KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

Calorie Restriction Using High-Fat/Low-Carbohydrate Diet Suppresses Liver Fat Accumulation and Pancreatic Beta-Cell Dedifferentiation in Obese Diabetic Mice.

In diabetes, pancreatic ß-cells gradually lose their ability to secrete insulin with disease progression. ß-cell dysfunction is a contributing factor to diabetes severity. Recently, islet cell heterogeneity, exemplified by ß-cell dedifferentiation and identified in diabetic animals, has attracted attention as an underlying molecular mechanism of ß-cell dysfunction. Previously, we reported ß-cell dedifferentiation suppression by calorie restriction, not by reducing hyperglycemia using hypoglycemic agents (including sodium-glucose cotransporter inhibitors), in an obese diabetic mice model (db/db). Here, to explore further mechanisms of the effects of food intake on ß-cell function, db/db mice were fed either a high-carbohydrate/low-fat diet (db-HC) or a low-carbohydrate/high-fat diet (db-HF) using similar calorie restriction regimens. After one month of intervention, body weight reduced, and glucose intolerance improved to a similar extent in the db-HC and db-HF groups. However, ß-cell dedifferentiation did not improve in the db-HC group, and ß-cell mass compensatory increase occurred in this group. More prominent fat accumulation occurred in the db-HC group livers. The expression levels of genes related to lipid metabolism, mainly regulated by peroxisome proliferator-activated receptor α and γ, differed significantly between groups. In conclusion, the fat/carbohydrate ratio in food during calorie restriction in obese mice affected both liver lipid metabolism and ß-cell dedifferentiation.

A rewiring of DNA replication mediated by MRE11 exonuclease underlies primed-to-naive cell de-differentiation.

Mouse embryonic stem cells (mESCs) in the primed pluripotency state, which resembles the post-implantation epiblast, can be de-differentiated in culture to a naive state that resembles the pre-implantation inner cell mass. We report that primed-to-naive mESC transition entails a significant slowdown of DNA replication forks and the compensatory activation of dormant origins. Using isolation of proteins on nascent DNA coupled to mass spectrometry, we identify key changes in replisome composition that are responsible for these effects. Naive mESC forks are enriched in MRE11 nuclease and other DNA repair proteins. MRE11 is recruited to newly synthesized DNA in response to transcription-replication conflicts, and its inhibition or genetic downregulation in naive mESCs is sufficient to restore the fork rate of primed cells. Transcriptomic analyses indicate that MRE11 exonuclease activity is required for the complete primed-to-naive mESC transition, demonstrating a direct link between DNA replication dynamics and the mESC de-differentiation process.

Uterine Leiomyosarcoma Associated With Perivascular Epithelioid Cell Tumor: A Phenomenon of Differentiation/Dedifferentiation and Evidence Suggesting Cell-of-Origin.

Perivascular epithelioid cell tumor (PEComa) is a mesenchymal tumor thought to originate from perivascular epithelioid cells (PECs). The normal counterpart to PEC, however, has not been identified in any human organ, and the debate as to whether PEComa is related to smooth muscle tumors has persisted for many years. The current series characterizes 4 cases of uterine leiomyosarcoma (LMS) coexisting with PEComas. All cases exhibited an abrupt transition from the LMS to PEComa components. The LMS component displayed typical spindled morphology and fascicular growth pattern and was diffusely positive for desmin and smooth muscle myosin heavy chain, completely negative for HMB-45 and Melan A, and either negative or had focal/weak expression of cathepsin K and GPNMB. In contrast, the PEComa tumor cells in case 1 contained glycogen or lipid-distended cytoplasm with a foamy appearance (low grade), and in cases 2, 3, and 4, they displayed a similar morphology characterized by epithelioid cells with eosinophilic and granular cytoplasm and high-grade nuclear atypia. Different from the LMS component, the epithelioid PEComa cells in all cases were focally positive for HMB-45, and diffusely immunoreactive for cathepsin K and GPNMB. Melan A was focally positive in cases 1 and 3. Loss of fumarate hydratase expression (case 1) and RB1 expression (cases 2, 3, 4) was identified in both LMS and PEComa components, indicating that they are clonally related. In addition, both components showed an identical TP53 p.R196* somatic mutation and complete loss of p53 and ATRX expression in case 2 and complete loss of p53 expression in case 3. We hypothesize that LMSs containing smooth muscle progenitor cells may give rise to divergent, lineage-specific PEComatous lesions through differentiation or dedifferentiation. While we do not dispute the recognition of PEComas as a distinct entity, we advocate the hypothesis that modified smooth muscle cells represent the origin of a subset of PEComas, and our case series provides evidence to suggest this theory.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

The transdifferentiation of human dedifferentiated fat cells into fibroblasts: An in vitro experimental pilot study.

