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Mentis N.,Diabeteszentrum Bad Lauterberg | Vardarli I.,Diabeteszentrum Bad Lauterberg | Kothe L.D.,Diabeteszentrum Bad Lauterberg | Holst J.J.,Panum Institute | And 4 more authors.
Diabetes | Year: 2011

OBJECTIVE - The incretin glucagon-like peptide 1 (GLP-1) exerts insulinotropic activity in type 2 diabetic patients, whereas glucose-dependent insulinotropic polypeptide (GIP) no longer does. We studied whether GIP can alter the insulinotropic or glucagonostatic activity of GLP-1 in type 2 diabetic patients. RESEARCH DESIGN AND METHODS - Twelve patients with type 2 diabetes (nine men and three women; 61 ± 10 years; BMI 30.0 ± 3.7 kg/m 2; HbA1c 7.3 ± 1.5%) were studied. In randomized order, intravenous infusions of GLP-1(7-36)-amide (1.2 pmol·kg -1 ·min-1), GIP (4 pmol·kg -1·min-1), GLP-1 plus GIP, and placebo were administered over 360 min after an overnight fast (≥1 day wash-out period between experiments). Capillary blood glucose, plasma insulin, C-peptide, glucagon, GIP, GLP-1, and free fatty acids (FFA) were determined. RESULTS - Exogenous GLP-1 alone reduced glycemia from 10.3 to 5.1 ± 0.2 mmol/L. Insulin secretion was stimulated (insulin, C-peptide, P < 0.0001), and glucagon was suppressed (P = 0.009). With GIP alone, glucose was lowered slightly (P = 0.0021); insulin and C-peptide were stimulated to a lesser degree than with GLP-1 (P < 0.001). Adding GIP to GLP-1 did not further enhance the insulinotropic activity of GLP-1 (insulin, P = 0.90; C-peptide, P = 0.85). Rather, the suppression of glucagon elicited by GLP-1 was antagonized by the addition of GIP (P = 0.008). FFA were suppressed by GLP-1 (P < 0.0001) and hardly affected by GIP (P = 0.07). CONCLUSIONS - GIP is unable to further amplify the insulinotropic and glucose-lowering effects of GLP-1 in type 2 diabetes. Rather, the suppression of glucagon by GLP-1 is antagonized by GIP. © 2011 by the American Diabetes Association.


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.


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.


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.


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.


Byskov A.G.,Copenhagen University | Hoyer P.E.,Panum Institute | Yding Andersen C.,Copenhagen University | Kristensen S.G.,Copenhagen University | And 2 more authors.
Human Reproduction | Year: 2011

Background: Conflicting Results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical Methods in the first trimester human ovary were present also in peri- and post-natal ovaries. Methods: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. Results: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. Conclusions: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla. © 2011 The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.


Hansen K.B.,Copenhagen University | Hansen K.B.,Panum Institute | Vilsboll T.,Copenhagen University | Bagger J.I.,Copenhagen University | And 2 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2012

Objective: The insulinotropic effect of the incretin hormones, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) is impaired in patients with type 2 diabetes. It remains unclear whether this impairment is a primary pathophysiological trait or a consequence of developing diabetes. Therefore, we aimed to investigate the insulinotropic effect of GIP and GLP-1 comparedwith placebo beforeandafter12dof glucose homeostatic dysregulation in healthy subjects. Research Design and Methods: The insulinotropic effect was measured using hyperglycemic clamps and infusion of physiological doses of GIP, GLP-1, or saline in 10 healthy Caucasian males before and after intervention using a high-calorie diet, sedentary lifestyle, and administration of prednisolone (37.5 mg once daily) for 12 d. Results: The intervention resulted in increased insulin resistance according to the homeostatic model assessment (1.2 ± 0.2 vs. 2.6 ± 0.5, P = 0.01), and glucose tolerance deteriorated as assessed by the area under curve for plasma glucose during a 75-g oral glucose tolerance test (730 ± 30 vs. 846 ± 57 mM for 2 h, P = 0.021). The subjects compensated for the change in insulin resistance by significantly increasing their postintervention insulin responses during saline infusion by 2.9 ± 0.5-fold (P = 0.001) but were unable to do so in response to incretin hormones (which caused insignificant increases of only 1.78 ± 0.3 and 1.38 ± 0.3-fold, P value not significant). Conclusions: These data show that impairment of the insulinotropic effect of both GIP and GLP-1 can be induced in healthy male subjects without risk factors for type 2 diabetes, indicating that the reduced insulinotropic effect of the incretin hormones observed in type 2 diabetes most likely is a consequence of insulin resistance and glucose intolerance rather than a primary event causing the disease. Copyright © 2012 by The Endocrine Society.


