Altered APP Processing in Insulin-Resistant Conditions Is Mediated by Autophagosome Accumulation via the Inhibition of Mammalian Target of Rapamycin Pathway

Insulin resistance, one of the major components of type 2 diabetes mellitus (T2DM), is a known risk factor for Alzheimer’s disease (AD), which is characterized by an abnormal accumulation of intra- and extracellular amyloid β peptide (Aβ). Insulin resistance is known to increase Aβ generation, but the underlying mechanism that links insulin resistance to increased Aβ generation is unknown. In this study, we examined the effect of high-fat diet–induced insulin resistance on amyloid precursor protein (APP) processing in mouse brains. We found that the induced insulin resistance promoted Aβ generation in the brain via altered insulin signal transduction, increased β- and γ-secretase activities, and accumulation of autophagosomes. These findings were confirmed in diabetic db/db mice brains. Furthermore, in vitro experiments in insulin-resistant SH-SY5Y cells and primary cortical neurons confirmed the alteration of APP processing by insulin resistance–induced autophagosome accumulation. Defects in insulin signal transduction affect autophagic flux by inhibiting the mammalian target of rapamycin pathway, resulting in altered APP processing in these cell culture systems. Thus, the insulin resistance that underlies the pathogenesis of T2DM might also trigger accumulation of autophagosomes, leading to increased Aβ generation, which might be involved in the pathogenesis of AD.Alzheimer’s disease (AD) is sometimes referred to as type 3 diabetes because of the shared risk factors for the two disorders (1,2). Because insulin plays an important role in maintaining normal brain function and in peripheral glucose metabolism (3), insulin dysregulation has harmful effects on brain function as well as on peripheral glucose regulation. A number of epidemiological studies have suggested that insulin resistance, characterized by failed glucose utilization, confers an approximate two- to threefold relative risk for AD (4). Several factors could help to explain this link, including insulin degrading enzyme (IDE) activity, mitochondrial dysfunction, inflammation, and oxidative stress (5). Although type 2 diabetes mellitus (T2DM) may be linked to AD via these factors, our understanding of the underlying mechanisms is limited.AD, the most common form of dementia, is characterized by senile plaques, neurofibrillary tangles, and neuronal loss (6). Senile plaques are extracellular deposits of amyloid-β peptide (Aβ); the deposits are associated with AD-related neurodegeneration (7). Aβ is a peptide of 40 or 42 amino acids derived predominantly from amyloid precursor protein (APP) upon sequential cleavage by β-secretase (β-site APP cleaving enzyme 1 [BACE1]) and the γ-secretase complex (8,9). The β- and γ-secretases reside predominantly in intracellular membrane compartments of the vacuolar apparatus, including autophagic vacuoles (AVs) (10). Several reports have shown that AVs exist in the brains of AD model mice and AD patients (10,11) and that they colocalize intimately with the γ-secretase complex, APP, and β-secretase–derived COOH-terminal fragment (β-CTF) (10,12). Several important signaling pathways, including the mammalian target of rapamycin (mTOR) pathway, AMP-activated protein kinase (AMPK) signaling, and the insulin/IGF-I signaling pathways, are reported to regulate AV formation (13,14). During activation of these signaling pathways, defects in the insulin signaling pathway (insulin resistance) induced abnormal autophagosome formation (15,16).During macroautophagy (hereafter referred to as autophagy), the pivotal processes required for survival of long-lived cytoplasmic constituents are degraded; it is the principal means by which cellular organelles and protein aggregates are turned over (17). Autophagosome formation is induced by the inhibition of the mTOR signal pathway (17). Autophagosomes and their contents undergo clearance upon fusion with endosomes (amphisomes) or lysosomes (autolysosomes) that contain proteases (18) (autophagic maturation process).Autophagosomes are one of the generation sites for Aβ, major toxic peptides in AD pathology (10). Because insulin resistance induces autophagosome accumulation and Aβ generation, and increased Aβ levels become the cause of AD, we wonder whether insulin resistance accelerates AD pathology via an autophagosome-induced increase in Aβ generation. To investigate accumulation of autophagosomes and altered APP processing under insulin-resistant conditions, we examined abnormalities in insulin signaling and in APP metabolism and expression levels in autophagy-related protein in mice fed a high-fat diet (HFD) and in diabetic db/db mice (19). To explore possible underlying links between insulin resistance, autophagosomes, and AD-like changes in vitro, human neuroblastoma SH-SY5Y cells and primary cortical neurons were subjected to prolonged exposure to high levels of insulin, leading to cellular insulin resistance (20). We present evidence that insulin resistance alters APP processing through autophagy activation and that insulin resistance–induced autophagosome accumulation is a potential link between AD and diabetes.     