BACKGROUND: Skin grafting is a common method of treating damaged skin; however, surgical complications may arise in patients with poor health. Currently, no effective conservative treatment is available for extensive skin loss. Mature adipocytes, which constitute a substantial portion of adipose tissue, have recently emerged as a potential source of stemness. When de-lipidated, these cells exhibit fibroblast-like characteristics and the ability to redifferentiate, offering homogeneity and research utility as "dedifferentiated fat cells." METHODS AND RESULTS: We conducted an in vitro study to induce fibroblast-like traits in the adipose tissue by transdifferentiating mature adipocytes for skin regeneration. Human subcutaneous fat tissues were isolated and purified from mature adipocytes that underwent a transformation process over 14 days of cultivation. Microscopic analysis revealed lipid degradation over time, ultimately transforming cells into fibroblast-like forms. Flow cytometry was used to verify their characteristics, highlighting markers such as CD90 and CD105 (mesenchymal stem cell markers) and CD56 and CD106 (for detecting fibroblast characteristics). Administering dedifferentiated fat cells with transforming growth factor-ß at the identified optimal differentiation concentration of 5 ng/mL for a span of 14 days led to heightened expression of alpha smooth muscle actin and fibronectin, as evidenced by RNA and protein analysis. Meanwhile, functional validation through cell sorting demonstrated limited fibroblast marker expression in both treated and untreated cells after transdifferentiation by transforming growth factor-ß. CONCLUSION: Although challenges remain in achieving more effective transformation and definitive fibroblast differentiation, our trial could pave the way for a novel skin regeneration treatment strategy.

Dedifferentiation in bone and soft tissue sarcomas: How do we define it? What is prognostically relevant?

Dedifferentiation traditionally is defined by descriptive criteria as a tumor showing an abrupt change in histology from a conventional, classic, low-grade appearing neoplasm to a tumor that is more cellular, pleomorphic and "high grade", with grading typically being performed by subjective criteria. The dedifferentiated areas range from areas with recognizable histologic differentiation which differs from the primary tumor (such as an osteosarcoma arising from a low-grade chondrosarcoma) to areas containing sarcomas without specific histologic differentiation (such as pleomorphic or spindle cell sarcoma). Many, but not all, dedifferentiated tumors are aggressive and associated with significantly shorter survival than their conventional counterparts, even grade 3 conventional tumors. As a result, dedifferentiated tumors are generally considered to be clinically aggressive and as a result, more aggressive surgery or the addition of (neo)adjuvant chemotherapy is often considered. However, long-term (greater than 20 year) survivors are reported in the most common dedifferentiated bone and soft tissue sarcomas. Moreover, use of mitotic criterion for defining dedifferentiation in dedifferentiated liposarcoma as well as grading (by the French system) have been found to be associated with survival. This paper reviews the literature on dedifferentiated chondrosarcoma, dedifferentiated liposarcoma, dedifferentiated chordoma and dedifferentiated parosteal osteosarcoma. As a result of that review, recommendations are advocated to identify evidence-based, objective diagnostic and grading criteria for dedifferentiation that are appropriate for each tumor type. Adding such criteria will improve consistency in diagnosis worldwide, allow easier comparison of clinical research performed on dedifferentiated tumors and help communicate (to patients and clinicians) the tumors with highest risk of clinically aggressive behavior, to allow appropriate and personalized treatment planning.

Cyb5r3 activation rescues secondary failure to sulfonylurea but not ß-cell dedifferentiation.