McLaughlin T.,Stanford University | Peck M.,Stanford University | Holst J.,Panum Institute | Deacon C.,Panum Institute
Journal of Clinical Endocrinology and Metabolism | Year: 2010

Context: Severe hypoglycemia after Roux-en-Y gastric bypass surgery (RYGB) is an increasingly recognized condition, characterized by neuroglycopenia and inappropriately elevated insulin concentrations that occur primarily in the postprandial state. Both pathophysiology and treatment of this disorder remain elusive, but it has been postulated that hyperplasia and/or hypertrophy of β-cells due to morbid obesity and/or postsurgical nesidioblastosis may contribute. Objective: The objective of this study was to elucidate the pathophysiology of this condition; specifically, we hypothesized that metabolic abnormalities were a function of altered nutrient transit through the gastrointestinal tract rather than anatomical changes to pancreatic β-cells that would lead to consistently high insulin secretion irrespective of nutrient transit route. Design/Setting/Subject/Outcome Measures: We describe a unique case wherein gastrostomy tube (GT) insertion into the remnant stomach reversed neuroglycopenic symptoms. This subject was admitted to a university hospital research center for standardized measurement of glucose, insulin, and incretin hormones including glucagon-like peptide-1, gastric-inhibitory peptide, and glucagon. Results: Standardized liquid meal administration via GT vs. oral route demonstrated complete reversal of severe metabolic abnormalities that included hypersecretion of insulin and GLP-1. Conclusion: Post-RYGB hyperinsulinemia and hypoglycemia result entirely from altered nutrient delivery rather than generalized hyperfunction of β-cells due to presurgical hypertrophy/hyperfunction or postsurgical nesidioblastosis. These findings support the use of GT for treatment of severe cases and have implications for surgical manipulations that may reverse/prevent this condition. Copyright © 2010 by The Endocrine Society.


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.


Danielsen E.M.,Panum Institute | Hansen G.H.,Panum Institute | Severinsen M.C.K.,Panum Institute
Toxicon | Year: 2014

Okadaic acid (OA) is a polyether fatty acid produced by marine dinoflagellates and the causative agent of diarrhetic shellfish poisoning. The effect of OA on apical endocytosis in the small intestine was studied in organ cultured porcine mucosal explants. Within 0.5-1 h of culture, the toxin caused hyper protein phosphorylation, but no detectable loss of cell polarity or cytoskeletal integrity of the enterocytes. Using a fluorescent membrane marker, FM dye, endocytosis from the brush border was affected by the toxin. Although constitutive uptake into subapical terminal web-localized early endosomes (TWEEs) occurred unimpeded in the presence of OA, FM condensed in larger subapical structures by 1 h, implying a perturbed endosomal trafficking/ maturation. The fluorescent lysosomotropic agent Lysotracker revealed induction of large lysosomal structures by OA. Endocytosis from the brush border was studied at the electron microscopic level using the membrane-impermeable marker Ruthenium Red (RR). Like FM dye, RR was taken up into TWEEs and multivesicular bodies (MVBs). However, OA induced the formation of a large number of lamellar bodies (LBs), a type of lysosome-related organelles. LBs are the hallmark of phospholipidosis, a pathological condition characterized by lysosomal phospholipid accumulation. Phospholipidosis is observed in acquired lysosomal storage diseases and is induced by a large number of cationic amphiphilic drugs. Unlike the latter, however, OA does not act by accumulating in acidic organelles, implying a different toxic mechanism of action. We propose that rapid induction of LBs, an indicator of phospholipidosis, should be included in the future toxicity profile of OA. © 2014 Elsevier Ltd. All rights reserved.

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