in-vivo-imaging-liver-spheroids-engrafted-anterior-chamber-mouse

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

Karolinska University Hospital

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

Type 2 diabetes mellitus is affecting more than 382 million people worldwide. Although much progress has been made, a comprehensive understanding of the underlying disease mechanism is still lacking. Here we report a role for the β-cell primary cilium in type 2 diabetes susceptibility. We find impaired glucose handling in young Bbs4(-/-) mice before the onset of obesity. Basal body/ciliary perturbation in murine pancreatic islets leads to impaired first phase insulin release ex and in vivo. Insulin receptor is recruited to the cilium of stimulated β-cells and ciliary/basal body integrity is required for activation of downstream targets of insulin signalling. We also observe a reduction in the number of ciliated β-cells along with misregulated ciliary/basal body gene expression in pancreatic islets in a diabetic rat model. We suggest that ciliary function is implicated in insulin secretion and insulin signalling in the β-cell and that ciliary dysfunction could contribute to type 2 diabetes susceptibility.

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents.

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell-specific reduction of miR-31-5p levels, both after intramuscular injection of IFNβ and in patients with Sjögren's syndrome (SjS). To interrogate the role of miR-31-51p in T cells we transfected human CD4 T cells with a miR-31-5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR-31-5p. Furthermore, treatment with IFN-α also increased the basal levels of human CD4 T-cell metabolism. In all, our results suggest that reduced levels of miR-31-5p in T cells of SjS patients support autoimmune T-cell responses during chronic type I IFN exposure.

miR-31 regulates energy metabolism and is suppressed in T cells from patients with Sjögren's syndrome.

Cell metabolism drives T cell functions, while platelets regulate overall CD4 T cell immune responses.

Platelet factor 4 enhances CD4 T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis.

Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity.

Lowering apolipoprotein CIII protects against high-fat diet-induced metabolic derangements.

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.

Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models.

Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation.

Cell metabolism has been shown to play an active role in regulation of stemness and fate decision. In order to identify favorable culture conditions for mesenchymal stromal cells (MSCs) prior to transplantation, this study aimed to characterize the metabolic function of MSCs from different developmental stages in response to different oxygen tension during expansion.

Diversity of respiratory parameters and metabolic adaptation to low oxygen tension in mesenchymal stromal cells.

Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.

Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology.

Pancreatic islets are micro-organs composed of a mixture of endocrine and non-endocrine cells, where the former secrete hormones and peptides necessary for metabolic homeostasis. Through vasculature and innervation the cells within the islets are in communication with the rest of the body, while they interact with each other through juxtacrine, paracrine and autocrine signals, resulting in fine-tuned sensing and response to stimuli. In this context, cellular protrusion in islet cells, such as primary cilia and filopodia, have gained attention as potential signaling hubs. During the last decade, several pieces of evidence have shown how the primary cilium is required for islet vascularization, function and homeostasis. These findings have been possible thanks to the development of ciliary/basal body specific knockout models and technological advances in microscopy, which allow longitudinal monitoring of engrafted islets transplanted in the anterior chamber of the eye in living animals. Using this technique in combination with optogenetics, new potential paracrine interactions have been suggested. For example, reshaping and active movement of filopodia-like protrusions of δ-cells were visualized in vivo, suggesting a continuous cell remodeling to increase intercellular contacts. In this review, we discuss these recent discoveries regarding primary cilia and filopodia and their role in islet homeostasis and intercellular islet communication.

Novel aspects of intra-islet communication: Primary cilia and filopodia.

Mitochondrial dynamics and bioenergetics are central to glucose-stimulated insulin secretion by pancreatic beta cells. Previously, we demonstrated that a disturbance in glucose-invoked fission impairs insulin secretion by compromising glucose catabolism. Here, we investigated whether the overexpression of mitochondrial fission regulator  in MIN6 cells can improve or rescue insulin secretion. Although  overexpression slightly improves the triggering mechanism of insulin secretion of the -knockdown cells and has no adverse effects on mitochondrial metabolism in wildtype MIN6 cells, the constitutive presence of  unexpectedly impairs insulin content, which leads to a reduction in the absolute values of secreted insulin. Coherent with previous studies in -overexpressing muscle cells, we found that the upregulation of ER stress-related genes (, , and ) possibly impacts insulin production in MIN6 cells. Collectively, we confirm the important role of  for the energy-coupling of insulin secretion but unravel off-targets effects by  overexpression on insulin content that warrant caution when manipulating  in disease therapy.

Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

Longitudinal monitoring of liver function in vivo is hindered by the lack of high-resolution non-invasive imaging techniques. Using the anterior chamber of the mouse eye as a transplantation site, we have established a platform for longitudinal in vivo imaging of liver spheroids at cellular resolution. Transplanted liver spheroids engraft on the iris, become vascularized and innervated, retain hepatocyte-specific and liver-like features and can be studied by in vivo confocal microscopy. Employing fluorescent probes administered intravenously or spheroids formed from reporter mice, we showcase the potential use of this platform for monitoring hepatocyte cell cycle activity, bile secretion and lipoprotein uptake. Moreover, we show that hepatic lipid accumulation during diet-induced hepatosteatosis is mirrored in intraocular in vivo grafts. Here, we show a new technology which provides a crucial and unique tool to study liver physiology and disease progression in pre-clinical and basic research.

Article	Year
Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects. The international journal of biochemistry & cell biology\| PubMed ID: 24814290	2014
Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents. Nature communications\| PubMed ID: 25374274	2014
miR-31 regulates energy metabolism and is suppressed in T cells from patients with Sjögren's syndrome. European journal of immunology\| PubMed ID: 30307034	2019
Platelet factor 4 enhances CD4 T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 32671959	2020
Lowering apolipoprotein CIII protects against high-fat diet-induced metabolic derangements. Science advances\| PubMed ID: 33712458	2021
Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models. Journal of cellular and molecular medicine\| PubMed ID: 33742502	2021
An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation. Nature communications\| PubMed ID: 34772928	2021
Diversity of respiratory parameters and metabolic adaptation to low oxygen tension in mesenchymal stromal cells. Metabolism open\| PubMed ID: 35528374	2022
Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology. Life science alliance\| PubMed ID: 36104081	2022
Novel aspects of intra-islet communication: Primary cilia and filopodia. Advances in biological regulation\| PubMed ID: 36266190	2023
Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells. International journal of molecular sciences\| PubMed ID: 36293194	2022
Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function. Nature communications\| PubMed ID: 38278787	2024

Noah Moruzzi

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