[Analysis of the Tumor Immune Microenvironment of Colorectal Cancer by Deep Learning-Based Imaging Cytometry].

The tumor immune microenvironment(TIME)of colorectal cancer contains indicators of unique therapeutic outcomes for each cancer patient. Deep learning-based imaging cytometry(DL-IC), which can obtain objective and reproducible cell- related information in tissue sections, has attracted attention as an analytical method for clarifying this indicator. This study demonstrates the validation process of Cu-Cyto, one of DL-IC, regarding cell identification accuracy. Acquisition of"spatial structure"information in TIME is useful for biomarker retrieval and contributes to precision oncology.

Deep Learning-based Image Cytometry Using a Bit-pattern Kernel-filtering Algorithm to Avoid Multi-counted Cell Determination.

BACKGROUND/AIM: In pathology, the digitization of tissue slide images and the development of image analysis by deep learning have dramatically increased the amount of information obtainable from tissue slides. This advancement is anticipated to not only aid in pathological diagnosis, but also to enhance patient management. Deep learning-based image cytometry (DL-IC) is a technique that plays a pivotal role in this process, enabling cell identification and counting with precision. Accurate cell determination is essential when using this technique. Herein, we aimed to evaluate the performance of our DL-IC in cell identification. MATERIALS AND METHODS: Cu-Cyto, a DL-IC with a bit-pattern kernel-filtering algorithm designed to help avoid multi-counted cell determination, was developed and evaluated for performance using tumor tissue slide images with immunohistochemical staining (IHC). RESULTS: The performances of three versions of Cu-Cyto were evaluated according to their learning stages. In the early stage of learning, the F1 score for immunostained CD8+ T cells (0.343) was higher than the scores for non-immunostained cells [adenocarcinoma cells (0.040) and lymphocytes (0.002)]. As training and validation progressed, the F1 scores for all cells improved. In the latest stage of learning, the F1 scores for adenocarcinoma cells, lymphocytes, and CD8+ T cells were 0.589, 0.889, and 0.911, respectively. CONCLUSION: Cu-Cyto demonstrated good performance in cell determination. IHC can boost learning efficiencies in the early stages of learning. Its performance is expected to improve even further with continuous learning, and the DL-IC can contribute to the implementation of precision oncology.

Patients younger than 40 years with colorectal cancer have a similar prognosis to older patients.

PURPOSE: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. In recent years, the proportion of patients diagnosed with CRC at younger ages has increased. The clinicopathological features and oncological outcomes in younger patients with CRC remain controversial. We aimed to analyze the clinicopathological features and oncological outcomes in younger patients with CRC. METHODS: We examined 980 patients who underwent surgery for primary colorectal adenocarcinoma between 2006 and 2020. Patients were divided into two cohorts: younger (< 40 years old) and older (≥ 40 years old). RESULTS: Of the 980 patients, 26 (2.7%) were under the age of 40 years. The younger group had more advanced disease (57.7% vs. 36.6%, p = 0.031) and more cases beyond the transverse colon (84.6% vs. 65.3%, p = 0.029) than the older group. Adjuvant chemotherapy was administered more frequently in the younger group (50% vs. 25.8%, p < 0.01). Relapse-free survival and overall survival were similar between the groups at all stages. Moreover, in stages II and III they were also comparable, regardless of the administration of adjuvant chemotherapy. CONCLUSIONS: Younger patients with CRC have a prognosis equivalent to that of older patients. Further studies are needed to establish the optimal treatment strategies for these patients.

Timing of Medium-Chain Triglyceride Consumption Modulates Effects in Mice with Obesity Induced by a High-Fat High-Sucrose Diet.

The prevalence of obesity is increasing worldwide, and obesity can cause type 2 diabetes, atherosclerosis, hypertension, cardiovascular disease, and cancer. Intake of medium-chain triglycerides (MCTs) containing medium-chain fatty acids reduces body fat and insulin resistance in rodents and humans. This study aimed to determine how the timing of MCT consumption affects obesity and metabolic dysfunction induced in mice by a high-fat high-sucrose diet (HFHSD). Mice received an HFHSD with or without MCT (M-HFHSD) during either the active or rest phase for 9 weeks. Significant reduction in body weight, white adipose tissue (WAT) weight, and adipocyte size in epididymal WAT (eWAT) and improved insulin sensitivity in mice fed with M-HFHSD during the active but not the rest phase were observed. The consumption of M-HFHSD during both active and rest phases increased glucose tolerance. Phosphorylated Akt was more abundant in the gastrocnemius muscles and eWAT of M-HFHSD-fed mice than in those fed HFHSD during the active phase. The mRNA and protein expression of lipogenic genes increased in the eWAT of mice fed M-HFHSD compared with those fed HFHSD. Feeding with M-HFHSD during the active phase significantly increased the abundance of phosphorylated Ser563 and 660 of hormone-sensitive lipase and its upstream protein kinase A in eWAT. These results indicated that the timing of consumption modulates the effects of MCT on eWAT hypertrophy and glucose and lipid metabolism in mice.

