Lipotoxicity is glucose-dependent in INS-1E cells but not in human islets and MIN6 cells

<p>Abstract</p> <p>Background</p> <p>Prolonged elevated levels of lipids have negative effects on beta-cell function and mass (lipotoxicity). To what extent exposure to high glucose concentration is important in the harmful effects of lipids (glucolipotoxicity) has been debated.</p> <p>Methods</p> <p>We addressed beta-cell lipotoxicity by measuring apoptosis in isolated intact control human islets and insulin-secreting cell lines MIN6 and INS-1E cultured in the presence of palmitate and low (5.5 mM) or high (25 mM) glucose for 48 hours.</p> <p>Results</p> <p>In both cell lines and human islets palmitate induced apoptosis after culture at low glucose. Palmitate-induced apoptosis was not increased after culture at high compared to low glucose in human islets and MIN6 cells but glucose-induced rise in apoptosis was observed in INS-1E cells. The rise in apoptosis in INS-1E cells was partially reversed by inclusion of AMPK-agonist AICAR. When CPT1-inhibitor etomoxir was included during culture at low glucose palmitate-triggered apoptosis was accentuated both in the islets and the cell lines. Palmitate oxidation in human islets and the cell lines was comparable after culture at low glucose. At high glucose, palmitate oxidation was reduced by 30% in human islets and MIN6 cells but by 80% in INS-1E cells. In INS-1E cells, AICAR increased oxidation of palmitate. Presence of etomoxir at low glucose decreased palmitate oxidation both in the islets and the cell lines.</p> <p>Conclusions</p> <p>In summary, lipotoxicity is evident not only in the presence of high but also low glucose concentrations. Additional effects of glucose are prominent in INS-1E but not in MIN6 cells and intact control human islets, which are able to efficiently oxidize fatty acids at high glucose and in this way avoid glucolipotoxicity.</p

Reduced levels of SCD1 accentuate palmitate-induced stress in insulin-producing β-cells

<p>Abstract</p> <p>Background</p> <p>Stearoyl-CoA desaturase 1 (SCD1) is an ER resident enzyme introducing a double-bond in saturated fatty acids. Global knockout of SCD1 in mouse increases fatty acid oxidation and insulin sensitivity which makes the animal resistant to diet-induced obesity. Inhibition of SCD1 has therefore been proposed as a potential therapy of the metabolic syndrome. Much of the work has focused on insulin target tissue and very little is known about how reduced levels of SCD1 would affect the insulin-producing β-cell, however. The aim of the present study was therefore to investigate how reduced levels of SCD1 affect the β-cell.</p> <p>Results</p> <p>Insulin-secreting MIN6 cells with reduced levels of SCD1 were established by siRNA mediated knockdown. When fatty acid oxidation was measured, no difference between cells with reduced levels of SCD1 and mock-transfected cells were found. Also, reducing levels of SCD1 did not affect insulin secretion in response to glucose. To investigate how SCD1 knockdown affected cellular mechanisms, differentially regulated proteins were identified by a proteomic approach. Cells with reduced levels of SCD1 had higher levels of ER chaperones and components of the proteasome. The higher amounts did not protect the β-cell from palmitate-induced ER stress and apoptosis. Instead, rise in levels of p-eIF2α and CHOP after palmitate exposure was 2-fold higher in cells with reduced levels of SCD1 compared to mock-transfected cells. Accordingly, apoptosis rose to higher levels after exposure to palmitate in cells with reduced levels of SCD1 compared to mock-transfected cells.</p> <p>Conclusions</p> <p>In conclusion, reduced levels of SCD1 augment palmitate-induced ER stress and apoptosis in the β-cell, which is an important caveat when considering targeting this enzyme as a treatment of the metabolic syndrome.</p

The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

BACKGROUND: The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. CONCLUSION: Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed

Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion

<p>Abstract</p> <p>Background</p> <p>Development of type 2 diabetes mellitus (T2DM) is characterized by aberrant insulin secretory patterns, where elevated insulin levels at non-stimulatory basal conditions and reduced hormonal levels at stimulatory conditions are major components. To delineate mechanisms responsible for these alterations we cultured INS-1E cells for 48 hours at 20 mM glucose in absence or presence of 0.5 mM palmitate, when stimulatory secretion of insulin was reduced or basal secretion was elevated, respectively.</p> <p>Results</p> <p>After culture, cells were protein profiled by SELDI-TOF-MS and 2D-PAGE. Differentially expressed proteins were discovered and identified by peptide mass fingerprinting. Complimentary protein profiles were obtained by the two approaches with SELDI-TOF-MS being more efficient in separating proteins in the low molecular range and 2D-PAGE in the high molecular range. Identified proteins included alpha glucosidase, calmodulin, gars, glucose-6-phosphate dehydrogenase, heterogenous nuclear ribonucleoprotein A3, lon peptidase, nicotineamide adenine dinucleotide hydrogen (NADH) dehydrogenase, phosphoglycerate kinase, proteasome p45, rab2, pyruvate kinase and t-complex protein. The observed glucose-induced differential protein expression pattern indicates enhanced glucose metabolism, defense against reactive oxygen species, enhanced protein translation, folding and degradation and decreased insulin granular formation and trafficking. Palmitate-induced changes could be related to altered exocytosis.</p> <p>Conclusion</p> <p>The identified altered proteins indicate mechanism important for altered β-cell function in T2DM.</p

Role of MAPK in apolipoprotein CIII-induced apoptosis in INS-1E cells

<p>Abstract</p> <p>Background</p> <p>Individuals with type 2 diabetes mellitus (T2DM) have elevated levels of circulating apolipoprotein CIII (apoCIII). ApoCIII plays an important role for plasma triglyceride levels and elevated levels of the apolipoprotein have been connected with dyslipidemia in T2DM subjects. In addition, apoCIII has been linked to enhanced β-cell apoptosis. The present study was undertaken to investigate apoptotic mechanisms induced by the apolipoprotein.</p> <p>Results</p> <p>ApoCIII (10 μg/ml) enhanced apoptosis 2-fold in insulin-producing INS-1E cells after 24 hours exposure to the apolipoprotein. At this time point phosphorylation of mitogen activated protein kinase (MAPK) p38 had doubled but ERK1/2 and JNK were not activated. Instead, ERK1/2 showed rapid and transient phosphorylation (2-fold after 0.5 hour). No JNK phosphorylation was observed. In support of a role of activation of not only p38 but also ERK1/2 in apoCIII-induced apoptosis, inclusion of p38 inhibitor SB203580 (10 μM) or ERK1/2 inhibitor PD98059 (100 μM) normalized apoptosis. Whereas influx of Ca²⁺was linked to apoCIII-induced ERK1/2 activation, pro-apoptotic protein CHOP/GADD of the unfolded protein response (UPR) was not affected by apoCIII.</p> <p>Conclusion</p> <p>It is suggested that elevated circulating apoCIII levels may contribute to β-cell apoptosis via activation of p38 and ERK1/2 in individuals with T2DM. Therapies aiming at normalizing levels of apoCIII could be beneficial not only for the function of the β-cell but also for cardiovascular protection.</p

UPR in palmitate-treated pancreatic beta-cells is not affected by altering oxidation of the fatty acid

<p>Abstract</p> <p>Background</p> <p>Elevated levels of lipids are detrimental for beta-cell function and mass. One of the mechanisms of how fatty acids induce apoptosis is development of the unfolded protein response (UPR). It is still far from understood how fatty acids activate the UPR, however.</p> <p>Methods</p> <p>We examined how palmitate-induced activation of the UPR was affected by altering the metabolism of the fatty acid in insulin-secreting INS-1E and MIN6 cell lines and intact human islets. To increase oxidation, we used low glucose (5.5 mM) or AICAR; and to reduce oxidation, we used high glucose (25 mM) or etomoxir. UPR was measured after 3, 24 and 48 hours of palmitate treatment.</p> <p>Results</p> <p>Modulation of palmitate oxidation by either glucose or the pharmacological agents did not affect palmitate-induced UPR activation.</p> <p>Conclusion</p> <p>Our finding suggests that other factors than oxidation of palmitate play a role in the activation of UPR in fatty acid-treated beta-cells.</p

