Yoshinori Shimamoto

Differential alterations in levels of hepatic microsomal cytochrome P450 isozymes following intracerebroventricular injection of bacterial lipopolysaccharide in rats

Abstract To investigate the effect of central inflammation due to bacterial infection, such as meningitis, on the activities of hepatic cytochromes P450 (CYPs), rats were injected intracerebroventricularly (i.c.v.) with 0.1 μ g of bacterial lipopolysaccharide (LPS). The LPS i.c.v. injection significantly decreased the total P450 contents (by 30% of the levels of control rats treated with saline i.c.v.), the contents of CYP1A (48%), 2B (54%), 2C11 (37%) and 3A (40%) and related drug metabolizing activities, 7-ethoxycoumarin O-deethylation (36%), imipramine N-demethylation (41%) and erythromycin N-demethylation (33%) in liver microsomes 24 h after the treatment. In contrast, intraperitoneal (i.p.) injection of LPS at the same dose as i.c.v. (0.1 μ g) did not significantly affect the hepatic microsomal contents of total P450 or the content of each individual CYP isozyme and its activity. CYP2D1 protein and the activity of imipramine 2-hydroxylase were not significantly decreased by LPS injection regardless of the route of administration. The inhibitory effects of 0.1 μg i.c.v. LPS on the activities of these CYPs were almost equal to those of 10 μg i.p. LPS, and 0.01 μg of i.c.v. LPS significantly decreased the activity of imipramine N-demethylase only. Therefore, the LPS i.c.v. injection resulted in CYP isozyme-selective inhibition at an ineffective dose when injected i.p.. It is suggested that a central inflammation, such as meningitis, differentially decreases the levels of hepatic CYP isozymes. A possible involvement is discussed of the central nervous system in this down-regulation.

Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: Possible involvement of immune cells in mesenteric adipose tissue

The anti-diabetic effects of Brazilian propolis were examined using ob/ob mice. Although repeated injection of an ethanol extract of Brazilian propolis (100 mg/kg, ip, twice a week for 12 weeks) did not affect body weight gain and food intake of ob/ob mice, blood glucose and plasma cholesterol levels were significantly attenuated. Moreover, the propolis extract partially restored glucose tolerance and insulin resistance, indicating anti-diabetic properties of the extract. The propolis-treated mice exhibited lower weight gain in mesenteric adipose tissue, while weight gains in inguinal and epididymal adipose tissues were not modulated. Flow cytometric and microscopic analyses suggested that the extract promoted accumulation of eosinophils into mesenteric and epididymal adipose tissues. Alternatively, the ratio of M1-like macrophages to M2-like macrophages in mesenteric adipose tissue was reduced by the propolis injection, coincident with the decrement of the number of interleukin-12A+ cells. Levels of M1 macrophage markers, such as Itgax and Il12b transcripts, were decreased in the vascular stromal fraction of mesenteric adipose tissue, whereas those of pan-macrophage markers Emr1 and Cd68 were not influenced. Microarray and subsequent gene ontology term analyses suggested that propolis attenuated immune activation in mesenteric adipose tissues. Taken together, this indicates that Brazilian propolis improves diabetes in ob/ob mice, presumably through modification of immune cells in mesenteric adipose tissues.

CENTRAL IL-1 DIFFERENTIALLY REGULATES PERIPHERAL IL-6 AND TNF SYNTHESIS

Centrally given interleukin (IL)-1 is known to induce a rapid rises in blood IL-6. To extend this and to examine the mechanism by which this occurs, the effects of intracerebroventricular (icv) injection of human recombinant IL-1β on mRNA expression of IL-6 and tumour necrosis factor (TNF) in the spleen and liver were examined in rats. Icv injection of IL-1 produced a rapid rise of the tissue mRNA levels of IL-6 and TNF in both organs, prior to and/or in parallel with an increase in their serum levels. Pretreatment with chlorisondamine, a ganglionic blocking agent, inhibited the IL-6 responses, while it had little influence on the TNF responses. The results suggest that brain IL-1 induces peripheral production of IL-6, but not of TNF, through autonomic nervous system activation.

