11 research outputs found
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 fĂĽr 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 durchgefĂĽhrt. Aus den Analysen ergab sich, dass chronisch
hohe Glukosekonzentrationen überwiegend zu metabolischen Veränderungen im
Glukosestoffwechsel fĂĽhren. 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