Albumin and Derived Neutrophil-to-Lymphocyte Ratio is a Novel Prognostic Factor for Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Multidisciplinary treatment combining neoadjuvant treatment (NAT) and surgery has slightly improved the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Although various biomarkers targeting nutrition and inflammation are associated with cancer prognosis, most studies have focused on conditions prior to NAT. Developing real-time and sensitive biomarkers that monitor changes in systemic conditions during NAT is important. We established a novel nutritional and inflammatory index, represented as the albumin to derived neutrophil-to-lymphocyte ratio (Alb-dNLR), and calculated the change in Alb-dNLR (ΔAlb-dNLR) during neoadjuvant chemotherapy (nCT). In this study, we aimed to evaluate whether ΔAlb-dNLR is associated with prognosis in patients with ESCC. METHODS: We investigated 172 patients who underwent nCT before esophagectomy between April 2010 and March 2018. The dNLR was calculated as the ratio of neutrophil count to (white blood cell count - neutrophil count), Alb-dNLR was calculated by dividing the serum albumin level by the dNLR, and ΔAlb-dNLR was evaluated by dividing the post-Alb-dNLR by the pre-Alb-dNLR. Patients were divided into 'high' and 'low' groups according to the ΔAlb-dNLR. RESULTS: Thirty-nine patients (22.7%) had a low ΔAlb-dNLR (≤ 0.8), and the 5-year overall survival (OS) rates in patients with low and high ΔAlb-dNLR were 38.1% and 53.6%, respectively (p = 0.0072). Multivariate analyses demonstrated that estimated blood loss (p = 0.044), pathological T stage (p = 0.0005), pathological N stage (p = 0.017), and ΔAlb-dNLR (p = 0.005) were independent prognostic factors for OS. CONCLUSIONS: ΔAlb-dNLR is a useful prognostic factor for OS in patients with ESCC receiving nCT.

Thoracic cavity-to-cage ratio is a predictor of technical difficulties in minimally invasive esophagectomy.

BACKGROUND: Technical difficulties are occasionally encountered when performing conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy in patients with a narrow thoracic cavity. Thoracic cavity-to-cage ratio is an indicator of thoracic cavity length. We hypothesized that the thoracic cavity-to-cage ratio could be a predictor of technical difficulties in conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy. METHODS: We evaluated 340 patients who underwent minimally invasive esophagectomy for esophageal squamous cell carcinoma between April 2010 and March 2021. Thoracic cavity-to-cage ratio was calculated as the diameter of the thoracic cavity to that of the thoracic cage at the brachiocephalic vein, tracheal bifurcation, and inferior right pulmonary vein levels. Moreover, thoracic cavity-to-cage ratio score, which is an indicator of the whole thoracic cavity length based on the thoracic cavity-to-cage ratio at the 3 levels, was defined. The thoracic procedure time was considered an indicator of surgical difficulty. RESULTS: We divided the patients into the conventional minimally invasive esophagectomy (n = 295) and robot-assisted minimally invasive esophagectomy (n = 45) groups. The patients in each group were divided into 2 cohorts according to median thoracic procedure time. Based on multivariate analysis, body mass index (P = .0007), clinical N stage (P = .0191), and thoracic cavity-to-cage ratio score (P = .0005) were independent factors for thoracic procedure time in the conventional minimally invasive esophagectomy group. Moreover, thoracic cavity-to-cage ratio at the tracheal bifurcation level (P = .0331) was the only independent factor for thoracic procedure time in the robot-assisted minimally invasive esophagectomy group. CONCLUSION: Thoracic cavity-to-cage ratio could be a predictor of technical difficulties in both conventional minimally invasive esophagectomy and robot-assisted minimally invasive esophagectomy.

