Adolescents with obesity treated with exenatide maintain endogenous GLP-1, reduce DPP-4, and improve glycemic control.

Background: GLP-1 receptor agonists (GLP-1RA) are increasingly used to treat adolescent obesity. However, the effect on endogenous GLP-1 secretory patterns following treatment in adolescents is unknown. The GLP-1RA exenatide was shown to significantly lower BMI and 2-hour glucose in adolescents with obesity, in the placebo-controlled, randomized controlled trial Combat-JUDO. The aim of this study was to evaluate effects of weekly injections of 2 mg exenatide extended release on secretory patterns of endogenous hormones during OGTT. Subjects and Measurements: This study was a pre-planned sub-study of the Combat-JUDO trial, set at the Pediatric clinic at Uppsala University Hospital, Sweden and Paracelsus Medical University, Austria. 44 adolescents with obesity were included and randomized 1:1 to treatment:placebo. 19 patients in the treatment group and 18 in the placebo group completed the trial. Before and after treatment, GLP-1, glucose, insulin, glucagon and glicentin levels were measured during OGTT; DPP-4 and proinsulin were measured at fasting. A per-protocol approach was used in the analyses. Results: Exenatide treatment did not affect GLP-1 levels during OGTT. Treatment significantly lowered DPP-4, proinsulin and the proinsulin-to-insulin ratio at fasting, increased glicentin levels but did not affect insulin, C-peptide or glucagon levels during OGTT. Conclusion: Weekly s.c. injections with 2 mg of exenatide maintains endogenous total GLP-1 levels and lowers circulating DPP-4 levels. This adds an argument in favor of using exenatide in the treatment of pediatric obesity. Clinical trial registration: clinicaltrials.gov, identifier NCT02794402.

[Unlicensed drugs in Iceland in the years 2020 and 2021 with comparison to Sweden in 2020].

INTRODUCTION: Prescriptions of unlicensed drugs along with public discussion have increased in recent years. The cause of this increase is unclear. MATERIAL AND METHODS: This is a retrospective descriptive study of applications of unlicensed drugs in Iceland in the years 2020 and 2021, as well as applications in Sweden for the year 2020. Information was collected on unlicensed prescription applications, registered drugs and stock-out time. Information was analyzed and categorized to describe the scope of unlicensed prescriptions in the two countries during the study time. RESULTS: In Iceland, 49.161 applications were approved in 2020 and 46.581 in 2021. The most common reason for using unlicensed products was that no registered drug was on the market with the same ATC-number (Anatomical Therapeutic Chemical code) and formulation. Shortage of drug was the reason for unlicensed prescription in 8.8% of cases in 2020 and 7.6% in 2021. The list of the 50 most prescribed drugs included the same drugs in 70% of the cases in both years. The five most prescribed drugs were the same in both years. In Sweden, 38.458 applications were approved in 2020. Of the most prescribed unlicensed drug, 46% were as no registered drug with same ATC-number and dosage form was marketed. CONCLUSION: Many unlicensed drugs, prescribed in Iceland, are the same year after year. Only a small part of the applications approved were due to shortage of drug. The use of drugs without marketing license in Iceland in 2020 was higher than in Sweden when adjusted for the size of the market and population. Getting the five most prescribed unlicensed drugs licensed in Iceland could reduce largely, the total of prescription of unlicensed drugs in Iceland.

Hyperglucagonemia in youth is associated with high plasma free fatty acids, visceral adiposity, and impaired glucose tolerance.

OBJECTIVE: To delineate potential mechanisms for fasting hyperglucagonemia in childhood obesity by studying the associations between fasting plasma glucagon concentrations and plasma lipid parameters and fat compartments. METHODS: Cross-sectional study of children and adolescents with obesity (n = 147) and lean controls (n = 43). Differences in free fatty acids (FFAs), triglycerides, insulin, and fat compartments (quantified by magnetic resonance imaging) across quartiles of fasting plasma glucagon concentration were analyzed. Differences in oral glucose tolerance test (OGTT) glucagon response was tested in high vs low FFAs, triglycerides, and insulin. Human islets of Langerhans were cultured at 5.5 mmol/L glucose and in the absence or presence of a FFA mixture with total FFA concentration of 0.5 mmol/L and glucagon secretion quantified. RESULTS: In children with obesity, the quartile with the highest fasting glucagon had higher insulin (201 ± 174 vs 83 ± 39 pmol/L, P < .01), FFAs (383 ± 52 vs 338 ± 109 µmol/L, P = .02), triglycerides (1.5 ± 0.9 vs 1.0 ± 0.7 mmol/L, P < .01), visceral adipose tissue volume (1.9 ± 0.8 vs 1.2 ± 0.3 dm3 , P < .001), and a higher prevalence of impaired glucose tolerance (IGT; 41% vs 8%, P = .01) than the lowest quartile. During OGTT, children with obesity and high insulin had a worse suppression of glucagon during the first 10 minutes after glucose intake. Glucagon secretion was 2.6-fold higher in islets treated with FFAs than in those not treated with FFAs. CONCLUSIONS: Hyperglucagonemia in childhood obesity is associated with hyperinsulinemia, high plasma FFAs, high plasma triglycerides, visceral adiposity, and IGT. The glucagonotropic effect of FFAs on isolated human islets provides a potential mechanism linking high fasting plasma FFAs and glucagon levels.

