Seiichiro Aoe

Role of the Toll-like Receptor 4/NF-κB Pathway in Saturated Fatty Acid–Induced Inflammatory Changes in the Interaction Between Adipocytes and Macrophages

Objective—Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-α derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. Methods and Results—Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-&kgr;B (NF-&kgr;B), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-&kgr;B markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFα production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the β3-adrenergic stimulation, resulted in the activation of the TLR4/NF-&kgr;B pathway. Conclusion—These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-&kgr;B activation.

Increased Adiponectin Secretion by Highly Purified Eicosapentaenoic Acid in Rodent Models of Obesity and Human Obese Subjects

Objectives—Fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) or n-3 PUFAs have been shown to reduce the incidence of coronary heart disease. Here we investigated the effect of highly purified eicosapentaenoic acid (EPA) on production of adiponectin, the only established antiatherogenic and antiinflammatory adipocytokine, in rodent models of obesity and human obese subjects. Methods and Results—We demonstrated that EPA increases adiponectin secretion in genetically obese ob/ob mice and high-fat diet–induced obese mice. In the in vitro coculture of adipocytes and macrophages, EPA reversed the coculture-induced decrease in adiponectin secretion at least in part through downregulation of tumor necrosis factor-&agr; in macrophages. We also showed significant increase in plasma adiponectin concentrations in human obese subjects after a 3-month treatment with EPA (1.8 g daily). Multivariate regression analysis revealed that EPA treatment is the only independent determinant of plasma adiponectin concentrations. Conclusion—This study demonstrates that EPA increases adiponectin secretion in rodent models of obesity and human obese subjects, possibly through the improvement of the inflammatory changes in obese adipose tissue. Because EPA has reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial, this study provides important insight into its therapeutic implication in obesity-related metabolic sequelae.

Milk basic protein: a novel protective function of milk against osteoporosis

Milk is recommended as an excellent calcium source for bone health. Moreover, milk is considered to contain other components effective for bone health. In our previous studies, using an unfractionated bone cell culture system, we found that milk whey protein, especially its basic fraction (milk basic protein [MBP]), suppressed bone resorption. In this present study, we investigated whether MBP could prevent bone loss in aged ovariectomized rats. Twenty-one 51-week-old female Sprague-Dawley rats were ovariectomized (ovx), and another seven rats received a sham operation (sham). After a 4-week recovery period, the ovx rats were separated into three groups, and they were then fed a control diet, a 0.01% MBP diet (0. 01% casein of the control diet replaced with MBP), or a 0.1% MBP diet for 17 weeks. The sham rats were fed the control diet. Bone mineral density (BMD) of the femur was measured by dual-energy X-ray absorptiometry in vivo. The BMD in the ovx-control group noticeably decreased during the experimental period in comparison with that in the sham group. However, the BMD in the OVX-0.1% MBP group was significantly higher than that in ovx-control group at weeks 12 and 16 (p < 0.05). After the 17-week feeding period, the breaking energy of the excised femur of all groups was determined by use of a three-point bending rheolometer. The breaking energy in the ovx-control group was significantly lower than that in the sham group (p < 0.05). However, the breaking energy in the ovx-0.1% MBP group was significantly higher than that of the ovx-control group (p < 0.05). Urinary deoxypyridinoline (D-Pyr) level of the ovx-control group was higher than that of the sham group, whereas the level of D-Pyr excretion in the ovx-0.01% MBP and ovx-0.1% MBP groups was significantly lower than that of the ovx-control group (p < 0.05). These results suggest that MBP suppresses the osteoclast-mediated bone resorption and prevents bone loss caused by ovariectomy. Moreover, we performed an in vitro study using isolated osteoclasts from rabbit bone to investigate the possible mechanism. MBP dose-dependently suppressed the number of pits formed by these osteoclasts. This result indicates that MBP suppresses bone resorption by its direct effects on osteoclasts. To our knowledge, this study provides the first evidence that MBP directly suppresses osteoclast-mediated bone resorption, resulting in the prevention of the bone loss that occurs in ovx rats.

Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis

In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.

Milk sphingomyelin accelerates enzymatic and morphological maturation of the intestine in artificially reared rats.

Objectives Sphingomyelin (SPM) is the dominant phospholipid, comprising 38% of total human milk phospholipids. Although little is known about the nutritional importance of SPM during the neonatal period, SPM may affect the growth and development of tissues in the newborn infant through mechanisms regulating cell proliferation and differentiation. We evaluated the effect of sphingomyelin (SPM) in artificially reared rats as a suitable model of gut maturation in the suckling infant. Methods Seven-day-old Sprague-Dawley rat pups were cannulated intragastrically and reared artificially on milk containing 0.5% SPM or 0.5% phosphatidylcholine (PC) for 1 week. Results Intestinal lactase activity in the SPM group was significantly lower than that in the control or PC group. Upon histologic examination, intestinal villi were found to be occupied with vacuolated cells in the control and the PC group, whereas the vacuolated cells were restricted to the tip of villi in the SPM group. The Auerbach nerve plexus area of the ileum in the SPM group was significantly greater, possibly due to accelerated development, than that in the control group or PC group. Conclusions The present results suggest that SPM, the dominant phospholipid in milk, plays an important role in neonatal gut maturation during the suckling period.

