Elevated levels of proinflammatory volatile metabolites in feces of high fat diet fed KK-Ay mice.

When the microfloral composition deteriorates, it triggers low-level chronic inflammation associated with several lifestyle-related diseases including obesity and diabetic mellitus. Fecal volatile organic compounds (VOCs) have been found to differ in gastrointestinal diseases as well as intestinal infection. In this study, to evaluate a potential association between the pathogenesis of lifestyle-related diseases and VOCs in the intestinal tract, fecal VOCs from obese/diabetic KK-Ay mice (KK) or controls (C57BL/6J mice; BL) fed a normal or high fat diet (NFD or HFD) were investigated using headspace sampler-GC-EI-MS. Principal component analysis (PCA) of fecal VOC profiles clearly separated the experimental groups depending on the mouse lineage (KK vs BL) and the diet type (NFD vs HFD). 16 s rRNA sequencing revealed that the PCA distribution of VOCs was in parallel with the microfloral composition. We identified that some volatile metabolites including n-alkanals (nonanal and octanal), acetone and phenol were significantly increased in the HFD and/or KK groups. Additionally, these volatile metabolites induced proinflammatory activity in the RAW264 murine macrophage cell line indicating these bioactive metabolites might trigger low-level chronic inflammation. These results suggest that proinflammatory VOCs detected in HFD-fed and/or diabetic model mice might be novel noninvasive diagnosis biomarkers for diabetes.

Intestinal microbial metabolite stercobilin involvement in the chronic inflammation of ob/ob mice.

It is crucial that the host and intestinal microflora interact and influence each other to maintain homeostasis and trigger pathological processes. Recent studies have shown that transplantation of the murine intestinal content to recipient germ-free mice enables transmission of the donor's phenotypes, such as low level chronic inflammation associated with lifestyle-related diseases. These findings indicate that intestinal bacteria produce some molecules to trigger pathological signals. However, fecal microbial metabolites that induce obesity and the type II diabetic phenotype have not been fully clarified. Here, we showed that the intestinal bacterial metabolite stercobilin, a pigment of feces, induced proinflammatory activities including TNF-α and IL-1ß induction in mouse macrophage RAW264 cells. Proinflammatory stercobilin levels were significantly higher in ob/ob mice feces than in the feces of control C57BL/6 J mice. Moreover, in this study, we detected stercobilin in mice plasma for the first time, and the levels were higher in ob/ob mice than that of C57BL/6 J mice. Therefore, stercobilin is potentially reabsorbed, circulated through the blood system, and contributes to low level chronic inflammation in ob/ob mice. Since, stercobilin is a bioactive metabolite, it could be a potentially promising biomarker for diagnosis. Further analyses to elucidate the metabolic rate and the reabsorption mechanism of stercobilin may provide possible therapeutic and preventive targets.

Identification of soybean peptide leginsulin variants in different cultivars and their insulin-like activities.

We have recently reported that green soybean cultivar, echigomidori, and not the yellow cultivar, fukuyutaka, is a rich source of hormone-like peptide leginsulin consisting of 37 amino acids (Leg_1_37, PDB 1JU8A) and its C-terminal glycine deletant, Leg_1_36. Green soybean is mature, but the color of the seedcoat and cotyledon remains green. Therefore, in this study, we examined the leginsulin content in different varieties of 11 colored soybeans (including green, yellow, red, brown and black) and edamame (immature soybean). Profile analysis of soybean constituents by LC-MS showed that Leg_1 (36 + 37) detected as a prominent peak in 3 green and 1 yellow soybean cultivar was the strongest contributor in principal component analysis, indicating Leg_1 is the most characteristic feature for distinguishing soybean cultivars. However, smaller amounts of leginsulin-like peptides, defined as Leg_2 and Leg_3, were detected in other samples. The cDNA sequences and LC-MS/MS analyses revealed that Leg_2 was a homologue of Leg_1 with three amino acid substitutions derived from SNPs, while Leg_3 was a Leg_1/Leg_2 paralog. Expression levels of Leg_1 were markedly higher than Leg_2 and Leg_3. Additionally, in glucose uptake assay, purified TRX-His-tag fused recombinant Leg_1_37 prepared by bacterial expression showed stronger insulin-like activities than other variants including Leg_2, Leg_3, and their Gly deletants in myotube-like differentiated L6 and C2C12 cells. These results suggest that dietary consumption of soybean seed, especially including a higher amount of Leg_1_37, could be useful for lowering of blood glucose.

Effects of Sanyaku and Its Constituent Diosgenin on the Fasted and Postprandial Hypertriacylglycerolemia in High-Fat-Diet-Fed KK- A y Mice.

