Interplay between mitochondria, primary cilium, diabetes and its complications

Diabetes is one of the major health problems of the 21st century. Dysfunction of the insulin secreting pancreatic β-cell together with insulin resistance is central to the pathogenesis of type 2 diabetes mellitus (T2DM). The cause of the disease and the underlying mechanisms linking hyperglycemia to diabetes complications are still unclear. This thesis is focused on two cellular organelles, the mitochondrion and the primary cilium, and their role in the pathophysiological mechanisms of diabetes and its complications. In the first paper, we studied the effect of hyperglycemia on cell biology and energy metabolism in human primary fibroblasts and endothelial cells. Acute hyperglycemia triggered a metabolic switch from mitochondrial respiration to aerobic glycolysis, which was persistent after prolonged exposure together with reduced ATP/ADP ratio without increase in reactive oxygen species (ROS). An acute decrease in mitochondrial transmembrane potential and cellular proliferation with changes in cytoskeletal reorganization was linked to the increased osmotic pressure induced by hyperglycemia. In the second and third papers we investigated the effect of hypoxia, a common feature in diabetes, and hyperoxia in pancreatic islets. Here, we found deleterious effects on mitochondrial content, respiration and glucose-stimulated insulin secretion. Preconditioning with the K+ATP channel opener diazoxide enhanced insulin release, HIF-1α and AMPK activation and improved β-cell survival in response to hypoxia. In the fourth paper, a role for the β-cell primary cilium in diabetes was reported. We found reduced first phase insulin secretion in ciliary defective cells and islets, and impaired glucose tolerance in a ciliopathy mouse model. These results were linked to impaired recruitment of insulin receptor A to the cilium, necessary for proper insulin signaling. Mitochondrial respiration and glucose uptake was unaffected by cilia impairment. Additionally, in vivo evidence of ciliary morphology alteration in the GK rat, a model of T2DM, supported a relationship between ciliary defect and T2DM. Preliminary results show that decreasing intracellular ATP and increasing mitochondrial ROS production impaired cilia morphology and/or number in two different cell types. Further, cilia were decreased in number with altered morphology in the kidneys of a mouse model of T2DM with diabetic nephropathy, characterized by increased ROS and altered mitochondrial metabolism. Finally, a reduction of 60-80% in mtDNA content (reported in diabetes) did not affect mitochondrial metabolism, respiration and energy production in two different cell types. In summary, mitochondrial dysfunctions during diabetes and its complications are most probably due to a combination of hyperglycemia and other factors such as hypoxia, depending on the cells and tissues involved. A proper ciliary/basal body function is necessary for insulin release and signaling in β-cell. Cilia morphology and number can be affected by mitochondrial dysfunction/ROS and thus related to diabetic complications such as diabetic nephropathy

Insulin Receptor Isoforms in Physiology and Metabolic Disease

Insulin receptors (IRs) are ubiquitously expressed and essential for all cell types. Their signaling cascades are connected to key pathways involved in cell metabolism, proliferation, and differentiation, amongst others. Thus, dysregulation of IR-mediated signaling can lead to diseases such as metabolic disorders. In mammals, the IR pre-mRNA is alternatively spliced to generate two receptor isoforms, IR-A and IR-B, which differ in 12 amino acids in the α-chain involved in ligand binding. Given the isoforms have different affinities for their ligands insulin, proinsulin, and insulin-like growth factors (IGFs), it is speculated that IR amount and splicing regulation might contribute to a change in IR-mediated effects and/or insulin resistance. The aim of this chapter is to increase awareness of this subject in the research fields of diseases characterized by disturbances in insulin signaling. Here, we will describe the IR isoform distribution and discuss the current knowledge of their expression and ligand binding affinities as well as their signaling in physiology and during obesity and type 2 diabetes in humans and animal models. Moreover, we will discuss the necessary steps to gain a better understanding on the function and regulation of the IR isoforms, which could result in future therapeutic approaches against IR-related dysfunction

Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells

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 Drp1 in MIN6 cells can improve or rescue insulin secretion. Although Drp1 overexpression slightly improves the triggering mechanism of insulin secretion of the Drp1-knockdown cells and has no adverse effects on mitochondrial metabolism in wildtype MIN6 cells, the constitutive presence of Drp1 unexpectedly impairs insulin content, which leads to a reduction in the absolute values of secreted insulin. Coherent with previous studies in Drp1-overexpressing muscle cells, we found that the upregulation of ER stress-related genes (BiP, Chop, and Hsp60) possibly impacts insulin production in MIN6 cells. Collectively, we confirm the important role of Drp1 for the energy-coupling of insulin secretion but unravel off-targets effects by Drp1 overexpression on insulin content that warrant caution when manipulating Drp1 in disease therapy