Milk Basic Protein (MBP) Increases Radial Bone Mineral Density in Healthy Adult Women

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.

Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.

Although macrophages are thought to be crucial for the pathogenesis of chronic inflammatory diseases, how they are involved in disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is poorly understood. Here we report the unique histological structure termed “hepatic crown-like structures (hCLS)” in the mouse model of human NASH; melanocortin-4 receptor deficient mice fed a Western diet. In hCLS, CD11c-positive macrophages aggregate to surround hepatocytes with large lipid droplets, which is similar to those described in obese adipose tissue. Histological analysis revealed that hCLS is closely associated with activated fibroblasts and collagen deposition. When treatment with clodronate liposomes effectively depletes macrophages scattered in the liver, with those in hCLS intact, hepatic expression of inflammatory and fibrogenic genes is unaffected, suggesting that hCLS is an important source of inflammation and fibrosis during the progression of NASH. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. We also observed increased number of hCLS in the liver of non-alcoholic fatty liver disease/NASH patients. Collectively, our data provide evidence that hCLS is involved in the development of hepatic inflammation and fibrosis, thereby suggesting its pathophysiologic role in disease progression from simple steatosis to NASH.

Effects of rice bran hemicellulose on 1,2‐dimethylhydrazine‐induced intestinal carcinogenesis in fischer 344 rats

The effect of rice bran hemicellulose (RBH) on 1,2-dimethylhydrazine- (DMH) induced intestinal carcinogenesis was studied in male Fischer 344 rats. Rats were fed a basal control diet or a diet containing 2% or 4% RBH at five weeks of age. At 6 weeks of age, all animals were given an intraperitoneal injection of DMH (20 mg/kg body wt) at weekly intervals for 20 weeks and autopsied 7 weeks after the last injection. The incidence of DMH-induced colon tumors was significantly lower in rats fed the 4% RBH diet than in rats fed the basal control diet (p < 0.05). The number of colon tumors per rat was also significantly lower in rats fed the 4% RBH diet than in rats fed the basal control diet (p < 0.05). The present study suggested that the water-soluble RBH played a preventive role in DMH-induced large bowel carcinogenesis in Fischer 344 rats.

Comparison of the effects of milk components and calcium source on calcium bioavailability in growing male rats

Abstract We compared the effects of milk components (lactose and milk protein) and calcium source on calcium bioavailability in growing male rats. Six-week-old male Sprague-Dawley rats were divided into four groups (Milk components (MC)+CaCO 3 , CaCO 3 , MC+WheyCa and WheyCa). Each group of rats was fed either a diet containing milk components plus CaCO 3 or whey Ca, or a milk components-free diet containing CaCO 3 or whey Ca for 46 days. Calcium bioavailability was defined by apparent calcium absorption rate and bone mineral density (BMD). Two-way ANOVA was used to estimate the effects of milk components and calcium source on calcium bioavailability. The apparent calcium absorption rate was estimated at 7 and 12 weeks of age. The type of dietary calcium source did not affect the calcium absorption rate in either period. However, the milk components significantly increased the apparent calcium absorption rate at 7 weeks of age, and tended to increase the apparent calcium absorption rate at 12 weeks of age (p=0.09). Moreover, the milk components increased the apparent magnesium absorption rate in both periods. The type of dietary calcium source did not affect the BMD of the femur or lumbar vertebrae (L3–L5). However, the milk components diet increased the BMD of femur and L3–L5. These data indicate that the administration of milk components is more effective for the enhancement of calcium bioavailability rather than the chemical form of calcium in growing rats.

Small Intestinal Goblet Cell Proliferation Induced by Ingestion of Soluble and Insoluble Dietary Fiber Is Characterized by An Increase in Sialylated Mucins in Rats

The study aimed to examine the effects of insoluble and soluble fibers on mucin sialylation and sulfation in the small intestine. First, diets containing soluble [konjac mannan (KM), psyllium, or guar gum; 50 g/kg) or insoluble (polystyrene foam, wheat bran, or cornhusk; 80 g/kg) fiber were fed to rats for 13 d. The fiber-fed groups had more goblet cells in the ileum than the fiber-free control group. High-iron diamine/alcian blue staining showed more sialylated mucin-producing cells in the fiber-fed groups than in the control, whereas sulfated mucin-producing cells were fewer (insoluble fibers) or unchanged (soluble fibers). Second, feeding KM (50 g/kg) and beet fiber (BF) (80 g/kg) diets for 7 d yielded a higher ileum Siat4C expression than the control, but Gal3ST2 and Gal3ST4 expression was comparable. Luminal mucin content correlated with sialic acid (r = 0.96; P < 0.001) or sulfate (r = 0.62; P < 0.01), but the slope of the sialic acid-derived equation was greater than that of the sulfate-derived equation, indicating a preferred increase in sialylated mucins. Third, rats were fed the control diet for 10 d while receiving antibiotic treatment. Analysis of the luminal mucin showed that sialylated mucins were more vulnerable to bacterial degradation than sulfated mucins. Finally, a study of bromo-deoxyuridine incorporation in rats fed a BF diet indicated that goblet cell proliferation accompanied by increased sialylated mucin appeared to be related to accelerated ileal epithelial cell migration. We conclude that intestinal goblet cell responses to insoluble and soluble fibers are characterized by increases in sialylated mucin production.