In this study, we examined the fasted and postprandial triacylglycerol (TG) levels in KK- A y mice fed a high-fat diet (HFD) or a HFD containing either 500 ppm (0.05%) of diosgenin or 500 ppm (0.05%) of diosgenin-containing Chinese yam sanyaku. Oral fat tolerance tests revealed that, not only in the fasting state but also after loading of lipid emulsion, plasma levels of TG were significantly reduced in sanyaku- and diosgenin- fed mice. Levels of fat oxidation, especially in the dark phase (from 7 p.m. to 7 a.m.), were increased in the sanyaku and diosgenin groups. Moreover mRNA levels of lipoprotein lipase and peroxisome proliferator-activated receptor γ, coactivator 1α were moderately upregulated in the liver of sanyaku- and diosgenin-ingested mice. These results suggest that consecutive ingestion of diosgenin or diosgenin-containing sanyaku at the dose achievable in a human diet potentially ameliorates fasted and postprandial hypertriacylglycerolemia, which could be associated with the improvement of TG metabolism.

FGF21 Alleviates Hepatic Endoplasmic Reticulum Stress under Physiological Conditions.

Fibroblast growth factor 21 (FGF21), mainly synthesized and secreted from the liver, is an endocrine FGF that regulates glucose and fatty acid metabolism to maintain whole body energy homeostasis. Gene expression of FGF21 was previously reported to be induced by endoplasmic reticulum (ER) stress through activating transcription factor 4 (ATF4). It has been reported that drug-induced ER stress is reduced by overexpression of FGF21. However, the function of endogenous FGF21 under physiological conditions such as the postprandial state remains unknown. Here, we examined the effects of both endogenous and exogenous FGF21 on postprandial hepatic ER stress. In mice, postprandial and tunicamycin-induced ER stress was significantly reduced by overexpression of FGF21 using a recombinant adenovirus. FGF21-deficient mice exhibited a more considerable increase in drug-induced ER stress target gene expression than wild-type mice. Following refeeding after fasting, FGF21 deficiency caused severe ER stress in the liver. The postprandial ER stress response was significantly reduced when hepatic FGF21 gene expression was increased by feeding a diet containing the soy protein ß-conglycinin which activates ATF4. Together, these results demonstrate that FGF21 reduces the increased expression of a subset of genes in the liver in response to ER stress and may correct metabolic disorders caused by ER stress.

BCL11B gene heterozygosity causes weight loss accompanied by increased energy consumption, but not defective adipogenesis, in mice.

BCL11B is a zinc finger-type transcription factor that regulates the development of the white adipose tissue (WAT), skin, central nervous system, and immune system. BCL11B is required for proper adipocyte differentiation, and BCL11B-/- embryos at E19.5 have very low amounts of the subcutaneous WAT. Here, we demonstrated that BCL11B+/- mice have lower body weight than BCL11B+/+ mice, whereas the expression of adipogenic marker genes in the WAT was comparable between BCL11B+/+ and BCL11B+/- mice. Histological analysis indicated that BCL11B+/- mice fed a high-fat diet have much smaller white adipocytes and lipid droplets in the WAT and liver, respectively. In addition, BCL11B+/- mice had increased energy consumption under both standard and high-fat diets. Thus, this study identifies BCL11B as a regulator of energy metabolism, and it is unlikely that BCL11B functions in the WAT contribute to energy metabolism in BCL11B+/- mice.

Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

Identification of BCL11B as a regulator of adipogenesis.

The differentiation of preadipocytes into adipocytes is controlled by several transcription factors, including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), which are known as master regulators of adipogenesis. BCL11B is a zinc finger-type transcription factor that regulates the development of the skin and central nervous and immune systems. Here, we found that BCL11B was expressed in the white adipose tissue (WAT), particularly the subcutaneous WAT and that BCL11B(-/-) mice had a reduced amount of subcutaneous WAT. During adipogenesis, BCL11B expression transiently increased in 3T3-L1 preadipocytes and mouse embryonic fibroblasts (MEFs). The ability for adipogenesis was reduced in BCL11B knockdown 3T3-L1 cells and BCL11B(-/-) MEFs, whereas the ability for osteoblastogenesis was unaffected in BCL11B(-/-) MEFs. Luciferase reporter gene assays revealed that BCL11B stimulated C/EBPß activity. Furthermore, the expression of downstream genes of the Wnt/ß-catenin signaling pathway was not suppressed in BCL11B(-/-) MEFs during adipogenesis. Thus, this study identifies BCL11B as a novel regulator of adipogenesis, which works, at least in part, by stimulating C/EBPß activity and suppressing the Wnt/ß-catenin signaling pathway.