Is the BRAFV600E mutation useful as a predictor of preoperative risk in papillary thyroid cancer?

ObjectiveRecent studies have shown that a BRAF^V600E reflects poor prognosis, mainly in Western countries. However, some clinicians in Japan have suggested that the BRAF^V600E mutation is not associated with a poor prognosis. Therefore, we investigated a relationship between BRAF^V600E mutation and clinicopathologic factors.MethodsFrom September 2008 to December 2009, we performed routine analysis of the BRAF^V600E mutation using thyroid cancer tissue from 424 patients who underwent thyroidectomy with cervical lymph node dissection.ResultsThe BRAF^V600E mutation was found in 335 of 424 cases (79%) and was higher in classic papillary thyroid carcinoma (PTC) (79.7%) than in the follicular variant of PTC (62.5%) (P = .019). On univariate analysis, the BRAF^V600E mutation was associated with extrathyroidal extension (P = .009) and variants of PTC (P = .019), but a high-risk Metastasis, Patient Age, Completeness of resection, local Invasion and Tumor Size (MACIS) score (≥ 6) (P = .146) and lymph node metastasis (P = .628) were not significantly associated with the BRAF^V600E mutation. Multivariate analysis showed that extrathyroidal extension is independently associated with the BRAF^V600E mutation (relative ratio: 2.466; 95% confidence interval, 1.213–5.011; P < .013).ConclusionIt is not clear that the BRAF^V600E mutation is useful for prediction of poor prognosis of PTC.     

Impact of coculture with ischemic preconditioned hepatocellular carcinoma cell line (Hep-G2) cells on insulin secreting function of rat insulin-secreting cell line (RIN-5F) cells

Introduction

Although Islet cell isolation and culture have been well developed, there has been little progress to prolong transplanted islet sruvival. Hepatic ischemia and insufficient neovascularization of islets are considered to be the barriers to long-term survival, Hepatocytes that survive ischemic injury have been reported to protect themeslves and regenerate using the IL-6 interleukin 6 and STAT3 pathways.

Materials and Methods

The hepatocellular carcinoma (Hep-G2) cell line preconditioned for 0, 2, 4, 6, and 24 hours in a hypoxic chamber, was cocultured with rat insulin-secreting celline (RIN-5F) cells. We measured cell viabilities, insulin secretion, and p-STAT3, IL-6, and NF-κB levels.

Results

Cocultured Hep-G2 and RIN-5F cells aggregated to form spheroids. Viabilities of Hep-G2 cells were no different after various ischemic preconditioning times, but insulin secretion increased in a time-dependent fashion with preconditioning. Western blotting showed p-STAT3, NF-κB, and IL-6 levels to increase with preconditioning time.

Conclusion

The IL-6/STAT3 pathway of Hep-G2 cells after ischemic injury showed beneficial effects on insulin secretion of RIN-5f cells cocultured with themselves.     

Hybrid cellular spheroids from hepatocellular carcinoma and insulin-secreting cell lines

During islet transplantation into the portal vein of the liver, the islet cells are expected to have complex interactions with hepatocytes. However, the mechanism underlying this interaction is not yet understood. Hence, we developed cellular complexes containing a mixture of human hepatocellular carcinoma cell line (Hep-G2) and rat insulin-secreting cell line (RIN-5F) by using a co-culture model and studied the function and morphology of the resultant hybrid cellular spheroids (HCSs). The RIN-5F and Hep-G2 cells were suspension cultured and, within 5 days of culture, the two types of cells aggregated to yield spheroids. The functionality of the thus formed HCSs was evaluated by measuring the levels of insulin and albumin in the culture supernatant. The HCSs retained their insulin- and albumin-secreting ability and their morphology, as revealed by immunohistological staining. The insulin and albumin levels secreted by the HCSs were considerably higher than those secreted by spheroids of single-cell origin. Generally, obtaining complexes from more than two types of cells is difficult. However, we were able to generate HCSs. We believe that this culture method could have various applications such as studying the in vitro cell-cell interactions and developing new cell transplantation models.     