Diabetes mellitus is characterized by insulin resistance and ß-cell failure. The latter involves impaired insulin secretion and ß-cell dedifferentiation. Sulfonylurea (SU) is used to improve insulin secretion in diabetes, but it suffers from secondary failure. The relationship between SU secondary failure and ß-cell dedifferentiation has not been examined. Using a model of SU secondary failure, we have previously shown that functional loss of oxidoreductase Cyb5r3 mediates effects of SU failure through interactions with glucokinase. Here we demonstrate that SU failure is associated with partial ß-cell dedifferentiation. Cyb5r3 knockout mice show more pronounced ß-cell dedifferentiation and glucose intolerance after chronic SU administration, high-fat diet feeding, and during aging. A Cyb5r3 activator improves impaired insulin secretion caused by chronic SU treatment, but not ß-cell dedifferentiation. We conclude that chronic SU administration affects progression of ß-cell dedifferentiation and that Cyb5r3 activation reverses secondary failure to SU without restoring ß-cell dedifferentiation.

Novel insight on IRE1 in the regulation of chondrocyte dedifferentiation through ER stress independent pathway.

Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression.

Differential histone acetylation and super-enhancer regulation underlie melanoma cell dedifferentiation.

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression.

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Elucidation of the anti-ß-cell dedifferentiation mechanism of a modified Da Chaihu Decoction by an integrative approach of network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Da Chaihu decoction (MDCH) is a traditional Chinese herbal prescription that has been used in the clinic to treat type 2 diabetes (T2D). Previous studies have confirmed that MDCH improves glycemic and lipid metabolism, enhances pancreatic function, and alleviates insulin resistance in patients with T2D and diabetic rats. Evidence has demonstrated that MDCH protects pancreatic ß cells via regulating the gene expression of sirtuin 1 (SIRT1) and forkhead box protein O1 (FOXO1). However, the detailed mechanism remains unclear. AIM OF THE STUDY: Dedifferentiation of pancreatic ß cells mediated by FOXO1 has been recognized as the main pathogenesis of T2D. This study aims to investigate the therapeutic effects of MDCH on T2D in vitro and in vivo to elucidate the potential molecular mechanisms. MATERIALS AND METHODS: To predict the key targets of MDCH in treating T2D, network pharmacology methods were used. A T2D model was induced in diet-induced obese (DIO) C57BL/6 mice with a single intraperitoneal injection of streptozotocin. Glucose metabolism indicators (oral glucose tolerance test, insulin tolerance test), lipid metabolism indicators (total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol), inflammatory factors (C-reactive protein, interleukin 6, tumor necrosis factor alpha), oxidative stress indicators (total antioxidant capacity, superoxide dismutase, malondialdehyde), and hematoxylin and eosin staining were analyzed to evaluate the therapeutic effect of MDCH on T2D. Immunofluorescence staining and quantification of FOXO1, pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), octamer-binding protein 4 (OCT4), neurogenin 3 (Ngn3), insulin, and SIRT1, and Western blot analysis of insulin, SIRT1, and FOXO1 were performed to investigate the mechanism by which MDCH inhibited pancreatic ß-cell dedifferentiation. RESULTS: The chemical ingredients identified in MDCH were predicted to be important for signaling pathways related to lipid metabolism and insulin resistance, including lipids in atherosclerosis, the advanced glycation end product receptor of the advanced glycation end product signaling pathway, and the FOXO signaling pathway. Experimental studies showed that MDCH improved glucose and lipid metabolism in T2D mice, alleviated inflammation and oxidative stress damage, and reduced pancreatic pathological damage. Furthermore, MDCH upregulated the expression levels of SIRT1, FOXO1, PDX1, and NKX6.1, while downregulating the expression levels of OCT4 and Ngn3, which indicated that MDCH inhibited pancreatic dedifferentiation of ß cells. CONCLUSIONS: MDCH has therapeutic effects on T2D, through regulating the SIRT1/FOXO1 signaling pathway to inhibit pancreatic ß-cell dedifferentiation, which has not been reported previously.

Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells / Investigar os efeitos do humor vítreo na proliferação e desdiferenciação de células NTERA2 diferenciadas

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.

Downregulation of Sirt3 contributes to ß-cell dedifferentiation via FoxO1 in type 2 diabetic mellitus.