Suppressive effects of processed aconite root on dexamethasone-induced muscle ring finger protein-1 expression and its active ingredients.

Processed aconite root (PA), the tuberous root of Aconitum carmichaelii prepared by autoclaving, is a crude drug used in Japanese traditional Kampo medicine and traditional Chinese medicine for the symptoms of kidney deficiency, that is related to the muscle atrophy in modern medicine. The objective of the present study is to evaluate the effectiveness of PA on muscle atrophy and to find its active ingredients using dexamethasone-induced muscle ring finger protein-1 (MuRF1) mRNA expression in murine myoblast C2C12 cells. Dexamethasone-induced MuRF1 expression was significantly suppressed by methanol-soluble part of boiling water extract of PA in a concentration-dependent manner with its IC50 value of 1.5 mg/ml. By the activity-guided fractionations of PA extract using the partition between organic solvents and its aqueous solution, the activity of PA did not transfer into the fraction containing aconitine-type diterpenoid alkaloids but into BuOH layer. Then, we found higenamine and salsolinol as the active ingredients in PA. Higenamine and salsolinol significantly suppressed dexamethasone-induced MuRF1 expression, and their IC50 values were 0.49 and 50 µM, respectively. The contents of higenamine and salsolinol in the decoctions of commercially available fourteen PA products are 0.12 and 14 µg/ml as the average values, and varied with the coefficient of variation (CV) values of 97 and 63%, respectively. Higenamine also significantly suppressed dexamethasone-induced mRNA expressions of muscle atrophy F-box protein (MAFbx)/atrogin1, casitas B-lineage lymphoma-b (Cbl-b), troponin, branched-chain amino acid aminotransferase 2 (BCAT2), and Bcl-2 binding and pro-apoptotic protein3 (Bnip3). Although the quality control of PA is regulated by the contents of diterpene alkaloids, salsolinol and higenamine can be used as the marker compounds to certificate the pharmacological activities of PA.

Combination of extramural venous invasion and lateral lymph node size detected with magnetic resonance imaging is a reliable biomarker for lateral lymph node metastasis in patients with rectal cancer.

PURPOSE: Pathological extramural venous invasion (EMVI) is defined as the active invasion of malignant cells into veins beyond the muscularis propria in colorectal cancer. It is associated with poor prognosis and increases the risk of disease recurrence. Specific findings on MRI (termed MRI-EMVI) are reportedly associated with pathological EMVI. In this study, we aimed to identify risk factors for lateral lymph node (LLN) metastasis related to rectal cancer and to evaluate whether MRI-EMVI could be a new and useful imaging biomarker to help LLN metastasis diagnosis besides LLN size. METHODS: We investigated 67 patients who underwent rectal resection and LLN dissection for rectal cancer. We evaluated MRI-EMVI grading score and examined the relationship between MRI-EMVI and LLN metastasis. RESULTS: Pathological LLN metastasis was detected in 18 cases (26.9%), and MRI-EMVI was observed in 32 cases (47.8%). Patients were divided into two cohorts, according to LLN metastasis. Multivariate analyses demonstrated that higher risk of LLN metastasis was significantly associated with MRI-EMVI (P = 0.0112) and a short lateral lymph node axis (≥ 5 mm) (P = 0.0002). The positive likelihood ratios of MRI-EMVI alone, LLN size alone, and the combination of both factors were 2.12, 4.84, and 16.33, respectively. Patients negative for both showed better 2-year relapse-free survival compared to other patients (84.4% vs. 62.1%, P = 0.0374). CONCLUSIONS: MRI-EMVI was a useful imaging biomarker for identifying LLN metastasis in patients with rectal cancer. The combination of MRI-EMVI and LLN size can improve diagnostic accuracy.

Reduced Number and Immune Dysfunction of CD4+ T Cells in Obesity Accelerate Colorectal Cancer Progression.