Glucose Appearance Rate Rather than the Blood Glucose Concentrations Explains Differences in Postprandial Insulin Responses between Wholemeal Rye and Refined Wheat Breads-Results from A Cross-Over Meal Study.

SCOPE: Ingestion of rye bread leads to lower postprandial plasma insulin concentrations than wheat bread ingestion, but most often not too different glucose profiles. The mechanism behind this discrepancy is still largely unknown. This study investigates whether glucose kinetics may explain the observed discrepancy. METHODS AND RESULTS: Nine healthy men participated in a crossover study, eating 50 g of available carbohydrates as either refined wheat (WB) or traditional wholemeal rye bread (WMR) during d-[6,6-2 H2 ]glucose infusion. Labeled glucose enrichment is measured by an HPLC-TOF-MS method. The calculated rate of glucose appearance (RaE) is significantly lower after ingestion of WMR during the initial 15 min postprandial period. Additionally, the 0-90 min RaE area under the curve (AUC) is significantly lower after ingestion of WMR, as is plasma gastric inhibitory polypeptide (GIP) at 60 and 90 min. Postprandial glycemic responses do not differ between the breads. Postprandial insulin is lower after ingestion of WMR at 45 and 60 min, as is the 0-90 min AUC. CONCLUSION: Ingestion of WMR elicits a lower rate of glucose appearance into the bloodstream compared with WB. This may explain the lower insulin response observed after rye bread ingestion, commonly known as the rye factor.

Oleate increase neutral lipid accumulation, cellular respiration and rescues palmitate-exposed GLP-1 secreting cells by reducing ceramide-induced ROS.

BACKGROUND: Fatty acids (FAs), and especially monounsaturated FAs (MUFAs) stimulate GLP-1 release. However, lipotoxicity is indicated in GLP-1 secreting cells following long-term exposure to elevated levels of saturated FAs (SFAs) in vivo and in vitro, where in vitro studies indicate that cosupplementation with MUFAs confers lipoprotection. SFAs and MUFAs differentially affect the fate of cells in ways that depend on the cell type, concentration and ratio of the FAs. The present study was designed to further elucidate the mechanisms underlying the effects of SFAs/MUFAs on GLP-1-producing cells in terms of lipotoxicity/lipoprotection and GLP-1 secretion. METHODS: Cultured GLP-1 secreting cells were exposed to hyperlipidemia simulated by SFA-albumin complexes where the molar ratio was 2:1. The cellular response to simulated hyperlipidemia was assessed in the presence/absence of MUFA cosupplementation by determining intracellular ceramide, ROS, neutral lipid accumulation, and cellular respiration. The role for cellular respiration in GLP-1 secretion in response to SFAs/MUFAs was assessed. RESULTS: Generation of intracellular ceramide mediate a detrimental increased in ROS production following long term exposure to SFAs in GLP-1-secreting cells. Cosupplementation with MUFAs increases cellular respiration, triglyceride synthesis, and the expression of ceramide kinase, while reducing ceramide synthesis and attenuating ROS production, caspase-3 activity and DNA fragmentation. Further, acute secretory effects of unsaturated FAs are independent of FAO, but mediated by a FFAR1 induced increase in cellular respiration. CONCLUSION: This study demonstrates novel data supporting effects of MUFAs on the ceramide biosynthetic pathway, triglyceride storage respiration and secretion in GLP-1 secreting cells. These findings may be of value for nutritional interventions, as well as for identification of novel targets, to help preserve L-cell mass and potentiate GLP-1 secretion in diabesity.

Lysophosphatidylcholine and its phosphorothioate analogues potentiate insulin secretion via GPR40 (FFAR1), GPR55 and GPR119 receptors in a different manner.

