A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids.

Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity.

A small molecule MST1/2 inhibitor accelerates murine liver regeneration with improved survival in models of steatohepatitis.

Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.

The Hippo kinase LATS2 impairs pancreatic ß-cell survival in diabetes through the mTORC1-autophagy axis.

Diabetes results from a decline in functional pancreatic ß-cells, but the molecular mechanisms underlying the pathological ß-cell failure are poorly understood. Here we report that large-tumor suppressor 2 (LATS2), a core component of the Hippo signaling pathway, is activated under diabetic conditions and induces ß-cell apoptosis and impaired function. LATS2 deficiency in ß-cells and primary isolated human islets as well as ß-cell specific LATS2 ablation in mice improves ß-cell viability, insulin secretion and ß-cell mass and ameliorates diabetes development. LATS2 activates mechanistic target of rapamycin complex 1 (mTORC1), a physiological suppressor of autophagy, in ß-cells and genetic and pharmacological inhibition of mTORC1 counteracts the pro-apoptotic action of activated LATS2. We further show a direct interplay between Hippo and autophagy, in which LATS2 is an autophagy substrate. On the other hand, LATS2 regulates ß-cell apoptosis triggered by impaired autophagy suggesting an existence of a stress-sensitive multicomponent cellular loop coordinating ß-cell compensation and survival. Our data reveal an important role for LATS2 in pancreatic ß-cell turnover and suggest LATS2 as a potential therapeutic target to improve pancreatic ß-cell survival and function in diabetes.

Inhibition of PHLPP1/2 phosphatases rescues pancreatic ß-cells in diabetes.

Pancreatic ß-cell failure is the key pathogenic element of the complex metabolic deterioration in type 2 diabetes (T2D); its underlying pathomechanism is still elusive. Here, we identify pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) as phosphatases whose upregulation leads to ß-cell failure in diabetes. PHLPP levels are highly elevated in metabolically stressed human and rodent diabetic ß-cells. Sustained hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) is the primary mechanism of the PHLPP upregulation linking chronic metabolic stress to ultimate ß-cell death. PHLPPs directly dephosphorylate and regulate activities of ß-cell survival-dependent kinases AKT and MST1, constituting a regulatory triangle loop to control ß-cell apoptosis. Genetic inhibition of PHLPPs markedly improves ß-cell survival and function in experimental models of diabetes in vitro, in vivo, and in primary human T2D islets. Our study presents PHLPPs as targets for functional regenerative therapy of pancreatic ß cells in diabetes.

LDHA is enriched in human islet alpha cells and upregulated in type 2 diabetes.

The lactate dehydrogenase isoform A (LDHA) is a key metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. Whereas LDHA is highly expressed in many tissues, its expression is turned off in the differentiated adult ß-cell within the pancreatic islets. The repression of LDHA under normal physiological condition and its inappropriate upregulation under a diabetogenic environment is well-documented in rodent islets/ß-cells but little is known about LDHA expression in human islet cells and whether its abundance is altered under diabetic conditions. Analysis of public single-cell RNA-seq (sc-RNA seq) data as well as cell type-specific immunolabeling of human pancreatic islets showed that LDHA was mainly localized in human α-cells while it is expressed at a very low level in ß-cells. Furthermore, LDHA, both at mRNA and protein, as well as lactate production is upregulated in human pancreatic islets exposed to chronic high glucose treatment. Microscopic analysis of stressed human islets and autopsy pancreases from individuals with type 2 diabetes (T2D) showed LDHA upregulation mainly in human α-cells. Pharmacological inhibition of LDHA in isolated human islets enhanced insulin secretion under physiological conditions but did not significantly correct the deregulated secretion of insulin or glucagon under diabetic conditions.

Neratinib protects pancreatic beta cells in diabetes.

The loss of functional insulin-producing ß-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic ß-cell death and dysfunction; its deficiency restores functional ß-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a ß-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves ß-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves ß-cell function, survival and ß-cell mass in type 1 (streptozotocin) and type 2 (obese Leprdb/db) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential ß-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes.

Matrix Metalloproteinase-3 is Key Effector of TNF-α-Induced Collagen Degradation in Skin.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

Loss of Deubiquitinase USP1 Blocks Pancreatic ß-Cell Apoptosis by Inhibiting DNA Damage Response.

Impaired pancreatic ß-cell survival contributes to the reduced ß-cell mass in diabetes, but underlying regulatory mechanisms and key players in this process remain incompletely understood. Here, we identified the deubiquitinase ubiquitin-specific protease 1 (USP1) as an important player in the regulation of ß-cell apoptosis under diabetic conditions. Genetic silencing and pharmacological suppression of USP1 blocked ß-cell death in several experimental models of diabetes in vitro and ex vivo without compromising insulin content and secretion and without impairing ß-cell maturation/identity genes in human islets. Our further analyses showed that USP1 inhibition attenuated DNA damage response (DDR) signals, which were highly elevated in diabetic ß-cells, suggesting a USP1-dependent regulation of DDR in stressed ß-cells. Our findings highlight a novel function of USP1 in the control of ß-cell survival, and its inhibition may have a potential therapeutic relevance for the suppression of ß-cell death in diabetes.

