Mayumi Nagashimada

CCR5 Plays a Critical Role in Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Both Macrophage Recruitment and M1/M2 Status

C-C motif chemokine receptor (CCR)2 and its ligand, monocyte chemoattractant protein (MCP)-1, are pivotal for adipose tissue macrophage (ATM) recruitment and the development of insulin resistance. However, other chemokine systems also may play a role in these processes. In this study, we investigated the role of CCR5 in obesity-induced adipose tissue inflammation and insulin resistance. We analyzed expression levels of CCR5 and its ligands in white adipose tissue (WAT) of genetically (ob/ob) and high-fat (HF) diet–induced obese (DIO) mice. Furthermore, we examined the metabolic phenotype of Ccr5−/− mice. CCR5 and its ligands were markedly upregulated in WAT of DIO and ob/ob mice. Fluorescence-activated cell sorter analysis also revealed that DIO mice had a robust increase in CCR5+ cells within ATMs compared with chow-fed mice. Furthermore, Ccr5−/− mice were protected from insulin resistance, glucose intolerance, and hepatic steatosis induced by HF feeding. The effects of loss of CCR5 were related to both reduction of total ATM content and an M2-dominant shift in ATM polarization. It is noteworthy that transplantation of Ccr5−/− bone marrow was sufficient to protect against impaired glucose tolerance. CCR5 plays a critical role in ATM recruitment and polarization and subsequent development of insulin resistance.

Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice

Mutations in the RET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. It has recently been shown that glial cell line-derived neurotrophic factor (GDNF) family receptor alpha1 (GFRalpha1), which binds to GDNF and activates RET, is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest-derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.

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.

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System

Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues. SIGNIFICANCE STATEMENT Elucidating the cellular identity of neuronal precursors provides novel insights into development and function of the nervous system. The enteric nervous system (ENS) is innervated richly by extrinsic nerve fibers, but little is known about the significance of extrinsic innervation to the structural integrity of the ENS. This report reveals that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate and differentiates into specific neuronal subtypes. SCP-specific ablation of the Ret gene leads to colonic oligoganglionosis, demonstrating a crucial role of SCP-derived neurogenesis in ENS development. Cross-lineage differentiation capacity in SCPs suggests their potential involvement in the development and pathology of a wide variety of neural crest-derived cell types.

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.

GDNF Signaling Levels Control Migration and Neuronal Differentiation of Enteric Ganglion Precursors

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development; however, the mechanisms by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop by radial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting neurotrophic factor for ENS development in a mouse model. GDNF promotes radial migration of SMG precursors. Interestingly, premigratory SMG precursors in the myenteric layer were distinguished from the surrounding neuronally differentiating cells by their lower activation of the GDNF-mediated MAPK pathway, suggesting that low activation of GDNF downstream pathways is required for the maintenance of the immature state. ENS precursors devoid of GDNF signaling during midgestation halt their migration, survive, and remain in an undifferentiated state over the long-term in vivo. Reactivation of GDNF signaling in these dormant precursors restores their migration and neuronal differentiation in gut organ culture. These findings suggest that pleiotropic function of GDNF is at least in part governed by modulating levels of intracellular activation of GDNF downstream pathways; high activation triggers neuronal differentiation, whereas low activation is crucial for the maintenance of progenitor state.

β-Cryptoxanthin alleviates diet-induced nonalcoholic steatohepatitis by suppressing inflammatory gene expression in mice.

Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.

Prevention and Reversal of Lipotoxicity-Induced Hepatic Insulin Resistance and Steatohepatitis in Mice by an Antioxidant Carotenoid, β- Cryptoxanthin

Excessive hepatic lipid accumulation promotes macrophages/Kupffer cells activation, resulting in exacerbation of insulin resistance and progression of nonalcoholic steatohepatitis (NASH). However, few promising treatment modalities target lipotoxicity-mediated hepatic activation/polarization of macrophages for NASH. Recent epidemiological surveys showed that serum β-cryptoxanthin, an antioxidant carotenoid, was inversely associated with the risks of insulin resistance and liver dysfunction. In the present study, we first showed that β-cryptoxanthin administration ameliorated hepatic steatosis in high-fat diet-induced obese mice. Next, we investigated the preventative and therapeutic effects of β-cryptoxanthin using a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat (CL) diet. After 12 weeks of CL diet feeding, β-cryptoxanthin administration attenuated insulin resistance and excessive hepatic lipid accumulation and peroxidation, with increases in M1-type macrophages/Kupffer cells and activated stellate cells, and fibrosis in CL diet-induced NASH. Comprehensive gene expression analysis showed that β-cryptoxanthin down-regulated macrophage activation signal-related genes significantly without affecting most lipid metabolism-related genes in the liver. Importantly, flow cytometry analysis revealed that, on a CL diet, β-cryptoxanthin caused a predominance of M2 over M1 macrophage populations, in addition to reducing total hepatic macrophage and T-cell contents. In parallel, β-cryptoxanthin decreased lipopolysaccharide-induced M1 marker mRNA expression in peritoneal macrophages, whereas it augmented IL-4-induced M2 marker mRNA expression, in a dose-dependent manner. Moreover, β-cryptoxanthin reversed steatosis, inflammation, and fibrosis progression in preexisting NASH in mice. In conclusion, β-cryptoxanthin prevents and reverses insulin resistance and steatohepatitis, at least in part, through an M2-dominant shift in macrophages/Kupffer cells in a lipotoxic model of NASH.

Novel Action of Carotenoids on Non-Alcoholic Fatty Liver Disease: Macrophage Polarization and Liver Homeostasis

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. It is characterized by a wide spectrum of hepatic changes, which may progress to non-alcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is considered a hepatic manifestation of metabolic syndrome; however, mechanisms underlying the onset and progression of NAFLD are still unclear. Resident and recruited macrophages are key players in the homeostatic function of the liver and in the progression of NAFLD to NASH. Progress has been made in understanding the molecular mechanisms underlying the polarized activation of macrophages. New NAFLD therapies will likely involve modification of macrophage polarization by restraining M1 activation or driving M2 activation. Carotenoids are potent antioxidants and anti-inflammatory micronutrients that have been used to prevent and treat NAFLD. In addition to their antioxidative action, carotenoids can regulate macrophage polarization and thereby halt the progression of NASH. In this review, we summarize the molecular mechanisms of macrophage polarization and the function of liver macrophages/Kupffer cells in NAFLD. From our review, we propose that dietary carotenoids, such as β-cryptoxanthin and astaxanthin, be used to prevent or treat NAFLD through the regulation of macrophage polarization and liver homeostasis.