Characterisation of pancreatic INS-1 insulinoma cells under chronic high glucose conditions as glucose toxicity model

Der Glukosestoffwechsel der pankreatischen Betazellen und die sich daraus ergebende Erhöhung des intrazellulären Konzentrationsverhältnisses von ATP zu ADP stellt das metabolische Signal für die Insulinsekretion dar. Im gesunden, normalen Zustand wird nach akuter Glukoseaufnahme das sezernierte Insulin durch dessen Neusynthese ersetzt. Dieser Prozess setzt eine funktionierende mRNA-Synthese voraus. Unter chronisch hyperglykämischen Bedingungen ist nicht nur die Insulintranskription, sondern auch die Proteinbiosynthese und Sekretion gestört. Daher war es Ziel der vorliegenden Arbeit Glukosestoffwechselwege zu identifizieren, die bei der Regulation der Insulingenexpression eine wesentliche Rolle spielen könnten. Des Weiteren sollte eine Assoziation zwischen Glukosemetabolismus und Proteinkinase- assoziierter Verminderung der Insulingenexpression gefunden werden. Zu diesem Zweck wurden an kultivierten pankreatischen Ratteninsulinomazellen (INS-1) Versuchsserien mit unterschiedlichen Fragestellungen durchgeführt. Anfänglich wurden anhand von Metabolitenprofilanalysen detaillierte Untersuchungen hinsichtlich der Glukoseverstoffwechselung unter chronisch hohen Glukosebedingungen durchgeführt. Aus den Analysen ergab sich, dass chronisch hohe Glukosekonzentrationen überwiegend zu metabolischen Veränderungen im Glukosestoffwechsel führen. Am deutlichsten waren Anreicherungen von glykolytischen und mitochondrialen Metaboliten, mit besonders kumulierenden Mengen von alpha-Ketoglutarat, sowie eine Akkumulation von Metaboliten des Pentosephosphatweges. Dabei war besonders eine zelluläre Anreicherung organischer Säuren auffallend. Eine starke Akkumulation der Pentosephosphatwegsmetaboliten Glucono-delta-lacton, 6-Phosphoglukonsäure und Glukonsäure und von Carbamylaspartat, initialer Teil des DNA/RNA Syntheseweges war nur bei chronisch hohen Glukosekonzentrationen zu verzeichnen. Die Spiegel dieser Metaboliten waren in INS-1 während der Kultivierung bei niedriger Glukose extrem niedrig beziehungsweise nicht detektierbar. Bei Glukonat handelt es sich um einen in pankreatischen Zellen neu identifizierten Metaboliten, dessen Herkunft als Glukoseabbauprodukt mit isotopenmarkierter [13C]Glukose nachgewiesen werden konnte. Dessen verstärkte Ausscheidung ins Medium könnte zum einen als Zeichen einer Stoffwechselüberbelastung gedeutet werden und könnte zum anderen eine Möglichkeit der Zelle aufzeigen, eine Anreicherung spezifischer Glukoseabbauprodukte zu kompensieren. Parallel zu den Metabolitenprofilanalysen sollte in INS-1 Zellen bei chronisch hohen Glukose, nach phänotypischer und genotypischer Charakterisierung, ein Proteinsignalweg, assoziiert mit einer Verminderung der Insulingenexpression, identifiziert werden. Voruntersuchungen zeigten, dass chronisch hohe Glukosekonzentrationen zu einer Verminderung der Insulingenexpression, assoziiert mit einer Verminderung der Insulingeninitiatoren MafA, Beta2, PDX-1 und des Differenzierungsfaktors Pax6 führten. Zusätzlich hatten chronisch hohe Glukosekonzentrationen eine Entdifferenzierung der Betazellen zur Folge, was sich in einer Induktion der Proliferation äußerte. Eine verstärkte Proliferation war möglicherweise auf eine erhöhte Aktivität der ERK1/2 Proteinkinase zurückzuführen. Die Hemmung der ERK1/2 Kinaseaktivität mit PD98059 resultierte in einer Erholung der Insulingenexpression und der positiven Insulingenregulatoren MafA, Beta2, PDX-1 und Pax6 nach chronisch hoher Glukoseexposition. Abschließend sollte geklärt werden, ob Metabolitenveränderungen an der Verminderung der Insulingenexpression beteiligt sein könnte und inwiefern die ERK1/2 Kinase dabei eine zentrale Rolle spielt. Eine Inhibierung der 6-Phosphoglukonsäuredehydrogenase des Pentosephosphatweges mit 6-AN resultierte ähnlich wie bei 16 mM Glukose in einer Akkumulation von Glucono-delta-lacton, 6-Phosphoglukonsäure und Glukonsäure und ging mit einer Verminderung der Insulingenexpression einher. Die Inhibierung der ERK1/2 Kinase hatte trotz chronisch hoher Glukosekonzentrationen keine Verminderung der Insulingenexpression zur Folge und resultierte in einer Verrinderung der Pentosephosphatwegmetaboliten Glucono-delta-lacton, 6-Phosphoglukonsäure und Glukonsäure, aber auch von alpha-Ketoglutarat. Die hier geschilderten Ergebnisse