Kentaro Ozawa

The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43

The gut microbiota affects nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. During feeding, gut microbes produce short-chain fatty acids, which are important energy sources for the host. Here we show that the short-chain fatty acid receptor GPR43 links the metabolic activity of the gut microbiota with host body energy homoeostasis. We demonstrate that GPR43-deficient mice are obese on a normal diet, whereas mice overexpressing GPR43 specifically in adipose tissue remain lean even when fed a high-fat diet. Raised under germ-free conditions or after treatment with antibiotics, both types of mice have a normal phenotype. We further show that short-chain fatty acid-mediated activation of GPR43 suppresses insulin signalling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of unincorporated lipids and glucose in other tissues. These findings establish GPR43 as a sensor for excessive dietary energy, thereby controlling body energy utilization while maintaining metabolic homoeostasis.

Prolonged Endoplasmic Reticulum Stress in Hypertrophic and Failing Heart After Aortic Constriction Possible Contribution of Endoplasmic Reticulum Stress to Cardiac Myocyte Apoptosis

Background—The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear. Methods and Results—Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)–positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12–, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels. Conclusions—These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.

ORP150/HSP12A protects renal tubular epithelium from ischemia-induced cell death

The 150 kDa oxygen‐regulated protein (ORP150) is an inducible endoplasmic reticulum (ER) chaperone with cytoprotective properties in settings of cell stress, such as ischemia/reperfusion (I/R). Renal tissue from patients with acute renal failure displayed strong induction of ORP150 in tubular epithelium. In a rodent model of renal I/R injury, ORP150 was expressed in both the ischemic and contralateral kidney, principally in the thick ascending loop of Henle (TAL) and distal tubules. Cultured renal epithelial cells exposed to hypoxic or hyperosmotic conditions displayed induction of ORP150. Renal tubular epithelial cells stably transfected with ORP150 sense or antisense cDNA displayed a strong correlation between ORP150 expression and vulnerability to hypoxic/osmotic stress; higher levels of ORP150 were protective, whereas lower levels increased susceptibility to cell death. Compared with nontransgenic controls, transgenic mice overexpressing ORP150 subjected to renal I/R displayed a blunted rise of serum creatinine and blood urea nitrogen, and enhanced survival of TAL, consistent with cytoprotection. In contrast, heterozygous ORP150+/− mice, with lower levels of ORP150, showed enhanced renal injury. These data are consistent with the possibility that ORP150 exerts cytoprotective effects in renal tubular epithelia subjected to I/R injury and suggest a key role for ER stress in the renal tubular response to acute renal failure.

ORP150/HSP12A Regulates Purkinje Cell Survival: A Role for Endoplasmic Reticulum Stress in Cerebellar Development

The endoplasmic reticulum (ER) stress response contributes to neuronal survival in ischemia and neurodegenerative processes. ORP150 (oxygen-regulated protein 150)/HSP12A (heat shock protein 12A), a novel stress protein located in the ER, was markedly induced in Purkinje cells maximally at 4-8 d after birth, a developmental period corresponding to their vulnerability to cell death. Both terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling analysis and immunostaining using anti-activated caspase-3 antibody revealed that transgenic mice with targeted neuronal overexpression of ORP150 (Tg ORP150) displayed diminished cell death in the Purkinje cell layer and increased numbers of Purkinje cells up to 40 d after birth (p < 0.01), compared with those observed in heterozygous ORP150/HSP12A-deficient (ORP150+/-) mice and wild-type littermates (ORP150+/+). Cultured Purkinje cells from Tg ORP150 mice displayed resistance to both hypoxia- and AMPA-induced stress. Behavioral analysis, using rotor rod tasks, indicated impairment of cerebellar function in Tg ORP150 animals, consistent with the concept that enhanced survival of Purkinje cells results in dysfunction. These data suggest that ER chaperones have a pivotal role in Purkinje cell survival and death and thus may highlight the importance of ER stress in neuronal development.

Short-chain fatty acid receptor GPR41-mediated activation of sympathetic neurons involves synapsin 2b phosphorylation

Synapsins are neuronal phosphoproteins that coat synaptic vesicles and are believed to function in the regulation of neurotransmitter release. The signaling mechanism for short‐chain free fatty acid (SCFA)‐stimulated NE release was examined using primary‐cultured mouse sympathetic cervical ganglion neurons. Pharmacological and knockdown experiments showed that activation of sympathetic neurons by SCFA propionate involves SCFA receptor GPR41 linking to Gβγ‐PLCβ3‐ERK1/2‐synapsin 2 signaling. Further, synapsin 2b directly interacts with activated ERK1/2 and can be phosphorylated on serine when SCFA activates sympathetic neurons.

S-nitrosylation regulates mitochondrial quality control via activation of parkin

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinsons disease.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

Proteomic analysis of the role of S‐nitrosoglutathione reductase in lipopolysaccharide‐challenged mice

S‐Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S‐nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S‐nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O6‐alkylguanine‐DNA alkyltransferase and promotes both spontaneous and carcinogen‐induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild‐type and GSNOR‐deficient (GSNOR−/−) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two‐dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR−/− mice compared to those in the wild‐type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR−/− mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR−/− mice a large increase in the expression of the pro‐inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide‐isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR−/− mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S‐nitrosylation of two members of the PDI protein family. These results suggest that S‐nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways.

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.