Lysophosphatidylcholine (LPC) is an endogenous ligand for GPR119 receptor, mediating glucose-stimulated insulin secretion (GSIS). We demonstrate that LPC facilitates GSIS in MIN6 pancreatic ß-cell line and murine islets of Langerhans by recognizing not only GPR119 but also GPR40 (free fatty acid receptor 1) and GPR55 activated by lysophosphatidylinositol. Natural LPCs are unstable when administered in vivo limiting their therapeutic value and therefore, we present phosphorothioate LPC analogues with increased stability. All the modified LPCs under study (12:0, 14:0, 16:0, 18:0, and 18:1) significantly enhanced GSIS. The 16:0 sulfur analogue was the most potent, evoking 2-fold accentuated GSIS compared to the native counterpart. Interestingly, LPC analogues evoked GPR40-, GPR55-and GPR119-dependent [Ca2+]i signaling, but did not stimulate cAMP accumulation as in the case of unmodified molecules. Thus, introduction of a phosphorothioate function not only increases LPC stability but also modulates affinity towards receptor targets and evokes different signaling pathways.

Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism.

Studies on the pathophysiology of type 2 diabetes mellitus (T2DM) have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our findings provide insight into how saturated fatty acids contribute to islet cell dysfunction by affecting the granule maturation process and confirmation in human islets of some previous findings from rodent islet and cell-line studies.

EndoC-ßH1 cells display increased sensitivity to sodium palmitate when cultured in DMEM/F12 medium.

Aims - Human pancreatic islets are known to die in response to the free fatty acid of sodium palmitate when cultured in vitro. This is in contrast to EndoC-ßH1 cells, which in our hands are not sensitive to the cell death-inducing effects sodium palmitate, making these cells seemingly unsuitable for lipotoxicity studies. However, the EndoC-ßH1 cells are routinely cultured in a nutrient mixture based on Dulbecco's Modified Eagle Medium (DMEM), which may not be the optimal choice for studies dealing with lipotoxicity. The aim of the present investigation was to define culture conditions that render EndoC-ßH1 cells sensitive to toxic effects of sodium palmitate. Methods - EndoC-ßH1 cells were cultured at standard conditions in either DMEM or DMEM/F12 culture medium. Cell death was analyzed using propidium iodide staining and flow cytometry. Insulin release and content was quantified using a human insulin ELISA. Results - We presently observe that substitution of DMEM for a DMEM/Ham's F12 mixture (50%/50% vol/vol) renders the cells sensitive to the apoptotic effects of sodium palmitate and sodium palmitate + high glucose leading to an increased cell death. Supplementation of the DMEM culture medium with linoleic acid partially mimicked the effect of DMEM/F12. Culture of EndoC-ßH1 cells in DMEM/F12 resulted also in increased proliferation, ROS production and insulin contents, but markers for metabolic stress, autophagy or amyloid deposits were unaffected. Conclusions - The culture conditions for EndoC-ßH1 cells can be modified so these cells display signs of lipotoxicity in response to sodium palmitate.

Initial hyperinsulinemia and subsequent ß-cell dysfunction is associated with elevated palmitate levels.

BACKGROUND: The prevalence of obesity-related diabetes in childhood is increasing and circulating levels of nonesterified fatty acids may constitute a link. Here, the association between palmitate and insulin secretion was investigated in vivo and in vitro. METHODS: Obese and lean children and adolescents (n = 80) were included. Palmitate was measured at fasting; insulin and glucose during an oral glucose tolerance test (OGTT). Human islets were cultured for 0 to 7 d in presence of 0.5 mmol/l palmitate. Glucose-stimulated insulin secretion (GSIS), insulin content and apoptosis were measured. RESULTS: Obese subjects had fasting palmitate levels between 0.10 and 0.33 mmol/l, with higher average levels compared to lean subjects. While obese children with elevated palmitate (>0.20 mmol/l) had accentuated insulin levels during OGTT, obese adolescents with high palmitate had delayed first-phase insulin response. In human islets exposed to palmitate for 2 d GSIS was twofold enhanced, but after 7 d attenuated. Intracellular insulin content decreased time-dependently in islets cultured in the presence of palmitate and cleaved caspase 3 increased. CONCLUSION: The rapid accentuated and delayed insulin secretory responses observed in obese children and adolescents, respectively, with high palmitate levels may reflect changes in islet secretory activity and integrity induced by extended exposure to the fatty acid.

Altered Plasma Levels of Glucagon, GLP-1 and Glicentin During OGTT in Adolescents With Obesity and Type 2 Diabetes.