verdeutlichen, dass hohe Glukosekonzentrationen langfristig eine, unter normalen Bedingungen nicht auftretende, intrazelluläre Anreicherung einer Vielzahl von Glukoseabbauprodukten in Betazellen zur Folge haben. Besonders auffällig ist ein kritischer Anstieg organischer Säuren, welche mit einer ERK1/2 Kinase - vemittelten Verminderung der Insulingenexpression assoziiert sein könnten.Glucose metabolism of pancreatic beta cells and the resulting increase of the intracellular ATP/ADP ratio are the metabolic signal for insulin secretion. Under normal nonpathological conditions the secreted insulin is replenished by newly synthesed insulin in a process which requires fully functioning mRNA synthesis. Under chronic hyperglycaemic conditions not only insulin transcription, but also protein synthesis and secretion is impaired. It was therefore the aim of this work to identify glucose metabolic pathways which could play an important role during regulation of insulin gene expression and in doing so investigate the potential association between glucose metabolism and a proteinkinase-associated reduction of insulin gene expression. Utilising metabolite profiling analyses of rat-derived cultivated pancreatic insulinoma cells (INS-1) experiments were undertaken with a view to shed light on the effects of chronic high glucose conditions on glucose metabolism. These analyses permitted the determination that such conditions led to wide-ranging changes to this system. Among the most dominant effects were the accumulation of glycolytic and mitochondrial metabolites, in particular of alpha- ketoglutarate as well as of metabolites of the pentose phosphate pathway (PPP) in addition to a cellular accumulation of a number of organic acids. A particularly great accumulation of the PPP metabolites glucono-delta-lactone, 6-phosphogluconic acid and gluconic acid in addition to a similar accumulation of carbamyl-aspartic acid, a precursor in the synthesis of DNA and RNA, all of which were only detectable at significant levels under chromic high glucose conditions. Gluconic acid can be considered a newly identified metabolite in pancreatic beta cells, the origin of which as a glucose degradation product was unambiguously determined using isotopically-labelled [13C]-glucose. The increased secretion of this compound into the growth medium could be interpreted as a metabolic overload and as a indication that cells compensate for this by eliminating specific glucose by-products. Parallel to these metabolite profiling analyses, the INS-1 cells were also phenotypically and genotypically characterised in an attempt to identify a protein signal pathway associated with insulin gene expression reduction. Preliminary experiments showed that chronic high glucose levels led to decreased insulin gene expression, associated with decreased levels of transcription factors, such as MafA, Beta2, PDX-1 and differentiation factors, such as PAX6. Additionally, chronic high glucose levels resulted in increased proliferation of INS-1 which was thought to be a result of an increased ERK1/2 protein kinase activity. Inhibition of ERK1/2 activity with PD98059 prevented chronic high glucose- induced reduction of insulin gene expression and of transcription factors MafA, Beta2, PDX-1 und Pax6. Finally, it was intented to clarify whether metabolite changes participated in the reduction of insulin gene expression and to what extent ERK1/2 kinase is involved. Inhibition of the PPP enzyme 6-phosphogluconic acid dehydrogenase at low glucose levels with 6-aminonicotinamide resulted in a similar accumulation of glucono-delta- lactone, 6-phosphogluconic acid and gluconic acid as observed under high glucose conditions and also resulted in a reduction of insulin gene expression. The inhibition of ERK1/2, which prevented a reduction of insulin gene expression, also prevented such an accumulation of the PPP metabolites glucono-delta-lactone, 6-phosphogluconic acid and gluconic acid, but also alpha-ketoglutarate under chronic high glucose conditions. The results outlined here indicate that high glucose levels in the long term cause the intracellular enhancement of a number of glucose metabolic by-products in beta cells, which are not apparent under normal conditions. Especially noticeable was a critical increase of organic acids, which might be associated with a ERK1/2 kinase – mediated reduction of insulin gene expression

