Hydroxylation site-specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR.

The cholesterol metabolites, oxysterols, play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

Bile acids acting as a feeding signal and functional foods mimicking bile acid function.

To elucidate the function of the bile acid-binding receptor TGR5 in skeletal muscle, we developed transgenic mice expressing human TGR5 in the skeletal muscle tissue. A significant increase in muscle mass was observed in these transgenic mice, whereas a decrease in muscle mass was observed in the TGR5-deficient mice. Following treadmill exercise, TGR5 gene expression increased in response to ER stress induced in skeletal muscle via an ER stress response motif present in its promoter region. Exercise and rapid postprandial elevation in blood bile acid concentrations can be considered the primary stimuli for the TGR5-mediated increase in skeletal muscle mass. We developed a scoring system to identify food ingredients with TGR5 agonist activity, and identified the citrus limonoid nomilin. Similar effects were observed for other triterpenoids in addition to nomilin. Cell culture and in vivo experiments demonstrated that these food factors increase protein synthesis and muscle mass.

HNF4α is required for Tkfc promoter activation by ChREBP.

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we showed that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of 2 hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and 1 carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in the livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.

[Advanced Gastric Cancer with Virchow's Lymph Node Metastasis-A Long-Term Survival Case].

A 36-year-old woman presented to our hospital with a complaint of melena. Examinations revealed type 3 gastric cancer with left supraclavicular lymph node(Virchow's node)and para-aortic lymph node(PAN)metastases. The patient was treated with S-1 and CDDP combination chemotherapy. After 2 courses of chemotherapy, the lymph node metastases were significantly reduced. Subsequently, a total gastrectomy with D2 plus PAN dissection was performed. Histopathological examination revealed the complete absence of cancer cells in both the primary lesion of the stomach and all dissected lymph nodes. No additional surgery or radiation therapy was performed for Virchow's node metastasis. Postoperatively, she received S-1 chemotherapy for 2.5 years. She remains well 9.5 years after the surgery, without any evidence of recurrent disease.

Lactic acid bacteria-derived γ-linolenic acid metabolites are PPARδ ligands that reduce lipid accumulation in human intestinal organoids.

Gut microbiota regulate physiological functions in various hosts, such as energy metabolism and immunity. Lactic acid bacteria, including Lactobacillus plantarum, have a specific polyunsaturated fatty acid saturation metabolism that generates multiple fatty acid species, such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and trans-fatty acids. How these bacterial metabolites impact host physiology is not fully understood. Here, we investigated the ligand activity of lactic acid bacteria-produced fatty acids in relation to nuclear hormone receptors expressed in the small intestine. Our reporter assays revealed two bacterial metabolites of γ-linolenic acid (GLA), 13-hydroxy-cis-6,cis-9-octadecadienoic acid (γHYD), and 13-oxo-cis-6,cis-9-octadecadienoic acid (γKetoD) activated peroxisome proliferator-activated receptor delta (PPARδ) more potently than GLA. We demonstrate that both γHYD and γKetoD bound directly to the ligand-binding domain of human PPARδ. A docking simulation indicated that four polar residues (T289, H323, H449, and Y473) of PPARδ donate hydrogen bonds to these fatty acids. Interestingly, T289 does not donate a hydrogen bond to GLA, suggesting that bacterial modification of GLA introducing hydroxy and oxo group determines ligand selectivity. In human intestinal organoids, we determined γHYD and γKetoD increased the expression of PPARδ target genes, enhanced fatty acid ß-oxidation, and reduced intracellular triglyceride accumulation. These findings suggest that γHYD and γKetoD, which gut lactic acid bacteria could generate, are naturally occurring PPARδ ligands in the intestinal tract and may improve lipid metabolism in the human intestine.

Identification of α-ionone, nootkatone, and their derivatives as TGR5 agonists.

TGR5 is a G-protein-coupled receptor that is activated by bile acids. The activation of TGR5 in brown adipose tissue (BAT) increases energy expenditure by increasing the expression level of thermogenesis-related genes, such as peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, uncoupling protein 1, and type II iodothyronine deiodinase. Therefore, TGR5 is a potential drug target in treating obesity and associated metabolic disorders. In this study, we identified the aroma compounds α-ionone and nootkatone as well as their derivatives as TGR5 agonists by using the luciferase reporter assay system. These compounds had little effect on the activity of the farnesoid X receptor, a nuclear receptor activated by bile acids. Mice fed 0.2% α-ionone containing high-fat diet (HFD) increased the thermogenesis-related gene expression level in BAT and suppressed weight gain compared with mice fed a normal HFD. These findings indicate that aromatic compounds with TGR5 agonist activity are promising chemicals to prevent obesity.

Prognostic significance of Ishii's sarcopenia screening score for patients undergoing curative surgery for obstructive colorectal cancer after intraluminal decompression.

