Toshihiko Iwanaga

Impact of UCP1 and β3AR gene polymorphisms on age-related changes in brown adipose tissue and adiposity in humans

Background:Brown adipose tissue (BAT) is involved in the regulation of whole-body energy expenditure and adiposity. The activity and prevalence of BAT decrease with age in humans.Objective:To examine the effects of single nucleotide polymorphisms of the genes for uncoupling protein 1 (UCP1) and β3-adrenergic receptor (β3AR), key molecules of BAT thermogenesis, on age-related decline of BAT activity and accumulation of body fat in humans.Methods:One hundred ninety-nine healthy volunteers (20–72u2009years old (y.o.)) underwent fluorodeoxyglucose-positron emission tomography (FDG-PET) and computed tomography (CT) after 2-h cold exposure to assess BAT activity. The visceral and subcutaneous fat areas at the abdominal level were estimated from the CT images. They were genotyped for −3826 A/G polymorphism of the UCP1 gene and 64 Trp/Arg mutation of the β3AR gene.Results:BAT was detected in 88 subjects out of 199 (44%), more in younger (⩽30u2009y.o., 55%) than older subjects (>40u2009y.o., 15%). BAT prevalence of older subjects tended to be lower in the UCP1 G/G group than the A allele group (A/A and A/G), and also in the β3AR Arg allele group (Trp/Arg and Arg/Arg) than the Trp/Trp group. When compared subjects who had two or more base substitutions on the two genes (the 2–4 allele group) with those who had less than two base substitutions (the 0–1 allele group), BAT prevalence was comparable in younger subjects (62% vs 50%) but lower in older subjects (0% vs 24%, P<0.05). Visceral fat area of the 2–4 allele group was higher than that of the 0–1 allele group (P<0.05) in older subjects, but not in younger subjects.Conclusion:UCP1 −3826 A/G and β3AR 64 Trp/Arg substitutions accelerate age-related decrease in BAT activity, and thereby may associate with visceral fat accumulation with age.

Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the β-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that β-carotene 15,15′-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

Differential Cellular Expression of Galectin Family mRNAs in the Epithelial Cells of the Mouse Digestive Tract

Galectin is an animal lectin that recognizes β-galactosides of glycoconjugates and is abundant in the gut. This study revealed the cellular expression of galectin subtypes throughout the mouse digestive tract by in situ hybridization. Signals for five subtypes (galectin-2, -3, -4/6, and -7) were detected exclusively in the epithelia. In the glandular stomach, galectin-2 and -4/6 were predominantly expressed from gastric pits to neck of gastric glands, where mucous cells were the main cellular sources. The small intestine exhibited intense, maturation-associated expressions of galectin-2, -3, and -4/6 mRNAs. Galectin-2 was intensely expressed from crypts to the base of villi, whereas transcripts of galectin-3 gathered at villous tips. Signals for galectin-4/6 were most intense at the lower half of villi. Galectin-2 was also expressed in goblet cells of the small intestine but not in those of the large intestine. In the large intestine, galectin-4/6 predominated, and the upper half of crypts simultaneously contained transcripts of galectin-3. Stratified epithelium from the lip to forestomach and anus intensely expressed galectin-7 with weak expressions of galectin-3. Because galectins in the digestive tract may be multi-functional, information on their cell/stage-specific expression contributes to a better understanding of the functions and pathological involvements of galectins.

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.

Immunohistochemical and in situ hybridization analysis of galectin-3, a β-galactoside binding lectin, in the urinary system of adult mice

Galectin is an animal lectin that has high affinity to β-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.

Dmbx1 is essential in agouti-related protein action

Dmbx1 is a paired-class homeodomain transcription factor. We show here that mice deficient in Dmbx1 exhibit severe leanness associated with hypophagia and hyperactivity and that isolation of a Dmbx1−/− mouse from its cohabitants induces self-starvation, sometimes leading to death, features similar to those of anorexia nervosa in humans. Interestingly, overexpression of agouti in Dmbx1−/− mice failed to induce aspects of the Ay/a phenotype, including hyperphagia, obesity, and diabetes mellitus. In Dmbx1−/− mice, administration of agouti-related protein increased cumulative food intake for the initial 6 h but significantly decreased it over 24- and 48-h periods. In addition, Dmbx1 was shown to be expressed at embryonic day 15.5 in the lateral parabrachial nucleus, the rostral nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus, and the reticular nucleus in the brainstem, all of which receive melanocortin signaling, indicating involvement of Dmbx1 in the development of the neural network for the signaling. Thus, Dmbx1 is essential for various actions of agouti-related protein and plays a role in normal regulation of energy homeostasis and behavior.