AIMS: FoxO1 is an important factor in the ß-cell differentiation in type 2 diabetes mellitus (T2DM). Sirt3 is found to be involved in FoxO1 function. This study investigated the role of Sirt3 in the ß-cell dedifferentiation and its mechanism. METHODS: Twelve-week-old db/db mice and INS1 cells transfected with Sirt3-specific short hairpin RNA (shSirt3) were used to evaluate the dedifferentiation of ß-cell. Insulin levels were measured by enzyme linked immunosorbent assay. The proteins of Sirt3, T-FoxO1, Ac-FoxO1 and differentiation indexes such as NGN3, OCT4, MAFA were determined by western blot or immunofluorescence staining. The combination of Sirt3 and FoxO1 was determined by the co-immunoprecipitation assay. The transcriptional activity of FoxO1 was detected by dual luciferase reporter assay. RESULTS: Both the in vivo and in vitro results showed that Sirt3 was decreased along with ß-cell dedifferentiation and decreased function of insulin secretion under high glucose conditions. When Sirt3 was knocked down in INS1 cells, increased ß-cell dedifferentiation and lowered insulin secretion were observed. This effect was closely related to the amount loss and the decreased deacetylation of FoxO1, which resulted in a reduction in transcriptional activity. CONCLUSION: Downregulation of Sirt3 contributes to ß-cell dedifferentiation in high glucose via FoxO1. Intervention of Sirt3 may be an effective approach to prevent ß-cell failure in T2DM.

Evidence of interactions among apoptosis, cell proliferation, and dedifferentiation in the rudiment during whole-organ intestinal regeneration in the sea cucumber.

Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the digestive tract. From the mesentery, a rudiment grows and gives rise to a new intestine within a few weeks. In the last decades, the cellular events that occur during intestinal regeneration have been characterized, including apoptosis, cell proliferation, and muscle cell dedifferentiation. Nevertheless, their contribution to the formation and early growth of the rudiment is still unknown. Furthermore, these cellular events' relationship and potential interdependence remain a mystery. Using modulators to inhibit apoptosis and cell proliferation, we tested whether rudiment growth or other regenerative cellular events like muscle cell dedifferentiation were affected. We found that inhibition of apoptosis by zVAD and cell proliferation by aphidicolin and mitomycin did not affect the overall size of the rudiment seven days post-evisceration (7-dpe). Interestingly, animals treated with aphidicolin showed higher levels of muscle cell dedifferentiation in the distal mesentery, which could act as a compensatory mechanism. On the other hand, inhibition of apoptosis led to a decrease in cell proliferation in the rudiment and a delay in the spatiotemporal progression of muscle cell dedifferentiation throughout the rudiment-mesentery structure. Our findings suggest that neither apoptosis nor cell proliferation significantly contributes to early rudiment growth during intestinal regeneration in the sea cucumber. Nevertheless, apoptosis may play an essential role in modulating cell proliferation in the rudiment (a process known as apoptosis-induced proliferation) and the timing for the progression of muscle cell dedifferentiation. These findings provide new insights into the role and relationship of cellular events during intestinal regeneration in an emerging regeneration model.

Ketogenic diet alleviates ß-cell dedifferentiation but aggravates hepatic lipid accumulation in db/db mice.