Single ingestion of soy ß-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects.

Soy protein ß-conglycinin has serum lipid-lowering and anti-obesity effects. We showed that single ingestion of ß-conglycinin after fasting alters gene expression in mouse liver. A sharp increase in fibroblast growth factor 21 (FGF21) gene expression, which is depressed by normal feeding, resulted in increased postprandial circulating FGF21 levels along with a significant decrease in adipose tissue weights. Most increases in gene expressions, including FGF21, were targets for the activating transcription factor 4 (ATF4), but not for peroxisome proliferator-activated receptor α. Overexpression of a dominant-negative form of ATF4 significantly reduced ß-conglycinin-induced increases in hepatic FGF21 gene expression. In FGF21-deficient mice, ß-conglycinin effects were partially abolished. Methionine supplementation to the diet or primary hepatocyte culture medium demonstrated its importance for activating liver or hepatocyte ATF4-FGF21 signaling. Thus, dietary ß-conglycinin intake can impact hepatic and systemic metabolism by increasing the postprandial circulating FGF21 levels.

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α.

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.

Consumption of soy protein isolate reduces hepatic SREBP-1c and lipogenic gene expression in wild-type mice, but not in FXR-deficient mice.

We examined to determine whether hepatic gene expression is affected in mice in which blood lipid levels remain unchanged fed soy protein isolate (SPI) for a short time. We also examined SPI-mediated effects in farnesoid X receptor (FXR)-deficient mice. Compared with casein, SPI affected the expression of various hepatic genes related to lipid metabolism in the wild-type mice. No effects of SPI were observed in the FXR-deficient mice, suggesting the importance of FXR. Hepatic peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) gene expression was reduced by SPI, and this might be associated with a decrease in FXR expression. Decreased FXR led to decreased expression of its target, the bile-salt export pump necessary for bile acid secretion and dietary lipid absorption. The earliest response to SPI was a decrease in hepatic sterol regulatory element-binding protein (SREBP)-1c mRNA, on day 3. SPI activated hepatic adenosine monophosphate-activated protein kinase (AMPK), which can lead to a reduction in SREBP-1c mRNA. These data indicate the importance of SREBP-1c and PGC-1α/FXR in SPI-mediated alterations in hepatic gene expression.

Glutamine stimulates the gene expression and processing of sterol regulatory element binding proteins, thereby increasing the expression of their target genes.

Here we show that the larger the amount of glutamine added to the medium, the more the expression of genes related to lipid homeostasis is promoted by the activation of sterol regulatory element binding proteins (SREBPs) at the transcriptional and post-translational levels in human hepatoma HepG2 cells. Glutamine increases the mRNA levels of several SREBP targets, including SREBP-2. The gene expression of SREBP-1a, a predominant form of SREBP-1 in most cultured cells and a target of the general transcription factor Sp1, is significantly augmented by glutamine via an increased binding of Sp1 to the SREBP-1a promoter. In contrast, the increased expression of SREBP targets including SREBP-2 is due to stimulation of the processing of SREBP proteins by glutamine. It is also shown that glutamine accelerates SREBP processing through increased transport of the SREBP/SREBP cleavage-activating protein complex from the endoplasmic reticulum to the Golgi apparatus. The processing of activating transcription factor 6 is activated by the same proteases as SREBPs in the Golgi in response to endoplasmic reticulum stress and is not induced by glutamine. Taken together, these results clearly demonstrate that glutamine brings about not only the induction of SREBP-1a transcription but also the stimulation of SREBP processing, thereby facilitating the gene expression of SREBP targets in cultured cells.

Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet.

TGR5 is a member of the G protein-coupled receptor family and is activated by bile acids (BAs). TGR5 is thought to be a promising drug target for metabolic diseases because the activation of TGR5 prevents obesity and hyperglycemia in mice fed a high-fat diet (HFD). In the present study, we identified a naturally occurring limonoid, nomilin, as an activator of TGR5. Unlike BAs, nomilin did not exhibit the farnesoid X receptor ligand activity. Although the nomilin derivative obacunone was capable of activating TGR5, limonin (the most abundant limonoid in citrus seeds) was not a TGR5 activator. When male C57BL/6J mice fed a HFD for 9 weeks were further fed a HFD either alone or supplemented with 0.2%w/w nomilin for 77 days, nomilin-treated mice had lower body weight, serum glucose, serum insulin, and enhanced glucose tolerance. Our results suggest a novel biological function of nomilin as an agent having anti-obesity and anti-hyperglycemic effects that are likely to be mediated through the activation of TGR5.