Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Control of pancreatic β cell regeneration by glucose metabolism

Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

Bace2 is a β cell-enriched protease that regulates pancreatic β cell function and mass

Decreased β cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human β cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented β cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of β cell maintenance and provide a rational strategy to inhibit this protease?for the expansion of functional pancreatic β cell mass.     

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.     

Pancreatic β cell regeneration induced by clinical and preclinical agents

Diabetes,one of the most common chronic diseases in the modern world,has pancreaticβcell deficiency as a major part of its pathophysiological mechanism.Pancreatic regeneration is a potential therapeutic strategy for the recovery ofβcell loss.However,endocrine islets have limited regenerative capacity,especially in adult humans.Almost all hypoglycemic drugs can protectβcells by inhibitingβcell apoptosis and dedifferentiation via correction of hyperglycemia and amelioration of the consequent inflammation and oxidative stress.Several agents,including glucagon-like peptide-1 andγ-aminobutyric acid,have been shown to promoteβcell proliferation,which is considered the main source of the regeneratedβcells in adult rodents,but with less clarity in humans.Pancreatic progenitor cells might exist and be activated under particular circumstances.Artemisinins andγ-aminobutyric acid can induceα-to-βcell conversion,although some disputes exist.Intestinal endocrine progenitors can transdeterminate into insulin-producing cells in the gut after FoxO1 deletion,and pharmacological research into FoxO1 inhibition is ongoing.Other cells,including pancreatic acinar cells,can transdifferentiate intoβcells,and clinical and preclinical strategies are currently underway.In this review,we summarize the clinical and preclinical agents used in different approaches forβcell regeneration and make some suggestions regarding future perspectives for clinical application.     

Diabetes mellitus and the β cell: the last ten years

Diabetes is a major global problem. During the past decade, the genetic basis of various monogenic forms of the disease, and their underlying molecular mechanisms, have been elucidated. Many genes that increase type 2 diabetes (T2DM) risk have also been identified, but how they do so remains enigmatic. Nevertheless, defective insulin secretion emerges as the main culprit in both monogenic and polygenic diabetes, with environmental and lifestyle factors, via obesity, accounting for the current dramatic increase in T2DM. There also have been significant advances in therapy, particularly for some monogenic disorders. We review here what ails the β cell and how its function may be restored.     

Inhibition of p38 mitogen-activated protein kinase exerts a hypoglycemic effect by improving β cell function via inhibition of β cell apoptosis in db/db mice

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in endoplasmic reticulum stress (ERS) and inflammation, which may play an important role in the pathogenesis of type 2 diabetes (T2DM). This study aimed to investigate whether p38 MAPK contributes to the pathogenesis of T2DM. 6-week-old female db/db mice were randomly assigned to Dmo and Dmi groups, and C57 mice were assigned as controls. The Dmi group was gavaged with the p38 MAPK inhibitor SB203580 for 9?weeks, and the effects on β cell dysfunction and apoptosis were investigated. db/db mice showed higher food intake, body mass, fasting glucose, and plasma insulin levels than C57 mice. After SB203580 administration, blood glucose was significantly lower. HOMA β and HOMA IR were improved. Islet mRNA expression levels of the ERS markers were lower. P38 MAPK inhibition reduced blood glucose and improved β cell function, at least in part by reducing β cell apoptosis.     

Cyclin D2 is sufficient to drive β cell self-renewal and regeneration

Diabetes results from an inadequate mass of functional β cells, due to either β cell loss caused by autoimmune destruction (type I diabetes) or β cell failure in response to insulin resistance (type II diabetes). Elucidating the mechanisms that regulate β cell mass may be key to developing new techniques that foster β cell regeneration as a cellular therapy to treat diabetes. While previous studies concluded that cyclin D2 is required for postnatal β cell self-renewal in mice, it is not clear if cyclin D2 is sufficient to drive β cell self-renewal. Using transgenic mice that overexpress cyclin D2 specifically in β cells, we show that cyclin D2 overexpression increases β cell self-renewal post-weaning and results in increased β cell mass. β cells that overexpress cyclin D2 are responsive to glucose stimulation, suggesting they are functionally mature. β cells that overexpress cyclin D2 demonstrate an enhanced regenerative capacity after injury induced by streptozotocin toxicity. To understand if cyclin D2 overexpression is sufficient to drive β cell self-renewal, we generated a novel mouse model where cyclin D2 is only expressed in β cells of cyclin D2^?/? mice. Transgenic overexpression of cyclin D2 in cyclin D2^?/? β cells was sufficient to restore β cell mass, maintain normoglycaemia, and improve regenerative capacity when compared with cyclin D2^?/? littermates. Taken together, our results indicate that cyclin D2 is sufficient to regulate β cell self-renewal and that manipulation of its expression could be used to enhance β cell regeneration.     

Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.     