OBJECTIVE: The aim of this study was to explore the effect of the ketogenic diet (KD) on ß-cell dedifferentiation and hepatic lipid accumulation in db/db mice. METHODS: After a 3-wk habituation, male db/db mice ages 8 wk were assigned into one of three groups: normal diet (ND), KD, and 75% calorie restriction (CR) group. Free access to a standard diet, a KD, and 75% of a standard diet, respectively, were given to each group. Additionally, sex-matched 8-wk-old C57BL/6 mice were used to construct a control (C) group. After a 4-wk dietary intervention, mouse body weight, fasting blood glucose (FBG), blood lipids, fasting insulin (FINS), glucose tolerance, and ß-hydroxybutyric acid level were measured. The morphologies of the islet and liver were observed by hematoxylin and eosin staining. Positive expressions of ß-cell-specific transcription factors in mouse islets were determined by double immunofluorescence staining. The size and number of lipid droplets in mouse liver were examined by Oil Red O staining. Real-time quantitative reverse transcription polymerase chain reaction detected relative levels of adipogenesis-associated and lipolysis-associated genes in mouse liver. Additionally, expressions of CD36 protein in the mouse liver were determined by immunohistochemical staining and Western blot. RESULTS: After a 4-wk dietary intervention, FBG, FINS, and glucose area under the curve in the KD group became significantly lower than in the ND group (all P < 0.05). Regular morphology of mouse islets was observed in the KD group, with an increased number of islet cells. The KD significantly reversed the decrease in ß-cell number, disarrangement of ß-cells, decline of ß/α-cell ratio, and downregulation of ß-cell-specific transcription factors in db/db mice. Serum levels of triacylglycerol, total cholesterol, and low-density lipoprotein cholesterol were comparable between the ND and KD groups. In contrast, serum triacylglycerol levels were significantly lower in the CR group than in the ND group (P < 0.05). Vacuolar degeneration and lipid accumulation in the liver were more prominent in the KD group than in the ND and CR groups. The mRNA levels of Pparα and Acox1 in the KD group were lower than those in the ND group, although no significant differences were detected. Relative levels of Cd36 and inflammatory genes in the mouse liver were significantly higher in the KD group than in the ND group (all P < 0.05). CONCLUSION: The KD significantly reduced FBG and FINS and improved glucose tolerance in db/db mice by upregulating ß-cell-specific transcription factors and reversing ß-cell dedifferentiation. However, the KD also induced hepatic lipid accumulation and aggravated inflammatory response in the liver of db/db mice.

Anti-Inflammatory Properties of Eugenol in Lipopolysaccharide-Induced Macrophages and Its Role in Preventing ß-Cell Dedifferentiation and Loss Induced by High Glucose-High Lipid Conditions.

Inflammation is a natural immune response to injury, infection, or tissue damage. It plays a crucial role in maintaining overall health and promoting healing. However, when inflammation becomes chronic and uncontrolled, it can contribute to the development of various inflammatory conditions, including type 2 diabetes. In type 2 diabetes, pancreatic ß-cells have to overwork and the continuous impact of a high glucose, high lipid (HG-HL) diet contributes to their loss and dedifferentiation. This study aimed to investigate the anti-inflammatory effects of eugenol and its impact on the loss and dedifferentiation of ß-cells. THP-1 macrophages were pretreated with eugenol for one hour and then exposed to lipopolysaccharide (LPS) for three hours to induce inflammation. Additionally, the second phase of NLRP3 inflammasome activation was induced by incubating the LPS-stimulated cells with adenosine triphosphate (ATP) for 30 min. The results showed that eugenol reduced the expression of proinflammatory genes, such as IL-1ß, IL-6 and cyclooxygenase-2 (COX-2), potentially by inhibiting the activation of transcription factors NF-κB and TYK2. Eugenol also demonstrated inhibitory effects on the levels of NLRP3 mRNA and protein and Pannexin-1 (PANX-1) activation, eventually impacting the assembly of the NLRP3 inflammasome and the production of mature IL-1ß. Additionally, eugenol reduced the elevated levels of adenosine deaminase acting on RNA 1 (ADAR1) transcript, suggesting its role in post-transcriptional mechanisms that regulate inflammatory responses. Furthermore, eugenol effectively decreased the loss of ß-cells in response to HG-HL, likely by mitigating apoptosis. It also showed promise in suppressing HG-HL-induced ß-cell dedifferentiation by restoring ß-cell-specific biomarkers. Further research on eugenol and its mechanisms of action could lead to the development of therapeutic interventions for inflammatory disorders and the preservation of ß-cell function in the context of type 2 diabetes.

Angiotensin-(1-7) Improves Islet ß-cell Dedifferentiation by Activating PI3K/Akt/FoxO1 Pathway.