Analysis of transplant outcomes after five or six human leukocyte antigen-mismatched living donor kidney transplantation

Background

Recently, the impact of human leukocyte antigen (HLA) mismatch (MM) on graft outcome has diminished since the introduction of potent immunosuppressive agents, whereas previous reports support the notion that greater numbers of HLA matches are beneficial. This study was undertaken to evaluate outcomes after five or six HLA-mismatched living donor kidney transplantations (LDKT).

Methods

The authors retrospectively reviewed graft function after 2687 LDKTs performed between June 1984 and February 2010. A database of 1364 living related and 1063 living-unrelated donor (LURD) kidney transplantations was used for this study. LURD kidney transplantations were classified into three groups; (1) zero to one HLA MM (n = 158); (2) two to four HLA MM (n = 851); and (3) five to six MM (n = 54). An acute rejection episode was diagnosed based on clinical deterioration of graft function or biopsy findings. Graft survival was calculated using the Kaplan-Meier method.

Results

Graft survivals in the zero to one HLA MM, two to four HLA MM, five to six HLA MM, and one-haplo MM LDKT were not significantly different. The rates of acute rejection episodes within 1 year after transplantation were similar irrespective of the HLA MM; (1) zero to one HLA MM (37.3%), (2) two to four HLA MM (35.3%), (3) five to six HLA MM (33.3%; P = .832).

Conclusions

Survival of five or six HLA-mismatched LDKTs was comparable to that of one-haplo MM and relatively well-matched LDKT. The study showed that the presence of five or six HLA MM was not a risk factor for graft survival after LDKT.     

A prospective short-term outcome study of a short metaphyseal fitting total hip arthroplasty

The purpose of this study was to determine whether the short, metaphyseal fitting femoral stem would achieve stable fixation without diaphyseal fixation. A total of 126 patients (144 hips) were included in the study, and their mean age was 53.9 years (26-65 years). The mean duration of follow-up was 4.5 years (4-5 years). The predominant diagnosis was osteonecrosis of femoral head (88 of 144 hips, or 61%). The mean preoperative Harris hip score was 45 points, which improved to 96 points by the final follow-up. Western Ontario and McMaster Universities Osteoarthritis score and patient's activity score were improved substantially at the final follow-up. This short, metaphyseal fitting cementless femoral component achieved stable fixation without diaphyseal fixation, and there was minimal stress-shielding bone resorption in the calcar region.     

Novel technique for intraoperative tumor localization during totally laparoscopic distal gastrectomy: endoscopic autologous blood tattooing

Background

Knowledge of the intraoperative location of lesions is a prerequisite for deciding the proper extent of gastric resection or the choice of anastomosis technique during totally laparoscopic distal gastrectomy (TLDG) for early gastric cancer (EGC). In this study we introduce a novel tumor localization method for TLDG: endoscopic blood tattooing.

Methods

Twenty-three consecutive patients scheduled for TLDG for EGC were enrolled in this prospective study. The day before surgery, 2–3?ml of autologous blood was injected into the gastric muscle layer at 3–4?cm proximal to the lesion during endoscopy.

Results

The study subjects consisted of 15 males and 8 females with a mean age of 61?±?10.4?years. During surgery, the endoscopic blood tattooed sites were successfully identified in all 23 patients. No complications associated with the procedure occurred, and no patient had microscopic residual tumor cells at the proximal resection margin, with a mean proximal margin length of 3.3?±?2.7?cm. Eighteen patients underwent TLDG with Billroth II anastomosis, four patients with Roux-en-Y gastrojejunostomy, and one patient with laparoscopic total gastrectomy. At final pathologic examinations, 20 patients were of stage IA and 3 were of stage IB according to the UICC TNM classification (6th ed.).

Conclusions

Endoscopic blood tattooing provides a simple and useful means of localizing lesions during TLDG for EGC. Although the superiority of this technique over other localization methods needs to be evaluated further, the authors recommend endoscopic blood tattooing as an alternative to other intraoperative localization methods for laparoscopic surgery for EGC.