Leveraging zebrafish to investigate pancreatic development, regeneration, and diabetes.

The zebrafish has become an outstanding model for studying organ development and tissue regeneration, which is prominently leveraged for studies of pancreatic development, insulin-producing ß-cells, and diabetes. Although studied for more than two decades, many aspects remain elusive and it has only recently been possible to investigate these due to technical advances in transcriptomics, chemical-genetics, genome editing, drug screening, and in vivo imaging. Here, we review recent findings on zebrafish pancreas development, ß-cell regeneration, and how zebrafish can be used to provide novel insights into gene functions, disease mechanisms, and therapeutic targets in diabetes, inspiring further use of zebrafish for the development of novel therapies for diabetes.

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis.

AIMS/HYPOTHESIS: Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. METHODS: In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. RESULTS: Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. CONCLUSIONS/INTERPRETATION: Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. DATA AVAILABILITY: Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398 ).

In vivo screen identifies a SIK inhibitor that induces ß cell proliferation through a transient UPR.

It is known that ß cell proliferation expands the ß cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for ß cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human ß cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in ß cells blocks the effect of HG-9-91-01 on ß cell proliferation. Single-cell transcriptomic analyses of mouse ß cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive ß cell proliferation.

Is sustainable extensive green roof realizable without irrigation in a temperate monsoonal climate? A case study in Beijing.

A strategy to combat the adverse effects of urbanization involves the installation of green roofs under different climatic conditions. The design and maintenance of green roof systems need to be tailored to the local climate. However, there is a scarcity of reports on the performance of plants under temperate monsoonal climatic conditions. This study follows the growth pattern of 28 species (18 non-succulent forbs and 10 succulents) grown at three substrate depths (10, 15, and 20 cm) over three years on an unirrigated extensive green roof, located in Beijing, China. The results of this study revealed that sustainable extensive green roof was realizable without irrigation in Beijing. In terms of plant adaptive strategies, the most successful plants in this study were the stress-tolerant species, followed by the ruderal species. While deeper substrate could facilitate the survival and performance of plants, substrate moisture content was more significant for the survival of plants in the dry and cold winter in Beijing. This study recommended the use of a substrate depth, which was at least 15 cm deep for unirrigated green roofs in Beijing.

Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. METHODS: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. RESULTS: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. CONCLUSION: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.

Resveratrol controlled the fate of porcine pancreatic stem cells through the Wnt/ß-catenin signaling pathway mediated by Sirt1.

Porcine pancreatic stem cells (PSCs) are considered promising transplant materials that may be used to treat diabetes, but some problems, such as insufficient cell number and low differentiation efficiency, should be solved before its clinical application. Resveratrol is a natural polyphenolic compound that can alleviate the complications of diabetes. In this study, we aimed to explore the specific effect of resveratrol on porcine PSCs. We treated porcine PSCs with 10 µM, 25 µM resveratrol to explore the effect of resveratrol on porcine PSCs. We found that 10 µM resveratrol improved the proliferation of porcine PSCs, increased the expression of A-ß-catenin (active ß-catenin), Pcna, C-Myc, Bcl-2 and sirtuin-1 (Sirt1), and decreased the expression of P53, Caspase3. While 25 µM resveratrol had almost opposite effect compared with 10 µM resveratrol group. The utilization of Dickkopf-related protein 1 (DKK1, Wnt signaling pathway inhibitor) and nicotinamide (Sirt1 inhibitor) suggested that resveratrol regulated cell proliferation by controlling Wnt signaling pathway and this effect was mediated by Sirt1. Our results further revealed that 10 µM resveratrol promoted the formation of ß-like cells regulated by Wnt/ß-catenin signal pathway. Relatively low-dose resveratrol could improve porcine PSCs fate. It lays theoretical foundation for diabetes treatment with cell transplantation in future.

Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active ß-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway.

Autophagy stimulated proliferation of porcine PSCs might be regulated by the canonical Wnt signaling pathway.

Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active ß-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated that autophagy stimulated proliferation of PSCs might be regulated by the canonical Wnt signaling pathway. Our results for the first time shed light on a role of autophagy for stimulating the proliferation of porcine PSCs.

miR-375 controls porcine pancreatic stem cell fate by targeting 3-phosphoinositide-dependent protein kinase-1 (Pdk1).

OBJECTIVES: miR-375 is one of the highly expressed microRNAs (miRNAs) found in pancreatic islets of both humans and mice. In this study, we investigated functions of miRNA miR-375 in porcine pancreatic stem cells (PSC). MATERIALS AND METHODS: We transfected mimic and inhibitor of miR-375 in PSCs to measure functional roles of the microRNA and its effects on cell cycle proliferation and cell differentiation were determined. Luciferase assays were also performed to reveal the target gene of miR-375. RESULTS: Overexpression of miR-375 suppressed proliferation, promoted apoptosis and inhibited differentiation into islet-like cells. PDK1 was identified as being a target of miR-375. Furthermore, we found that overexpression of miR-375 inhibited activation of the PDK1-AKT signalling pathway. CONCLUSION: miR-375 directly targeted PDK1 in porcine PSCs, suppressing cell proliferation and differentiation into islet-like cells.

Establishment of a porcine pancreatic stem cell line using T-REx(™) system-inducible Wnt3a expression.

OBJECTIVES: Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T-REx(™) system, to study mechanisms of Wnt3a during PSC proliferation and differentiation. MATERIALS AND METHODS: Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR (QRT-PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs. RESULTS: Wnt3a-expressing clones regulated by T-REx(™) were successfully obtained. Wnt3a and GFP expression were strictly regulated by Dox in a time- and dose-dependent manner. Furthermore, we found that Wnt3a-expressing porcine PSCs induced by Dox exhibited raised proliferative potential. After Dox stimulation, expression of PCNA, C-MYC and active ß-catenin were higher, but were down-regulated after Dkk1 addition. CONCLUSION: We established a porcine PSC line that dynamically expressed Wnt3a, and we found that Wnt3a promoted PSC proliferative potential. This inducible expression system thus provides an important tool for further study on porcine PSC development and differentiation.