Mechanism of decrease in levels of hepatic P450 isozymes induced by intracerebral endotoxin: independence from sympathetic nervous and adrenocortical systems

Abstract We previously reported that lipopolysaccharide (LPS) injected intracerebroventricularly (i.c.v.) at an ineffective dose (0.1 μg/rat) decreased the drug metabolizing activities and related cytochrome P450 (CYP) isozymes in rat liver microsomes when injected intraperitoneally (i.p.). The dose study (doses <0.1 μg intracerebrally and >0.1 μg i.p.), which was carried out to examine how much more effective is i.c.v.-LPS than i.p.-LPS, showed that the pattern of alteration of expression of CYP isozymes induced by i.c.v.-LPS was different from that caused by i.p.-LPS at an effective dose (10 μg/rat). These results indicate that the decrease in hepatic CYP isozymes caused by i.c.v.-LPS could not be explained by the LPS leaked from the brain, suggesting that the decrease in hepatic CYP isozymes by i.c.v.-LPS may be caused by a central action of LPS. In this study, the possible involvement of sympathetic nervous and adrenocortical systems in the down-regulation of CYP isozymes by i.c.v.-LPS was investigated using surgical or chemical sympathetecomized or adrenalectomized rats. The norepinephrine (NE) content in the liver in rats with surgical hepatic sympathetectomy was reduced by 88% compared with that of sham-operated rats that received i.c.v.-saline and 85% compared with that of sham-operated rats that received i.c.v.-LPS, indicating that hepatic denervation was successful. The NE content in the liver in rats chemically sympathetectomized by two i.p. injections of 6-hydroxydopamine (40 mg/kg each time) 1 and 2 days before i.c.v. injection was reduced by 82% in i.c.v.-saline-treated and by 74% in i.c.v.-LPS-treated groups compared with that in rats pretreated with i.p.-saline. These results indicate that sympathetic NE terminals were effectively removed. Intracerebroventricular LPS decreased the total P450 content and the activities of CYP dependent drug metabolizing enzymes, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), imipramine N-demethylase (IMND) and erythromycin N-demethylase (ERND) after 24 h in both sympathetectomized rats and non-denervated rats. Adrenalectomy (ADX) reduced the level of corticosterone in serum by 81% compared to sham-operated rats, indicating that adrenalectomy was successful. ADX did not inhibit the decrease in the total P450 content and the metabolism of drugs induced by i.c.v.-LPS, but more profoundly emphasized the inhibitory effect of i.c.v.-LPS than the sham-operation. These results suggest that the sympathetic nervous systems both directly and indirectly innervating the liver do not participate in the primary mechanism of the decrease in the activities of CYP isozymes in rat liver microsomes induced by i.c.v.-LPS. Also, the adrenal glands, especially the adrenocortical system, play a suppressive role in the decrease in CYP isozymes caused by i.c.v.-LPS.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2‐69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI‐1/SERPINE1 genes were increased by 4‐and 1.8‐fold, respectively, after short hairpin RNA‐mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2‐69 in human myeloid cell lines. Supernatants derived from USP2‐KD cells induced IL6 (~ 6‐fold) and SAA3 (~ 15‐fold) in 3T3‐L1 adipocytes to suggest the anti‐inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2‐69 transgenic mice fed a high‐fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ~40% decrease in transcription of aP2 and PAI‐1. The aP2 locus exhibited elevated chromatin accessibility (>2.1‐fold), methylation of histone H3 lysine 4 (>4.5‐fold), and acetylation of histone H4 (>2.5‐fold) in USP2‐KD cells. Transfection of isopeptidase‐mutated USP2‐69 did not alter chromatin conformation on the aP2 locus in USP2‐KD cells. Our results suggest that USP2‐69 suppresses meta‐inflammatory molecules involved in the development of type‐2 diabetes.—Kitamura, H., Kimura, S., Shimamoto, Y., Okabe, J., Ito, M., Miyamoto, T., Naoe, Y., Kikuguchi, C., Meek, B., Toda, C., Okamoto, S., Kanehira, K., Hase, K., Watarai, H., Ishizuka, M., El‐Osta, A., Ohara, O., Miyoshi, I., Ubiquitin‐specific protease 2–69 in macrophages potentially modulates metainflammation. FASEB J. 27, 4940–4953 (2013). www.fasebj.org