Obesity, a known risk factor for various types of cancer, reduces the number and function of cytotoxic immune cells in the tumor immune microenvironment (TIME). However, the impact of obesity on CD4+ T cells remains unclear. Therefore, this study aimed to clarify the impact of obesity on CD4+ T cells in the TIME. A tumor-bearing obese mouse model was established by feeding with 45% high-fat diet (HFD), followed by inoculation with a colon cancer cell line MC38. Tumor growth was significantly accelerated compared to that in mice fed a control diet. Tumor CD4+ T cells showed a significant reduction in number and an increased expression of programmed death-1 (PD-1), and decreased CD107a expression and cytokine such as IFN-γ and TNF-α production, indicating dysfunction. We further established CD4+ T cell-depleted HFD-fed model mice, which showed reduced tumor infiltration, increased PD-1 expression in CD8+ T cells, and obesity-induced acceleration of tumor growth in a CD4+ T cell-dependent manner. These findings suggest that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells to ultimately accelerate the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity.

Albumin-Derived NLR Score is a Novel Prognostic Marker for Esophageal Squamous Cell Carcinoma.

BACKGROUND: Multidisciplinary treatment for esophageal squamous cell carcinoma (ESCC) has improved outcomes, but the prognosis for ESCC remains poor. Nutritional and inflammatory indicators are reported to be associated with cancer prognosis. The combination of albumin and the derived neutrophil-to-lymphocyte ratio (Alb-dNLR) score was established to measure the immune system and nutritional status. The authors hypothesized that the Alb-dNLR score could be a new reliable prognostic factor for ESCC patients. METHODS: The study evaluated 269 patients who underwent esophagectomy between April 2010 and March 2018, including 185 patients who received neoadjuvant chemotherapy. The Alb-dNLR score was calculated using serum albumin and the dNLR. The dNLR was calculated as neutrophils to (leukocyte-neutrophil count). The cutoff values of the albumin and dNLR for overall survival (OS) were determined using the receiver operating characteristic curve. Patients were divided into "high" and "low" groups according to the Alb-dNLR score. RESULTS: A high Alb-dNLR score was found in 61 cases (22.7%). The 5-year OS was 34% in the high Alb-dNLR group and 66.2% in the low Alb-dNLR group (p < 0.0001). The 5-year cause-specific survival (CSS) was 51.5% in the high Alb-dNLR group and 74.7% in the low Alb-dNLR group (p < 0.0001). Multivariate analyses demonstrated that the Alb-dNLR score was an independent prognostic factor for OS (hazard ratio [HR], 2.198; 95% confidence interval [CI], 1.460-3.263; p = 0.0002) and CSS (HR, 1.733; 95% CI, 1.035-2.835; p = 0.0371). CONCLUSIONS: The Alb-dNLR score is an extremely useful, easy-to-use parameter to predict OS and CSS for ESCC patients.

Risk Factors for Outlet Obstruction in Patients with Diverting Ileostomy Following Rectal Surgery.

OBJECTIVES: Anastomotic leakage is one of the most severe complications of rectal cancer surgery. A diverting ileostomy was constructed for the purpose of reducing anastomotic failure risk. Outlet obstruction (OO) is one of the complications of diverting stoma that results in a lack of fecal discharge from the stoma. Detailed etiologies and preventive measures for outlet obstruction have not yet been identified. METHODS: We studied 125 patients who underwent rectal resection, anastomosis, and elective ileostomy. We evaluated the incidence of outlet obstruction and looked for any relationship between perioperative factors and outlet obstruction. RESULTS: Outlet obstruction was detected in 20 cases (16.0%). Outlet obstruction occurred 9 days after surgery in most cases. Inserting a decompressing tube improved obstructive symptoms in 4 days. Patients were divided into two cohorts according to the occurrence of outlet obstruction. Postoperative hospital stay was longer in the outlet obstruction group (19 vs. 15 days; p = 0.0003). A multivariate analysis identified that younger patients, a postoperative thicker rectus abdominis muscle at the stoma passage and high output syndrome were independent risk factors for outlet obstruction. CONCLUSIONS: Younger patients, a postoperative thicker rectus abdominis muscle at stoma passage and high output syndrome were independent risk factors for outlet obstruction.

A phase II study of the safety of olanzapine for oxaliplatin based chemotherapy in coloraectal patients.