The role of autophagy in endoplasmic reticulum stress-induced pancreatic β cell death

In pancreatic β-cells, the endoplasmic reticulum (ER) is the crucial site for insulin biosynthesis, as this is where the protein-folding machinery for secretory proteins is localized. Perturbations to ER function of the β-cell, such as those caused by high levels of free fatty acid and insulin resistance, can lead to an imbalance in protein homeostasis and ER stress, which has been recognized as an important mechanism for type 2 diabetes. Macroautophagy (hereafter referred to as autophagy) is activated as a novel signaling pathway in response to ER stress. In this review, we outline the mechanism of ER stress-mediated β-cell death and focus on the role of autophagy in ameliorating ER stress. The development of drugs to take advantage of the potential protective effect of autophagy in ER stress, such as glucagon like peptide-1, will be a promising avenue of investigation.     

The relevance of AMP-activated protein kinase in insulin-secreting β cells: a potential target for improving β cell function?

Journal of Physiology and Biochemistry - AMP-activated protein kinase (AMPK) is present in different kinds of metabolically active cells. AMPK is an important intracellular energy sensor and plays...     

Estrogen receptor β signaling regulates the progression of Chinese non-small cell lung cancer

Prospective studies have found that the risk of non-small cell lung cancer (NSCLC) has close relationship with estrogen. The effects of estrogens are mediated via two estrogen receptor (ER) isoforms, that is, ER alpha (ERα) and ER beta (ERβ). ERα in NSCLC has been evaluated mostly by immunohistochemistry. However, our previous study showed that ERβ was also highly expressed in Chinese NSCLC. But the roles of ERβ in Chinese NSCLC have not been clarified as yet. So in the present study, two Chinese lung adenocarcinoma cell lines, SPC-A1 and LTEP-a2, were used and the role of ERβ in lung tumorigenesis was focused to be investigated by in vitro and in vivo experiments. The results showed that over-expressed ERβ can promote the development of NSCLC, while siRNAs targeting ERβ gene can inhibit growth of NSCLC cells and induce apoptosis of these cells via mitochondrial depolarization and caspase-3 activation. These results indicated that ERβ plays an important role in development of Chinese NSCLC. This suggests that ERβ deactivation or down-regulation may possess potential therapeutic utility for the treatment of lung cancer.     

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic β cell death

There is a growing evidence of the role of autophagy in pancreatic β cell homeostasis. During development of type 2 diabetes, β cells are required to supply the increased demand of insulin. In such a stage, β cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, β cell death and overt diabetes. In this study, we used insulin secretion-deficient β cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived β cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased β cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient β cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in β cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.     

Dyrk1A induces pancreatic β cell mass expansion and improves glucose tolerance

Type 2 diabetes is caused by a limited capacity of insulin-producing pancreatic β cells to increase their mass and function in response to insulin resistance. The signaling pathways that positively regulate functional β cell mass have not been fully elucidated. DYRK1A (also called minibrain/MNB) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. A significant amount of data implicates DYRK1A in brain growth and Down syndrome, and recent data indicate that Dyrk1A haploinsufficient mice have a low functional β cell mass. Here we ask whether Dyrk1A upregulation could be a way to increase functional β cell mass.

We used mice overexpressing Dyrk1A under the control of its own regulatory sequences (mBACTgDyrk1A). These mice exhibit decreased glucose levels and hyperinsulinemia in the fasting state. Improved glucose tolerance is observed in these mice as early as 4 weeks of age. Upregulation of Dyrk1A in β cells induces expansion of β cell mass through increased proliferation and cell size. Importantly, mBACTgDyrk1A mice are protected against high-fat-diet-induced β cell failure through increase in β cell mass and insulin sensitivity.

These studies show the crucial role of the DYRK1A pathway in the regulation of β cell mass and carbohydrate metabolism in vivo. Activating the DYRK1A pathway could thus represent an innovative way to increase functional β cell mass.     

Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.     

Structural and functional properties of meprin β metalloproteinase with regard to cell signaling

The metalloproteinase meprin β plays an important role during collagen I deposition in the skin, mucus detachment in the small intestine and also regulates the abundance of different cell surface proteins such as the interleukin-6 receptor (IL-6R), the triggering receptor expressed on myeloid cells 2 (TREM2), the cluster of differentiation 99 (CD99), the amyloid precursor protein (APP) and the cluster of differentiation 109 (CD109). With that, regulatory mechanisms that control meprin β activity and regulate its release from the cell surface to enable access to distant substrates are increasingly important. Here, we will summarize factors that alternate meprin β activity and thereby regulate its proteolytic activity on the cell surface or in the supernatant. We will also discuss cleavage of the IL-6R and TREM2 on the cell surface and compare it to CD109. CD109, as a substrate of meprin β, is cleaved within the protein core, thereby releasing defined fragments from the cell surface. At last, we will also summarize the role of proteases in general and meprin β in particular in substrate release on extracellular vesicles.