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Kappe C.,Karolinska Institutet | Holst J.J.,Panum Institute | Zhang Q.,Karolinska Institutet | Sjoholm T.,Karolinska Institutet
Biochemical and Biophysical Research Communications | Year: 2012

Background: Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where the antidiabetic drug metformin conferred protection from lipoapoptosis. The aim of the present study was to identify mechanisms involved in mediating lipotoxicity and metformin lipoprotection in GLP-1 secreting cells. These signaling events triggered by simulated hyperlipidemia may underlie reduced GLP-1 secretion in diabetic subjects, and metformin lipoprotection by metformin could explain elevated plasma GLP-1 levels in diabetic patients on chronic metformin therapy. The present study may thus identify potential molecular targets for increasing endogenous GLP-1 secretion through enhanced viability of GLP-1 secreting cells in diabetic hyperlipidemia and obesity. Methods: We have studied molecular mechanisms mediating lipotoxicity and metformin-induced lipoprotection in GLP-1-secreting L-cells in vitro, using the murine GLUTag cell line as a model. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Caspase-3 activity was used as a measure of GLUTag cell apoptosis. ROS production was determined using a fluorescent probe, and the activation of intracellular signaling pathways was assessed by Western blotting. Results: Palmitate increased ROS production in GLP-1 secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 and inhibition of p38 using the p38 inhibitor SB203580 significantly reduced palmitate-induced caspase-3 activity. Pre-incubation of palmitate with metformin further increased palmitate induced ROS production, while significantly reducing the expression of p38. Conclusion: This study demonstrates that palmitate induces ROS production and that the palmitate induced lipotoxicity is the result of increased ROS production, where the ROS sensitive MKK3/6-p38 pathway mediates lipoapoptosis of GLP-1-secreting cells. Further, in the presence of simulated hyperlipidemia, metformin increases ROS production. However, metformin significantly decreases the expression of p38, indicating that metformin mediated lipoprotection involves reduced activity of the p38 signaling pathway. © 2012 Elsevier Inc. Source

Skov J.,Aarhus University Hospital | Skov J.,Novo Nordisk AS | Dejgaard A.,Novo Nordisk AS | Frokiaer J.,Aarhus University Hospital | And 4 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2013

Introduction: Glucagon-like peptide-1 (GLP-1) is an incretin hormone with multiple actions in addition to control of glucose homeostasis. GLP-1 is known to cause natriuresis in humans, but the effects on basic renal physiology are still partly unknown. Subjects and Methods: Twelve healthy young males were examined in a randomized, controlled, double-blinded, single-day, crossover trial to evaluate the effects of 2 hours GLP-1 infusion on kidney functions. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were assessed with 51Cr-EDTA and 123I-hippuran, respectively, using a constant infusion renal clearance technique based on timed urine sampling. Results: GLP-1 had no significant effect on either GFR [+ 1.9%, 95% confidence interval (-0.8; 4.6%)] or RPF [+2.4%, 95% confidence interval (-3.6; 8.8%)]. Fractional urine excretion of lithium increased 9% (P = .013) and renal sodium clearance increased 40% (P = .007). Angiotensin II decreased 19% (P = .003), whereas renin, aldosterone, and the urinary excretion of angiotensinogen showed no significant changes. GLP-1 did not affect blood pressure but induced a small transient increase in heart rate. Conclusion: The results indicate that although GLP-1 markedly reduces proximal tubule sodium reabsorption, the acute effects on GFR and RPF are very limited in healthy humans. The finding of GLP-1's ability to reduce angiotensin II concentration is novel and should be further elucidated. Copyright © 2013 by The Endocrine Society. Source

Ford T.W.,University of Nottingham | Meehan C.F.,Panum Institute | Kirkwood P.A.,University College London
Journal of Neurophysiology | Year: 2014

