唐澤 直義

Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow-and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders.

Rhabdomyolysis is one of the main causes of community-acquired acute kidney injury (AKI). Although inflammation is involved in the pathogenesis of rhabdomyolysis-induced AKI (RIAKI), little is known about the mechanism that triggers inflammation during RIAKI. Recent evidence has indicated that sterile inflammation triggered by tissue injury can be mediated through multiprotein complexes called the inflammasomes. Therefore, we investigated the role of NLRP3 inflammasomes in the pathogenesis of RIAKI using a glycerol-induced murine rhabdomyolysis model. Inflammasomerelated molecules were upregulated in the kidney of RIAKI. Renal tubular injury and dysfunction preceded leukocyte infiltration into the kidney during the early phase of RIAKI, and they were markedly attenuated in mice deficient in NLRP3, ASC, caspase-1, and interleukin (IL)-1 beta compared with those in wild-type mice. No difference in leukocyte infiltration was observed between wild-type and NLRP3-deficient mice. Furthermore, NLRP3 deficiency strikingly suppressed the expression of renal injury markers and inflammatory cytokines and apoptosis of renal tubular cells. These results demonstrated that NLRP3 inflammasomes contribute to inflammation, apoptosis, and tissue injury during the early phase of RIAKI and provide new insights into the mechanism underlying the pathogenesis of RIAKI.

Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in N mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3(-/-) and Asc(-/-) mice phenotypes were dependent on the balance between interleukin (IL)-1 a and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.

Objective-Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. We previously showed that the inflammasome, which regulates the caspase-1-dependent interleukin-1 beta production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation. Approach and Results-Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe(-/-) mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1 beta release in macrophages, and this activation was mediated through an angiotensin type I receptor/mitochondria-derived reactive oxygen species-dependent pathway. Conclusions-Our results demonstrate the importance of the NLRP3 inflammasome in the initial inflammatory responses in AAA formation, indicating its potential as a novel therapeutic target for preventing AAA progression.

Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor(PPAR) alpha promoter in an autoloop fashion and is crucial for the ligand transactivation of PPAR alpha by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.

Inflammation plays a crucial role in the pathophysiologicat characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a muttiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X(7)-potassium efflux and reactive oxygen species dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.

Objective: Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis. Methods and results: Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe(-/-)Casp1(-/-) mice were significantly reduced compared to those in Apoe(-/-) mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe(-/-)Casp1(-/-) mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1 beta in the plaques as well as plasma levels of IL-1 beta, IL-1 alpha, IL-6, CCL2, and TNF-alpha, in Apoe(-/-)Casp1(-/-) mice were lower than those in Apoe(-/-) mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1 beta and IL-1 alpha, in macrophages. Conclusion: Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis. (c) 2012 Elsevier Inc. All rights reserved.

Objective-Elovl6, a long-chain fatty acid elongase, is a rate-limiting enzyme that elongates saturated and monounsaturated fatty acids and has been shown to be related to obesity-induced insulin resistance via modification of fatty acid composition. In this study, we investigated the roles of Elovl6 in foam cell formation in macrophages and atherosclerosis in mice. Methods and Results-To investigate the roles of Elovl6 in macrophages in the progression of atherosclerosis, we transplanted bone marrow cells of wild-type or Elovl6(-/-) mice into irradiated LDL-R(-/-) mice that were fed a western diet. Aortic atherosclerotic lesion areas and infiltration of macrophages were significantly smaller in Elovl6(-/-) bone marrow cells-transplanted LDL-R(-/-) mice than in wild-type. Accumulation of esterified cholesterol on exposure to acetylated-LDL was less severe in peritoneal macrophages from Elovl6(-/-) mice than those from wild-type. Cholesterol efflux and expression of cholesterol efflux transporters were increased in Elovl6(-/-) macrophages, although no difference in uptake of acetylated-LDL was found between the two groups. On analysis of fatty acid composition of the esterified cholesterol fraction in macrophages, n-6 polyunsaturated fatty acids were decreased by absence of Elovl6. Conclusion-These findings suggest that Elovl6 in macrophages may contribute to foam cell formation and progression of atherosclerosis. (Arterioscler Thromb Vasc Biol. 2011;31:1973-1979.)

Objective-Sterol regulatory element-binding protein-1 (SREBP-1) is nutritionally regulated and is known to be a key transcription factor regulating lipogenic enzymes. The goal of this study was to evaluate the roles of SREBP-1 in dyslipidemia and atherosclerosis. Methods and Results-Transgenic mice that overexpress SREBP-1c in the liver and SREBP-1-deficient mice were crossed with low-density lipoprotein receptor (LDLR)-deficient mice, and the plasma lipids and atherosclerosis were analyzed. Hepatic SREBP-1c overexpression in LDLR-deficient mice caused postprandial hypertriglyceridemia, increased very-low-density lipoprotein (VLDL) cholesterol, and decreased high-density lipoprotein cholesterol in plasma, which resulted in accelerated aortic atheroma formation. Conversely, absence of SREBP-1 suppressed Western diet-induced hyperlipidemia in LDLR-deficient mice and ameliorated atherosclerosis. In contrast, bone marrow-specific SREBP-1 deficiency did not alter the development of atherosclerosis. The size of nascent VLDL particles secreted from the liver was increased in SREBP-1c transgenic mice and reduced in SREBP-1-deficient mice, accompanied by upregulation and downregulation of phospholipid transfer protein expression, respectively. Conclusion-Hepatic SREBP-1c determines plasma triglycerides and remnant cholesterol and contributes to atherosclerosis in hyperlipidemic states. Hepatic SREBP-1c also regulates the size of nascent VLDL particles. (Arterioscler Thromb Vasc Biol. 2011;31:1788-1795.)

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high- fat diet or mated to leptin- deficient ob/ ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate- 2 and suppression of hepatic protein kinase C e activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.