH. Overall, astaxanthin was more effective at both preventing and treating NASH compared with vitamin E in mice. Furthermore, astaxanthin improved hepatic steatosis and tended to ameliorate the progression of NASH in biopsy-proven human subjects. These results suggest that astaxanthin might be a novel and promising treatment for NASH.