Glutamate dehydrogenase, insulin secretion and Type 2 Diabetes. A new means to protect the pancreatic β-cell?

In this issue of Journal of Endocrinology, Dr Han and colleagues report a protective effect of the glutamate dehydrogenase-activator BCH under diabetes-like conditions that impair β-cell function in both a pancreatic β-cell line and in db/db mice. Based on these observations, the authors suggest that BCH could serve as novel treatment modality in Type 2 Diabetes. The present commentary discusses the importance of the findings. Some additional questions are raised, that may be addressed in future investigations, as is some concern regarding BCH treatment of β-cell failure

Chronic high glucose and pyruvate levels differentially affect mitochondrial bioenergetics and fuel-stimulated insulin secretion from clonal INS-1 832/13 cells.

Glucotoxicity in pancreatic β-cells is a well-established pathogenetic process in Type 2 Diabetes. It has been suggested that metabolism-derived reactive oxygen species perturb the β-cell transcriptional machi-nery. Less is known about altered mitochondrial function in this condition. We used INS-1 832/13 cells cultured for 48 h in 2.8 mM glucose (low-G), 16.7 mM glucose (high-G) or 2.8 mM glucose plus 13.7 mM pyruvate (high-P) to identify metabolic perturbations. High-G cells showed decreased responsiveness, relative to low-G cells, with respect to mitochondrial membrane hyperpolarization, plasma membrane depolarization and insulin secretion, when stimulated acutely with 16.7 mM glucose or 10 mM pyruvate. In contrast, high-P cells were functionally unimpaired, eliminating chronic provision of saturating mitochondrial substrate as a cause of glucotoxicity. Although cellular insulin content was depleted in high-G cells, relative to low-G and high-P cells, cellular functions were largely recovered following a further 24 h culture in low-G medium. After 2 h at 2.8 mM glucose, high-G cells did not retain increased levels of glycolytic or TCA-cycle intermediates, but nevertheless displayed increased glycolysis, increased respiration and an increased mitochondrial proton leak relative to low-G and high-P cells. This notwithstanding, titration of low-G cells with low protonophore concen-trations, monitoring respiration and insulin secretion in parallel, showed that the perturbed insulin secretion of high-G cells could not be accounted for by increased proton leak. The present study supports the idea that glucose-induced disturbances of stimulus-secretion coupling by extra-mitochondrial metabolism upstream of pyruvate, rather than exhaustion from metabolic overload, underlie glucotoxicity in insulin-producing cells

Unique and Shared Metabolic Regulation in Clonal β-cells and Primary Islets Derived from Rat Revealed by Metabolomics Analysis.

As models for β-cell metabolism, rat islets are, to some extent, a, heterogeneous cell-population stressed by the islet isolation procedure, while rat-derived clonal β-cells exhibit a tumor-like phenotype. To describe to what extent either of these models reflect normal cellular metabolism, we compared metabolite profiles and gene expression in rat islets and the INS-1 832/13 line, a widely used clonal β-cell model. We found that insulin secretion and metabolic regulation provoked by glucose were qualitatively similar in these β-cell models. However, rat islets exhibited a more pronounced glucose-provoked increase of glutamate, glycerol-3-phosphate, succinate and lactate levels while INS-1 832/13 cells showed a higher glucose-elicited increase in glucose-6-phosphate, alanine, isocitrate, and α-ketoglutarate levels. Glucose induced a decrease in levels of γ-aminobutyrate (GABA) and aspartate in rat islets and INS-1 832/13 cells, respectively. Genes with cellular functions related to proliferation and the cell cycle were more highly expressed in the INS-1 832/13 cells. Most metabolic pathways that were differentially expressed included GABA metabolism, in line with altered glucose responsiveness of GABA. Also, lactate dehydrogenase A, which is normally expressed at low levels in mature β-cells, was more abundant in rat islets than in INS-1 832/13 cells, confirming the finding of elevated glucose-provoked lactate production in the rat islets. Overall, our results suggest that metabolism in rat islets and INS-1 832/13 cells is qualitatively similar, albeit with quantitative differences. Differences may be accounted for by cellular heterogeneity of islets and proliferation of the INS-1 832/13 cells

Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.

We have previously identified Transcription Factor B1 Mitochondrial (TFB1M) as a Type 2 Diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D we created a β-cell specific knockout of Tfb1 m, which gradually developed diabetes. Prior to the onset of diabetes, β-Tfb1 m(-/-) mice exhibited retarded glucose clearance due to impaired insulin secretion. β-Tfb1 m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules, and displayed reduced β-cell mass. Moreover, mitochondria in Tfb1 m-deficient β-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine-dimethylation of 12S ribosomal RNA (rRNA). Here, we found that levels of TFB1M and mitochondrial encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in β-Tfb1 m(-/-) islets. Furthermore, levels of reactive oxygen species in response to cellular stress were increased while induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+)-cells in the islets. Taken together, our findings demonstrate that Tfb1 m-deficiency in β-cells caused mitochondrial dysfunction and subsequently diabetes due to combined loss of β-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of β-cell function and mass, the hallmarks of T2D