CONTEXT: Proglucagon-derived hormones are important for glucose metabolism, but little is known about them in pediatric obesity and type 2 diabetes mellitus (T2DM). OBJECTIVE: Fasting and postprandial levels of proglucagon-derived peptides glucagon, GLP-1, and glicentin in adolescents with obesity across the glucose tolerance spectrum were investigated. DESIGN: This was a cross-sectional study with plasma hormone levels quantified at fasting and during an oral glucose tolerance test (OGTT). SETTING: This study took place in a pediatric obesity clinic at Uppsala University Hospital, Sweden. PATIENTS AND PARTICIPANTS: Adolescents with obesity, age 10-18 years, with normal glucose tolerance (NGT, n = 23), impaired glucose tolerance (IGT, n = 19), or T2DM (n = 4) and age-matched lean adolescents (n = 19) were included. MAIN OUTCOME MEASURES: Outcome measures were fasting and OGTT plasma levels of insulin, glucagon, active GLP-1, and glicentin. RESULTS: Adolescents with obesity and IGT had lower fasting GLP-1 and glicentin levels than those with NGT (0.25 vs 0.53 pM, P < .05; 18.2 vs 23.6 pM, P < .01) and adolescents with obesity and T2DM had higher fasting glucagon levels (18.1 vs 10.1 pM, P < .01) than those with NGT. During OGTT, glicentin/glucagon ratios were lower in adolescents with obesity and NGT than in lean adolescents (P < .01) and even lower in IGT (P < .05) and T2DM (P < .001). CONCLUSIONS: Obese adolescents with IGT have lowered fasting GLP-1 and glicentin levels. In T2DM, fasting glucagon levels are elevated, whereas GLP-1 and glicentin levels are maintained low. During OGTT, adolescents with obesity have more products of pancreatically than intestinally cleaved proglucagon (ie, more glucagon and less GLP-1) in the plasma. This shift becomes more pronounced when glucose tolerance deteriorates.

Free fatty acid receptor 1 (FFAR1/GPR40) signaling affects insulin secretion by enhancing mitochondrial respiration during palmitate exposure.

Fatty acids affect insulin secretion via metabolism and FFAR1-mediated signaling. Recent reports indicate that these two pathways act synergistically. Still it remains unclear how they interrelate. Taking into account the key role of mitochondria in insulin secretion, we attempted to dissect the metabolic and FFAR1-mediated effects of fatty acids on mitochondrial function. One-hour culture of MIN6 cells with palmitate significantly enhanced mitochondrial respiration. Antagonism or silencing of FFAR1 prevented the palmitate-induced rise in respiration. On the other hand, in the absence of extracellular palmitate FFAR1 agonists caused a modest increase in respiration. Using an agonist of the M3 muscarinic acetylcholine receptor and PKC inhibitor we found that in the presence of the fatty acid mitochondrial respiration is regulated via Gαq protein-coupled receptor signaling. The increase in respiration in palmitate-treated cells was largely due to increased glucose utilization and oxidation. However, glucose utilization was not dependent on FFAR1 signaling. Collectively, these results indicate that mitochondrial respiration in palmitate-treated cells is enhanced via combined action of intracellular metabolism of the fatty acid and the Gαq-coupled FFAR1 signaling. Long-term palmitate exposure reduced ATP-coupling efficiency of mitochondria and deteriorated insulin secretion. The presence of the FFAR1 antagonist during culture did not improve ATP-coupling efficiency, however, it resulted in enhanced mitochondrial respiration and improved insulin secretion after culture. Taken together, our study demonstrates that during palmitate exposure, integrated actions of fatty acid metabolism and fatty acid-induced FFAR1 signaling on mitochondrial respiration underlie the synergistic action of the two pathways on insulin secretion.

FFAR1 is involved in both the acute and chronic effects of palmitate on insulin secretion.

Free fatty acids (FFAs) have pleiotropic effects on the pancreatic ß-cell. Although acute exposure to FFAs stimulates glucose-stimulated insulin secretion (GSIS), prolonged exposure impairs GSIS and causes apoptosis. FFAs exert their effects both via intracellular metabolism and interaction with the FFA receptor 1 (FFAR1/GPR40). Here we studied the role of FFAR1 in acute and long-term effects of palmitate on GSIS and insulin content in isolated human islets by using the FFAR1 agonist TAK-875 and the antagonist ANT203. Acute palmitate exposure potentiated GSIS approximately 3-fold, whereas addition of the antagonist decreased this potentiation to approximately 2-fold. In the absence of palmitate, the agonist caused a 40% increase in GSIS. Treatment with palmitate for 7 days decreased GSIS to 70% and insulin content to 25% of control level. These negative effects of long-term exposure to palmitate were ameliorated by FFAR1 inhibition and further aggravated by additional stimulation of the receptor. In the absence of extracellularly applied palmitate, long-term treatment with the agonist caused a modest increase in GSIS. The protective effect of FFAR1 inhibition was verified by using FFAR1-deficient MIN6 cells. Improved ß-cell function by the antagonist was paralleled by the decreased apoptosis and lowered oxidation of palmitate, which may represent the potential mechanisms of protection. We conclude that FFAR1 in the pancreatic ß-cell plays a substantial role not only in acute potentiation of GSIS by palmitate but also in the negative long-term effects of palmitate on GSIS and insulin content.