Functional analysis of transcriptional repressor Otx3/Dmbx1

Otx3/Dmbx1 is a member of paired class homeodomain transcription factors. In this study, we found that Otx3/Dmbx1 represses the Otx2‐mediated transactivation by forming heterodimer with Otx2 on the P3C (TAATCCGATTA) sequence in vitro. The 156 amino acid region (residues 1–156) of Otx3/Dmbx1 is required for its repressor activity, and interacts directly with Otx2. Co‐localization of Otx3/Dmbx1 and Otx2 in brain sections was confirmed by in situ hybridization. These data suggest that Otx3/Dmbx1 represses Otx2‐mediated transcription in the developing brain. We also identified the consensus binding sequence [TAATCCGATTA and TAATCC(N2‐4)TAATCC] of Otx3/Dmbx1.

Cellular expression of Noc2, a Rab effector protein, in endocrine and exocrine tissues in the mouse.

Noc2 is a Rab effector which participates in regulated exocytosis. It is expressed abundantly in endocrine cells but at low levels in exocrine tissues. Noc2-deficient mice, however, exhibit marked accumulation of secretory granules in exocrine cells rather than endocrine cells. In the present study, we investigated localization of Noc2 immunohistochemically in various endocrine and exocrine tissues in normal mice. Western blotting detected a Noc2-immunoreactive band of 38xa0kDa in isolated pancreatic islets, the adrenal gland, pituitary gland, and thyroid gland. Immunostaining for Noc2 labeled endocrine cells in the adrenal medulla and adenohypophysis, pancreatic islet cells, thyroid parafollicular cells, and gut endocrine cells, supporting the notion that Noc2 is a Rab effector protein shared by amine/peptide-secreting endocrine cells. Besides endocrine tissues, granular ducts in salivary glands contained Noc2. Although immunostaining failed to detect Noc2 in acinar cells of all exocrine glands examined, reverse transcriptase-polymerase chain reaction analysis detected the mRNA expression in exocrine pancreas. Ultrastructurally, Noc2 immunoreactivity was associated with the limiting membrane of granules in both pancreatic endocrine and salivary duct exocrine cells. The cellular and subcellular localizations of Noc2 should yield key information on its functional significance as well as account for the phenotype in Noc2-deficient mice.

Galectin-1 and galectin-3 in the corpus luteum of mice are differentially regulated by prolactin and prostaglandin F2α.

Galectin-1 and galectin-3, β-galactoside-binding lectins, are specifically expressed in the regressing corpus luteum (CL) of mice; however, their function remains unclear. In this study, we examined the effects of prolactin (PRL) and prostaglandin F(2) (α) (PGF(2) (α)), two main regulatory molecules of mouse CL function, on galectin expression. In situ hybridization analysis clearly demonstrated an initial increase in galectin-1 in the newly formed CL (CLN) after postpartum ovulation 48 u200ah after compulsory weaning. This was accompanied by a decline in 3β-hydroxysteroid dehydrogenase (3β-HSD) and LH receptor (LH-R) expression, suggesting a withdrawal of PRL stimulation. At 72 u200ah after the weaning, the expression of both galectins in CLN was remarkably increased, being associated with an intense expression of progesterone degradation enzyme (20α-HSD). Compulsory weaning did not significantly alter both galectin expression in the remaining CL of pregnancy (CLP), while PGF(2) (α) strongly upregulated both galectin expression only in the remaining CLP, which lacked LH-R in postpartum mice. Administration of bromocriptine, an antagonist for PRL secretion, to nonpregnant cyclic mice induced an accumulation of galectin-1 - but not galectin-3 - in all CL of various generations, and additional PRL treatment reduced its accumulation, suggesting a direct suppressive effect of PRL on galectin-1 expression. Although the function and regulatory mechanism of galectin in the CL is not fully understood, PGF(2) (α) is an excellent candidate that regulates galectin expression, but its effect may be abolished by LH-R-mediated signal. PRL withdrawal seems to be necessary for an initiation of luteolysis and the following PGF(2) (α)-induced galectin expression.