Selection of suitable reference genes for mRNA quantification studies using common marmoset tissues

The common marmoset (Callithrix jacchus) is increasingly being used as a non-human primate animal model in biomedical research. To perform accurate quantitative analysis of gene expression by quantitative reverse transcription polymerase chain reaction, reliable reference genes should be selected. In this study, we evaluated the expressions of 11 widely used reference genes: ACTB, ATP5F1, B2M, GAPDH, HPRT1, PGK1, PPIA, RN18S1, RPLP0, TBP and UBC in 12 tissues and five brain areas of healthy common marmosets. NormFinder and geNorm indicated that the most suitable reference genes for cross-sectional studies of the 17 tissues were RN18S1 and RPLP0. Conversely, ACTB and PPIA were the most suitable for analyzing brain samples; however, the expression of PGK1 fluctuated among brain areas. These results indicate that suitable reference genes differ between the tissues examined. This study provides fundamental information for gene expression studies of the common marmoset and highlights the importance of validating reference genes before quantification of target mRNAs.

Brazilian propolis extract increases leptin expression in mouse adipocytes.

We investigated the anti-obesity effects of Brazilian green propolis ethanol extract using a mouse model of obesity. Repeated intraperitoneal injection of propolis (100 mg/kg twice a week) caused feeding suppression in C57BL/6 mice, whereas this treatment had negligible effects on C57BL/6 ob/ob mice. Since C57BL/6 ob/ob mice have a missense mutation in the Lep gene, leptin is likely to contribute to the propolis-induced feeding suppression. We found that propolis treatment indeed clearly increased leptin mRNA production in the visceral adipose tissues. Moreover, propolis extract directly elevated leptin expression in differentiated 3T3-L1 adipocytes. Artepillin C, an important organic compound found in Brazilian green propolis, failed to induce leptin mRNA in 3T3-L1 cells. Compounds other than artepillin C in Brazilian propolis must thus cause leptin induction in adipocytes, possibly resulting in the suppression of feeding and obesity.

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.

Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages. USP2 knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated from Usp2a transgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 in TNF, CXCL8, CCL4, and IL6 promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.

In situ hybridization study of CYP2D mRNA in the common marmoset brain.

The common marmoset is a non-human primate that has increasingly employed in the biomedical research including the fields of neuroscience and behavioral studies. Cytochrome P450 (CYP) 2D has been speculated to be involved in psycho-neurologic actions in the human brain. In the present study, to clarify the role of CYP2D in the marmoset brain, we investigated the expression patterns of CYP2D mRNA in the brain using in situ hybridization (ISH). In addition, to identify the gene location of CYP2D19, a well-studied CYP2D isoform in the common marmoset, a fluorescence in situ hybridization (FISH) study was performed. Consistent with findings for the human brain, CYP2D mRNA was localized in the neuronal cells of different brain regions; e.g., the cerebral cortex, hippocampus, substantia nigra, and cerebellum. FISH analysis showed that the CYP2D19 gene was located on chromosome 1q, which is homologous to human chromosome 22 on which the CYP2D6 gene exists. These results suggest that CYP2D in the marmoset brain may play the same role as human CYP2D6 in terms of brain actions, and that the CYP2D19 gene is conserved in a syntenic manner. Taken together, these findings suggest that the common marmoset is a useful model for studying psychiatric disorders related to CYP2D dysfunction in the brain.