PURPOSE: Sarcopenia influences the short- and long-term outcomes of various medical conditions including malignancy. Ishii's screening test estimates the probability of sarcopenia based on a score calculated by three simple variables: age, grip strength, and calf circumference. We investigated the clinical significance of Ishii's score for patients with non-metastatic obstructive colorectal cancer (OCRC) who underwent curative surgery after intraluminal decompression. METHODS: Ishii's score was calculated in 79 patients with OCRC. Muscle volume loss and decreased muscle quality were evaluated by computed tomography (CT) images as skeletal muscle index (SMI) and intramuscular adipose tissue content (IMAC), respectively. RESULTS: There were 46 men and 33 women, with a median age of 70 years old. The cutoff value for Ishii's score was 155.1 and 15 patients were in the high-score group. The high-score group was significantly associated with worse time to recurrence (TTR) and overall survival (OS), and a high Ishii's score was an independent negative prognostic factor for TTR (hazard ratio = 2.93, P = 0.015). A high Ishii's score was significantly associated with a low SMI value but not with the IMAC value. CONCLUSION: A high Ishii's score was independently associated with poorer TTR in patients with non-metastatic OCRC.

A decreased preoperative platelet-to-lymphocyte ratio, systemic immune-inflammation index, and pan-immune-inflammation value are associated with the poorer survival of patients with a stent inserted as a bridge to curative surgery for obstructive colorectal cancer.

PURPOSE: Inflammation is one of the hallmarks of cancer, and inflammation-based markers that are calculated easily from laboratory results have shown predictive abilities. We investigated the prognostic values of the preoperative platelet-to-lymphocyte ratio (PLR), systemic immune-inflammation index (SII), and pan-immune-inflammation value (PIV) in patients with non-metastatic obstructive colorectal cancer (OCRC) and a self-expandable metallic stent inserted as a bridge to curative surgery. METHODS: The subjects of this retrospective study were 86 patients with pathological stage I to III OCRC. We examined the associations of these biomarkers with short- and long-term outcomes. RESULTS: Multivariate analyses revealed that a preoperative PLR < 149, SII < 597, and PIV < 209 were independently associated with poorer relapse-free survival (RFS) (P = 0.007, P < 0.001, and P = 0.002, respectively) and that a PIV < 209 was independently associated with poorer cancer-specific survival (P = 0.030). A platelet count < 240 was significantly associated with worse RFS, whereas the lymphocyte count was not. Pre-stenting PLR < 221 was an independent poor prognostic factor for RFS (P = 0.045). CONCLUSION: This study showed that decreased preoperative PLR, SII, PIV, and pre-stenting PLR were associated with poorer RFS, contrary to the findings of most previous studies. Our results suggest that platelets and obstruction contributed primarily to the opposite relationships, which might provide new insight into the possible pathophysiology of platelet-tumor interactions generated in the OCRC environment.

Pathophysiological levels of GDF11 activate Smad2/Smad3 signaling and induce muscle atrophy in human iPSC-derived myocytes.

Skeletal muscle mass is negatively regulated by several TGF-ß superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.

Insulin-induced genes INSIG1 and INSIG2 mediate oxysterol-dependent activation of the PERK-eIF2α-ATF4 axis.

Insulin-induced genes (INSIGs) encode endoplasmic reticulum-resident proteins that regulate intracellular cholesterol metabolism. Oxysterols are oxygenated derivatives of cholesterol, some of which orchestrate lipid metabolism via interaction with INSIGs. Recently, it was reported that expression of activating transcription factor-4 (ATF4) was induced by certain oxysterols; the precise of mechanism is unclear. Herein, we show that INSIGs mediate ATF4 upregulation upon interaction with oxysterol. Oxysterols that possess a high affinity for INSIG, such as 27- and 25-hydroxycholesterol (25HC), markedly induced the increase of ATF4 protein when compared with other oxysterols. In addition, ATF4 upregulation by these oxysterols was attenuated in INSIG1/2-deficient Chinese hamster ovary cells and recovered by either INSIG1 or INSIG2 rescue. Mechanistic studies revealed that the binding of 25HC to INSIG is critical for increased ATF4 protein via activation of protein kinase RNA-activated-like ER kinase and eukaryotic translation initiation factor 2α. Knockout of INSIG1 or INSIG2 in human hepatoma Huh7 cells attenuated ATF4 protein upregulation, indicating that only one of the endogenous INSIGs, unlike overexpression of intrinsic INSIG1 or INSIG2, was insufficient for ATF4 induction. Furthermore, ATF4 proactively upregulated the cell death-inducible gene expression, such as Chop, Chac1, and Trb3, thereby markedly reducing cell viability with 25HC. These findings support a model whereby that INSIGs sense an increase in oxysterol in the endoplasmic reticulum and induce an increase of ATF4 protein via the protein kinase RNA-activated-like ER kinase-eukaryotic translation initiation factor 2α pathway, thereby promoting cell death.