Olanzapine has exhibited efficacy as an antiemetic agent when used with 5-HT3 receptor antagonists, dexamethasone, and NK1 receptor antagonists for patients receiving highly emetogenic chemotherapy. In addition, several studies have reported the efficacy or safety of olanzapine in patients receiving moderately emetogenic chemotherapy, including carboplatin, irinotecan, and oxaliplatin. However, no reports of olanzapine use have focused on patients receiving oxaliplatin-based chemotherapy. Therefore, we analyzed the safety of antiemetic therapy using olanzapine, palonosetron, aprepitant, and dexamethasone in colorectal cancer patients undergoing oxaliplatin-based chemotherapy. This study was a prospective phase II single-institution study of 40 patients (median age 60 years, 23 patients were male). The primary endpoint was the incidence of adverse events, and the exploratory endpoints were the rate of chemotherapy-induced nausea and vomiting. Almost all patients (90%) had a performance status of 0. All patients received the scheduled antiemetic therapy. The most common adverse event was somnolence (n = 7 patients, 17.5%). All adverse events were grade 1. Thirty-six patients were included in the exploratory analysis of efficacy. No patients experienced vomiting during the first 120 h after chemotherapy, and complete response and complete control were both 86.1%. The rate of total control was 55.6% during the same time period. Olanzapine use with 5-HT3 receptor antagonists, dexamethasone, and NK1 receptor antagonists was safe for colorectal cancer patients receiving oxaliplatin-based chemotherapy.

Maternal fish oil supplementation ameliorates maternal high-fructose diet-induced dyslipidemia in neonatal mice with suppression of lipogenic gene expression in livers of postpartum mice.

Maternal fructose consumption during pregnancy and lactation is associated with metabolic dysregulation in offspring. We tested the hypothesis that fish oil (FO) supplementation during pregnancy and lactation improves fructose-induced metabolic dysregulation in postpartum dams and offspring mice. We therefore aimed to determine the effects of FO supplementation on metabolic disruption in neonatal mice and dams induced by a maternal high-fructose diet (HFrD). The weight of the offspring of dams fed with HFrD on postnatal day 5 was significantly low, but this was reversed by adding FO to the maternal diet. Feeding dams with HFrD significantly increased plasma concentrations of triglycerides, uric acid, and total cholesterol, and decreased free fatty acid concentrations in offspring. Maternal supplementation with FO significantly suppressed HFrD-induced hypertriglyceridemia and hyperuricemia in the offspring. Maternal HFrD induced remarkable mRNA expression of the lipogenic genes Srebf1, Fasn, Acc1, Scd1, and Acly in the postpartum mouse liver without affecting hepatic and plasma lipid levels. Although expression levels of lipogenic genes were higher in the livers of postpartum dams than in those of nonmated mice, HFrD feeding increased the hepatic lipid accumulation in nonmated mice but not in postpartum dams. These findings suggest that although hepatic lipogenic activity is higher in postpartum dams than nonmated mice, the lipid consumption is enhanced in postpartum dams during pregnancy and lactation. Maternal FO supplementation obviously suppressed the expression of these lipogenic genes. These findings coincide with reduced plasma triglyceride concentrations in the offspring. Therefore, dietary FO apparently ameliorated maternal HFrD-induced dyslipidemia in offspring by suppressing maternal lipogenic gene expression and/or neonatal plasma levels of uric acid.

Decreased CT-number in the pancreatic parenchyma is a reliable imaging biomarker of the presence of malignancies in patients with high-risk intraductal papillary mucinous neoplasm.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is a premalignant cystic neoplasm of the pancreas and is frequently detected in imaging investigations. A proportion of the patients with IPMN develop malignancies including high-grade dysplasia and invasive carcinoma. To predict the presence of malignancies in IPMN, constant imaging follow-up is usually required. Pancreatic steatosis (PS) has been recently identified as a facilitating factor for pancreatic cancer, and can be predicted through computed tomography (CT). We hypothesized that the CT-number of the pancreatic parenchyma could be a new reliable imaging biomarker for IPMN patients. METHODS: Eighty-six patients undergoing pancreatectomy for IPMN were investigated. Using preoperative CT, the pancreatic index (PI) was calculated by dividing the CT-number of the pancreas by that of the spleen. RESULTS: Malignancies were pathologically detected in 63 cases (73.3%). Patients were divided into two cohorts according to the presence of malignancies and were compared for various factors including the PI scores. The comparison of the two cohorts detected significant differences in two parameters (CA19-9 and PI score), and the PI score was the most sensitive biomarker to predict the presence of malignancies in patients showing high-risk stigmata of IPMN. CONCLUSIONS: Pancreatic CT-number is an additional reliable imaging biomarker in distinguishing patients with IPMN having malignancies when investigating the patients showing high-risk stigmata.