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

Objective 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. Materials and methods We cultured human fetal cardiac MSCs and human adult bone-marrow MSCs for a week under hypoxia (3% O2) and normoxia (20% O2). We performed mitochondrial characterization and assessed oxygen consumption- and extracellular acidification-rates (OCR and ECAR) in addition to oxygen-sensitive respiration and mitochondrial complex activities, using both the Seahorse and Oroboros systems. Results Adult and fetal MSCs displayed similar basal respiration and mitochondrial amount, however fetal MSCs had lower spare respiratory capacity and apparent coupling efficiency. Fetal MSCs expanded in either hypoxia or normoxia demonstrated similar acidification rates, while adult MSCs downregulated their aerobic glycolysis in normoxia. Acute decrease in oxygen tension caused a higher respiratory inhibition in adult compared to fetal MSCs. In both sources of MSCs, minor changes in complex activities in normoxic and hypoxic cultures were found. Conclusions In contrast to adult MSCs, fetal MSCs displayed similar respiration and aerobic glycolysis at different O2 culture concentrations during expansion. Adult MSCs adjusted their respiration to glycolytic activities, depending on the culture conditions thus displaying a more mature metabolic function. These findings are relevant for establishing optimal in vitro culturing conditions, with the aim to maximize engraftment and therapeutic outcome.CC BY-NC-ND 4.0Corresponding author: Department of Surgical Sciences, Uppsala University, 751 85, Uppsala, Sweden. E-mail address: [email protected] (K.-H. Grinnemo).Available online 3 February 2022, Version of Record 5 February 2022The project was funded by Karolinska Institute-Mayo Clinic Collaborative Grant 2013; The Swedish Research Council young investigator: 2013–3590; Stockholm county; The Swedish Research Council; The Family Erling-Persson Foundation; ERC-2018-AdG (834860 EYELETS); Uppsala county; Uppsala County Association against Heart and Lung Diseases; and Higher Education of the Russian Federation (agreement no. 075-15-2020-899).</p

Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories

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

To characterize molecular changes during cell type transitions, the authors develop a method to simultaneously measure protein expression and thermal stability changes. They apply this approach to study differences between human pluripotent stem cells, their progenies, parental and allogeneic cells. 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

A Water Soluble CoQ 10 Formulation Improves Intracellular Distribution and Promotes Mitochondrial Respiration in Cultured Cells

Background: Mitochondria are both the cellular powerhouse and the major source of reactive oxygen species. Coenzyme Q 10 plays a key role in mitochondrial energy production and is recognized as a powerful antioxidant. For these reasons it can be argued that higher mitochondrial ubiquinone levels may enhance the energy state and protect from oxidative stress. Despite the large number of clinical studies on the effect of CoQ10 supplementation, there are very few experimental data about the mitochondrial ubiquinone content and the cellular bioenergetic state after supplementation. Controversial clinical and in vitro results are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability. Principal Findings: We measured the cellular and mitochondrial ubiquinone content in two cell lines (T67 and H9c2) after supplementation with a hydrophilic CoQ10 formulation (QterH) and native CoQ10. Our results show that the water soluble formulation is more efficient in increasing ubiquinone levels. We have evaluated the bioenergetics effect of ubiquinone treatment, demonstrating that intracellular CoQ10 content after Qter supplementation positively correlates with an improved mitochondrial functionality (increased oxygen consumption rate, transmembrane potential, ATP synthesis) and resistance to oxidative stress. Conclusions: The improved cellular energy metabolism related to increased CoQ 10 content represents a strong rationale fo

Effect of Qter® treatment on ATP, protein content and cell growth in H9c2 cells.

<p>H9c2 cells were treated up to 72 hours with 100 nM Qter® and the ATP content was measured at 24, 48 and 72 hours by HPLC analysis (A). Panel B shows the intracellular ATP content after 24 hours treatment with 100 nM Qter® or native CoQ₁₀, measured using luminescence ATP detection assay. Data are reported as arbitrary luminometric units and normalized on total protein content. (Values are means ± S.D.,n = 5, * p≤0.01 vs control). H9c2 cells treated with 100 nM Qter up to 72 hours were assayed for protein content at 24, 48 and 72 hours. Protein content was evaluated by Lowry method (C), (Values are means ± S.D., n = 5, * p≤0.05 vs. control). Cell growth was assessed by trypan blue exclusion method (D).</p