BACKGROUND: Islet ß-cell dedifferentiation may be the main cause of reduced insulin secretion. Angiotensin-(1-7) [Ang-(1-7)] can attenuate high glucose-induced apoptosis and dedifferentiation of pancreatic ß-cell, but the specific signal transduction pathway and mechanism are not yet clear. OBJECTIVES: This study aimed to investigate the effects of Ang-(1-7) on high glucose-induced islet ß-cell dedifferentiation by activating the phosphatidylinositol-3-kinase/Protein kinase B/ Forkhead box transcription factor O1 (PI3K/Akt/FoxO1) signaling pathway. METHODS: The mouse islet ß-cell line MIN6 cells were passaged and cultured and randomly divided into five groups: control (Con) group, high glucose (HG) group, HG with Ang-(1-7) group, HG with Ang-(1-7) and specific MasR antagonist A-779 group, and HG with Ang-(1-7) and PI3K inhibitor LY294002 group. After 48 hours, glucose-stimulated insulin secretion (GSIS) was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of ß-cell-specific factors (Pancreatic duodenal homeobox-1 (Pdx1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A(MafA)) and endocrine progenitor cell-specific factors (Octamer binding transcription factor 4(Oct4), Nanog) were measured by Real Time-PCR and Western blot. The factors of protein expression levels of PI3K/Akt/FoxO1 signaling pathway (Akt, p-Akt, Fox- O1, p-FoxO1) were determined by Western blot. RESULTS: We observed for the first time that high glucotoxicity can induce dedifferentiation of pancreatic islet ß-cell, causing a decrease in insulin secretion levels and expression of Pdx1, MafA, p-- FoxO1, and p-Akt and an increase in expression of Oct4 and Nanog. After Ang-(1-7) intervention, insulin secretion levels and expression of Pdx1, MafA, p-FoxO1 and p-Akt were increased, and the levels of Oct4 and Nanog were reduced. However, A-779 and LY294002 could reverse this effect. During these processes, the total Akt and total FoxO1 expression did not change significantly. CONCLUSION: Ang-(1-7) may prevent high glucose-induced pathological dedifferentiation of pancreatic ß-cell by activating the PI3K/Akt/FoxO1 signaling pathway.

Angiotensin (1-7) reverses glucose-induced islet ß cell dedifferentiation by Wnt/ß-catenin/FoxO1 signalling pathway.

INTRODUCTION: Recent studies have shown that a decline in isletß cells quality is due to ß-cell dedifferentiation, not only ß-cell apoptosis. Angiotensin (1-7) [Ang(1-7)] could attenuate high glucose-induced apoptosis and dedifferentiation of pancreaticß cells by combining with MAS receptors. However, the mechanism of such action has not been elucidated. Recent studies have revealed that Wnt/ß-catenin and forkhead box transcription factor O1 (FoxO1) are associated with ß-cell dedifferentiation. Our study aims to explore whether the effects of Ang(1-7)on islet b cell dedifferentiation are mediated through the Wnt/ß-catenin/FoxO1 pathway. MATERIAL AND METHODS: Isletß cells were divided into 6 groups: a control group, a high-glucose group, high glucose with Ang(1-7) group, high-glucose with Ang(1-7) and A779 group, high-glucose with angiotensin(1-7) and CHIR99021 group, and high-glucose with CHIR99021 group. A779 is a kind of MAS receptor antagonist that blocks the action of Ang(1-7), and CHIR99021 is a Wnt pathway activator. The morphology of pancreaticß cells was observed in each group after 48 hours of intervention. ß-cell insulin secretory function and expressions of relevant factors were measured. RESULTS: Compared with the control group, the cell morphology became degraded in the high-glucose group and the capability of insulin secretion was reduced. Meanwhile, the expressions of matureß cells markers [pancreatic and duodenal homeobox 1 (Pdx1) and MAF BZIP transcription factor A (MafA)] were reduced, while the expressions of endocrine progenitor cells makers [octamer-binding transcription factor 4 (Oct4) and Nanog] were increased. The addition of CHIR99021 resulted in profound deep destruction ofß cells compared with the high-glucose group. However, such changes were dramatically reversed following the treatment of Ang(1-7). The addition of A779 significantly inhibited the improvement caused by Ang(1-7). CONCLUSION: Ang(1-7) can effectively reverseß cell dedifferentiation through Wnt/ß-catenin/FoxO1 pathway. It might be a new strategy for preventing and treating diabetes.