Internal intercostal and abdominal motoneurons are strongly coactivated during expiration. We investigated whether that synergy was paralleled by synergistic Group I reflex excitation. Intracellular recordings were made from motoneurons of the internal intercostal nerve of T8 in anesthetized cats, and the specificity of the monosynaptic connections from afferents in each of the two main branches of this nerve was investigated. Motoneurons were shown by antidromic excitation to innervate three muscle groups: external abdominal oblique [EO; innervated by the lateral branch (Lat)], the region of the internal intercostal muscle proximal to the branch point (IIm), and muscles innervated from the distal remainder (Dist). Strong specificity was observed, only 2 of 54 motoneurons showing excitatory postsynaptic potentials (EPSPs) from both Lat and Dist. No EO motoneurons showed an EPSP from Dist, and no IIm motoneurons showed one from Lat. Expiratory Dist motoneurons fell into two groups. Those with Dist EPSPs and none from Lat (group A) were assumed to innervate distal internal intercostal muscle. Those with Lat EPSPs (group B) were assumed to innervate abdominal muscle (transversus abdominis or rectus abdominis). Inspiratory Dist motoneurons (assumed to innervate interchondral muscle) showed Dist EPSPs. Stimulation of dorsal ramus nerves gave EPSPs in 12 instances, 9 being in group B Dist motoneurons. The complete absence of heteronymous monosynaptic Group I reflex excitation between muscles that are synergistically activated in expiration leads us to conclude that such connections from muscle spindle afferents of the thoracic nerves have little role in controlling expiratory movements but, where present, support other motor acts. © 2014 the American Physiological Society. Source

Deacon C.F.,Panum Institute | Marx N.,RWTH Aachen
Expert Review of Cardiovascular Therapy | Year: 2012

Glucagon-like peptide (GLP)-1 agonists and dipeptidyl peptidase-4 inhibitors are two classes of drugs that have been approved for treatment of Type 2 diabetes mellitus, based upon the glucose-lowering actions of the gastrointestinal hormone GLP-1. However, GLP-1 receptors are also present in cardiovascular tissues. Data from animal and in vitro studies suggest that GLP-1 may have cardioprotective effects and improve myocardial and endothelial dysfunction. Clinical data demonstrating cardiovascular effects are more limited, and there is some evidence that incretin-based therapies may be associated with improvements in cardiovascular risk factors. Large prospective cardiovascular outcome trials are underway to examine the cardiovascular safety of incretin-based therapies, and may reveal whether these agents are associated with any reduction in cardiovascular adverse events in patients with Type 2 diabetes mellitus. © 2012 Expert Reviews Ltd. Source

Kappe C.,Karolinska Institutet | Patrone C.,Karolinska Institutet | Holst J.J.,Panum Institute | Zhang Q.,Karolinska Institutet | Sjoholm A.,Karolinska Institutet
Journal of Gastroenterology | Year: 2013

Background Metformin is the most frequently prescribed drug for treatment of type 2 diabetes. It improves insulin resistance and glycemia by reducing hepatic gluconeogenesis. In addition, diabetic patients on metformin therapy have elevated levels of the insulinotropic hormone glucagon- like peptide-1 (GLP-1) and metformin has been shown to regulate the expression of the GLP-1R in the pancreas. Methods We have studied the direct long-term effects of metformin on apoptosis, and function of GLP-1-secreting L cells in vitro, using the murine GLUTag cell line as a model. The apoptosis of GLUTag cells was detected by DNA-fragment assay and caspase-3 activity determination. GLP-1 secretion was determined using ELISA and the expression of proglucagon mRNA was assessed by reverse transcription polymerase chain reaction. The activation of intracellular messengers was determined using western blotting. Results Metformin significantly decreased lipotoxicityinduced apoptosis in conjunction with increased phosphorylated AMPK. Metformin also countered the JNK2 activation evoked by lipotoxicity. In addition, long-term metformin treatment stimulated GLP-1 secretion. Conclusion This study demonstrates that metformin protects against lipoapoptosis (possibly by blocking JNK2 activation), and enhances GLP-1 secretion from GLP-1- producing cells in vitro. These direct effects of the drug might explain the elevated plasma GLP-1 levels seen in diabetic patients on chronic metformin therapy. The findings may also be harnessed to therapeutic advantage in efforts aiming at enhancing endogenous GLP-1 secretion in type 2 diabetic patients. © Springer 2012. Source

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