Hippo Signaling: Key Emerging Pathway in Cellular and Whole-Body Metabolism.

The evolutionarily conserved Hippo pathway is a key regulator of organ size and tissue homeostasis. Its dysregulation is linked to multiple pathological disorders. In addition to regulating development and growth, recent studies show that Hippo pathway components such as MST1/2 and LATS1/2 kinases, as well as YAP/TAZ transcriptional coactivators, are regulated by metabolic pathways and that the Hippo pathway controls metabolic processes at the cellular and organismal levels in physiological and metabolic disease states such as obesity, type 2 diabetes (T2D), nonalcoholic fatty liver disease (NAFLD), cardiovascular disorders, and cancer. In this review we summarize the connection between key Hippo components and metabolism, and how this interplay regulates cellular metabolism and metabolic pathways. The emerging function of Hippo in the regulation of metabolic homeostasis under physiological and pathological conditions is highlighted.

An SCFFBXO28 E3 Ligase Protects Pancreatic ß-Cells from Apoptosis.

Loss of pancreatic ß-cell function and/or mass is a central hallmark of all forms of diabetes but its molecular basis is incompletely understood. ß-cell apoptosis contributes to the reduced ß-cell mass in diabetes. Therefore, the identification of important signaling molecules that promote ß-cell survival in diabetes could lead to a promising therapeutic intervention to block ß-cell decline during development and progression of diabetes. In the present study, we identified F-box protein 28 (FBXO28), a substrate-recruiting component of the Skp1-Cul1-F-box (SCF) ligase complex, as a regulator of pancreatic ß-cell survival. FBXO28 was down-regulated in ß-cells and in isolated human islets under diabetic conditions. Consistently, genetic silencing of FBXO28 impaired ß-cell survival, and restoration of FBXO28 protected ß-cells from the harmful effects of the diabetic milieu. Although FBXO28 expression positively correlated with ß-cell transcription factor NEUROD1 and FBXO28 depletion also reduced insulin mRNA expression, neither FBXO28 overexpression nor depletion had any significant impact on insulin content, glucose-stimulated insulin secretion (GSIS) or on other genes involved in glucose sensing and metabolism or on important ß-cell transcription factors in isolated human islets. Consistently, FBXO28 overexpression did not further alter insulin content and GSIS in freshly isolated islets from patients with type 2 diabetes (T2D). Our data show that FBXO28 improves pancreatic ß-cell survival under diabetogenic conditions without affecting insulin secretion, and its restoration may be a novel therapeutic tool to promote ß-cell survival in diabetes.

Ageing potentiates diet-induced glucose intolerance, ß-cell failure and tissue inflammation through TLR4.

Ageing and obesity are two major risk factors for the development of type 2 diabetes (T2D). A chronic, low-grade, sterile inflammation contributes to insulin resistance and ß-cell failure. Toll-like receptor-4 (TLR4) is a major pro-inflammatory pathway; its ligands as well as downstream signals are increased systemically in patients with T2D and at-risk individuals. In the present study we investigated the combined effects of high fat/high sucrose diet (HFD) feeding, ageing and TLR4-deficiency on tissue inflammation, insulin resistance and ß-cell failure. In young mice, a short-term HFD resulted in a mildly impaired glucose tolerance and reduced insulin secretion, together with a ß-cell mass compensation. In older mice, HFD further deteriorated insulin secretion and induced a significantly impaired glucose tolerance and augmented tissue inflammation in adipose, liver and pancreatic islets, all of which was attenuated by TLR4 deficiency. Our results show that ageing exacerbates HFD-induced impairment of glucose homeostasis and pancreatic ß-cell function and survival, and deteriorates HFD-induced induction of mRNA expression of inflammatory cytokines and pro-inflammatory macrophage markers. TLR4-deficiency protects against these combined deleterious effects of a high fat diet and ageing through a reduced expression of inflammatory products in both insulin sensitive tissues and pancreatic islets.

mTORC2 Signaling: A Path for Pancreatic ß Cell's Growth and Function.

The mechanistic target of rapamycin (mTOR) signaling pathway is an evolutionary conserved pathway that senses signals from nutrients and growth factors to regulate cell growth, metabolism and survival. mTOR acts in two biochemically and functionally distinct complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which differ in terms of regulatory mechanisms, substrate specificity and functional outputs. While mTORC1 signaling has been extensively studied in islet/ß-cell biology, recent findings demonstrate a distinct role for mTORC2 in the regulation of pancreatic ß-cell function and mass. mTORC2, a key component of the growth factor receptor signaling, is declined in ß cells under diabetogenic conditions and in pancreatic islets from patients with type 2 diabetes. ß cell-selective mTORC2 inactivation leads to glucose intolerance and acceleration of diabetes as a result of reduced ß-cell mass, proliferation and impaired glucose-stimulated insulin secretion. Thereby, many mTORC2 targets, such as AKT, PKC, FOXO1, MST1 and cell cycle regulators, play an important role in ß-cell survival and function. This indicates mTORC2 as important pathway for the maintenance of ß-cell homeostasis, particularly to sustain proper ß-cell compensatory response in the presence of nutrient overload and metabolic demand. This review summarizes recent emerging advances on the contribution of mTORC2 and its associated signaling on the regulation of glucose metabolism and functional ß-cell mass under physiological and pathophysiological conditions in type 2 diabetes.