Laparoscopic anatomical liver resection using indocyanine green fluorescence imaging.

BACKGROUND: Anatomical liver resections guided by a demarcation line after portal staining or inflow clamping of the target area have been established as essential methods for curative treatment of hepatocellular carcinoma (HCC) and have subsequently been applied to other malignancies. However, laparoscopic anatomical liver resection (LALR) procedures are very difficult to reproduce, and the confirmation of demarcation of the hepatic segment on a monitor is also challenging. Recently, indocyanine green (ICG) fluorescence imaging has been used to identify hepatic tumors and segmental boundaries during hepatectomy. Herein, we describe LALR using ICG fluorescence imaging. METHODS: Three patients underwent pure LALR using ICG fluorescence imaging at our institute. One patient underwent anatomical partial liver resection for HCC, another underwent segmentectomy 3 for metastatic liver cancer, and the third underwent right anterior sectionectomy for HCC. To visualize hepatic perfusion and the demarcation line by negative staining using an optical imaging system, 2.5 mg ICG was injected intravenously during surgery following clamping or closure of the proximal Glissonean pedicles. RESULTS: For all three cases, ICG fluorescent imaging clearly delineated the demarcation lines and allowed identification of intersegmental planes to some extent because the tumor-bearing hepatic region became non-fluorescing parenchyma during parenchymal transection. This allowed surgeons to recognize the direction and guide the transection of the liver parenchyma when performing LALR. CONCLUSION: LALR using ICG fluorescence imaging is a feasible procedure for resection of the tumor-bearing hepatic region and facilitates visualization of the demarcation line and identification of the boundaries of the hepatic sections.

A resected case of gallbladder metastasis with symptoms of acute cholecystitis in multiple metastatic ductal carcinoma of the breast.

Gallbladder metastasis from breast cancer, especially from ductal carcinoma, is rare. Herein, we report a rare case of gallbladder metastasis from ductal carcinoma of the breast that was diagnosed after laparoscopic cholecystectomy (LC) for acute cholecystitis. A 78-year-old woman presented with right upper abdominal tenderness and positive Murphy's sign during chemotherapy for advanced multiple metastases of the breast cancer. Abdominal ultrasonography and computed tomography showed a slightly thickened gallbladder wall and two calculi. After a diagnosis of acute calculous cholecystitis was established, LC was performed. Pathological examination revealed poorly differentiated adenocarcinoma infiltrating the submucosal and subserosal layer over the entire gallbladder, and a lymph node metastasis in the gallbladder neck. Immunohistochemical examination revealed that the tumor cells tested positive for estrogen receptor and negative for progesterone receptor, which was consistent with primary breast cancer. The patient was uneventfully discharged without abdominal pain 7 days later. Although she subsequently underwent several chemotherapies, she died 16 months later. In conclusion, gallbladder metastasis should be considered in patients with multiple metastatic breast cancer who present with signs or symptoms of cholecystitis. Moreover, LC should be considered to relieve the symptoms of cholecystitis for improved prognosis, even in a patient with multiple metastases.

Involvement of Cbl-b-mediated macrophage inactivation in insulin resistance.

Aging and overnutrition cause obesity in rodents and humans. It is well-known that obesity causes various diseases by producing insulin resistance (IR). Macrophages infiltrate the adipose tissue (AT) of obese individuals and cause chronic low-level inflammation associated with IR. Macrophage infiltration is regulated by the chemokines that are released from hypertrophied adipocytes and the immune cells in AT. Saturated fatty acids are recognized by toll-like receptor 4 (TLR4) and induce inflammatory responses in AT macrophages (ATMs). The inflammatory cytokines that are released from activated ATMs promote IR in peripheral organs, such as the liver, skeletal muscle and AT. Therefore, ATM activation is a therapeutic target for IR in obesity. The ubiquitin ligase Casitas b-lineage lymphoma-b (Cbl-b) appears to potently suppress macrophage migration and activation. Cbl-b is highly expressed in leukocytes and negatively regulates signals associated with migration and activation. Cbl-b deficiency enhances ATM accumulation and IR in aging- and diet-induced obese mice. Cbl-b inhibits migration-related signals and SFA-induced TLR4 signaling in ATMs. Thus, targeting Cbl-b may be a potential therapeutic strategy to reduce the IR induced by ATM activation. In this review, we summarize the regulatory functions of Cbl-b in ATMs.

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.