mTORC1 Signaling: A Double-Edged Sword in Diabetic ß Cells.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of metabolic and nutrient cues that integrates environmental inputs into downstream signaling pathways to control cellular metabolism, growth, and survival. While numerous in vitro and in vivo studies reported the positive functions of mTORC1 in the regulation of ß cell survival and proliferation under physiological conditions, more recent work demonstrates the opposite in the long term; this is exemplified by the constitutive inappropriate hyper-activation of mTORC1 in diabetic islets or ß cells under conditions of increased ß cell stress and metabolic demands. These recent findings uncover mTORC1's importance as an emerging significant player in the development and progression of ß cell failure in type 2 diabetes and suggest that mTORC1 may act as a "double edge sword" in the regulation of ß cell mass and function in response to metabolic stress such as nutrient overload and insulin resistance.

Reciprocal regulation of mTOR complexes in pancreatic islets from humans with type 2 diabetes.

AIMS/HYPOTHESIS: Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of nutritional status at the cellular and organismic level. While mTORC1 mediates beta cell growth and expansion, its hyperactivation has been observed in pancreatic islets from animal models of type 2 diabetes and leads to beta cell loss. We sought to determine whether such mTORC1 activation occurs in humans with type 2 diabetes or in metabolically stressed human islets and whether mTORC1 blockade can restore beta cell function of diabetic islets. METHODS: Human islets isolated from non-diabetic controls and individuals with type 2 diabetes, as well as human islets and INS-1E cells exposed to increased glucose (22.2 mmol/l), were examined for mTORC1/2 activity by western blotting analysis of phosphorylation of mTORC1 downstream targets ribosomal protein S6 kinase 1 (S6K1), S6 and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and mTORC2 downstream targets Akt and N-myc downstream regulated 1 (NDRG1). mTORC1/2 complexes' integrity was assessed by immunoprecipitation and subsequent western blot analysis. Cell-type specific expression of activated mTORC1 in human islets was examined by immunostaining of pS6 (Ser 235/236) in human islet sections. Beta cell function was measured by glucose-stimulated insulin secretion (GSIS). RESULTS: While mTORC2 signalling was diminished, mTORC1 activity was markedly increased in islets from patients with type 2 diabetes and in islets and beta cells exposed to increased glucose concentrations. Under high-glucose conditions in metabolically stressed human islets, we identified a reciprocal regulation of different mTOR complexes, with functional upregulation of mTORC1 and downregulation of mTORC2. pS6 immunostaining showed beta cell-specific upregulation of mTORC1 in islets isolated from patients with type 2 diabetes. Inhibition of mTORC1-S6K1 signalling improved GSIS and restored mTORC2 activity in islets from patients with type 2 diabetes as well as in islets isolated from diabetic db/db mice and mice fed a high-fat/high-sucrose diet. CONCLUSIONS/INTERPRETATION: Our data show the aberrant mTORC1 activity in islets from patients with type 2 diabetes, in human islets cultured under diabetes-associated increased glucose conditions and in diabetic mouse islets. This suggests that elevated mTORC1 activation is a striking pathogenic hallmark of islets in type 2 diabetes, contributing to impaired beta cell function and survival in the presence of metabolic stress.

Proproliferative and antiapoptotic action of exogenously introduced YAP in pancreatic ß cells.

Loss of functional pancreatic ß cells is a hallmark of both type 1 and 2 diabetes. Identifying the pathways that promote ß cell proliferation and/or block ß cell apoptosis is a potential strategy for diabetes therapy. The transcriptional coactivator Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, is a key regulator of organ size and tissue homeostasis by modulating cell proliferation and apoptosis. YAP is not expressed in mature primary human and mouse ß cells. We aimed to identify whether reexpression of a constitutively active form of YAP promotes ß cell proliferation/survival. Overexpression of YAP remarkably induced ß cell proliferation in isolated human islets, while ß cell function and functional identity genes were fully preserved. The transcription factor forkhead box M1 (FOXM1) was upregulated upon YAP overexpression and necessary for YAP-dependent ß cell proliferation. YAP overexpression protected ß cells from apoptosis triggered by multiple diabetic conditions. The small redox proteins thioredoxin-1 and thioredoxin-2 (Trx1/2) were upregulated by YAP; disruption of the Trx system revealed that Trx1/2 was required for the antiapoptotic action of YAP in insulin-producing ß cells. Our data show the robust proproliferative and antiapoptotic function of YAP in pancreatic ß cells. YAP reconstitution may represent a disease-modifying approach to restore a functional ß cell mass in diabetes.