Evaluation of fasting plasma insulin and proxy measurements to assess insulin sensitivity in horses

BackgroundProxies are mathematical calculations based on fasting glucose and/or insulin concentrations developed to allow prediction of insulin sensitivity (IS) and beta -cell response. These proxies have not been evaluated in horses with insulin dysregulation. The first objective of this study was to evaluate how fasting insulin (FI) and proxies for IS (1/Insulin, reciprocal of the square root of insulin (RISQI) and the quantitative insulin sensitivity check index (QUICKI)) and beta -cell response (the modified insulin-to-glucose ratio (MIRG) and the homeostatic model assessment of beta -cell function (HOMA-beta)) were correlated to measures of IS (M index) using the euglycemic hyperinsulinemic clamp (EHC) in horses with insulin resistance (IR) and normal IS. A second objective was to evaluate the repeatability of FI and proxies in horses based on sampling on consecutive days. The last objective was to investigate the most appropriate cut-off value for the proxies and FI.ResultsThirty-four horses were categorized as IR and 26 as IS based on the M index. The proxies and FI had coefficients of variation (CVs)= 0.89). All proxies and FI were good predictors of the M index (r=0.76-0.85; P9.5 mu IU/mL for FI.ConclusionsAll proxies and FI provided repeatable estimates of horses' IS. However, there is no advantage of using proxies instead of FI to estimate IR in the horse. Due to the heteroscedasticity of the data, proxies and FI in general are more suitable for epidemiological studies and larger clinical studies than as a diagnostic tool for measurement of IR in individual horses

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

BackgroundGLP-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 MeasurementsThis 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.ResultsExenatide 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.ConclusionWeekly 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 registrationclinicaltrials.gov, identifier NCT0279440

Therapist-guided internet-based psychodynamic therapy versus cognitive behavioural therapy for adolescent depression in Sweden: a randomised, clinical, non-inferiority trial

BACKGROUND: Adolescent major depressive disorder (MDD) is highly prevalent and associated with lifelong adversity. Evidence-based treatments exist, but accessible treatment alternatives are needed. We aimed to compare internet-based psychodynamic therapy (IPDT) with an established evidence-based treatment (internet-based cognitive behavioural therapy [ICBT]) for the treatment of adolescents with depression. METHODS: In this randomised, clinical trial, we tested whether IPDT was non-inferior to ICBT in the treatment of adolescent MDD. Eligible participants were 15-19 years old, presenting with a primary diagnosis of MDD according to DSM-5. Participants were recruited nationwide in Sweden through advertisements on social media, as well as contacts with junior and senior high schools, youth associations, social workers, and health-care providers. Adolescents who scored 9 or higher on the Quick Inventory of Depressive Symptomatology for Adolescents (QIDS-A17-SR) in an initial online screening were contacted by telephone for a diagnostic assessment using the Mini International Neuropsychiatric Interview. Participants were randomly assigned to ICBT or IPDT. Both interventions comprised eight self-help modules delivered over 10 weeks on a secure online platform. The primary outcome was change in depression severity measured weekly by the QIDS-A17-SR. Primary analyses were based on an intention-to-treat sample including all participants randomly assigned. A non-inferiority margin of Cohen's d=0·30 was predefined. The study is registered at ISRCTN, ISRCTN12552584. FINDINGS: Between Aug 19, 2019, and Oct 7, 2020, 996 young people completed screening; 516 (52%) were contacted for a diagnostic interview. 272 participants were eligible and randomly assigned to ICBT (n=136) or IPDT (n=136). In the ICBT group, 51 (38%) of 136 participants were classified as remitted, and 54 (40%) of 136 participants were classified as remitted in the IPDT group. Within-group effects were large (ICBT: within-group d=1·75, 95% CI 1·49 to 2·01; IPDT: within-group d=1·93, 1·67 to 2·20; both p<0·0001). No statistically significant treatment difference was found in the intention-to-treat analysis. Non-inferiority for IPDT was shown for the estimated change in depression during treatment (d=-0·18, 90% CI -0·49 to 0·13; p=0·34). All secondary outcomes showed non-significant between-group differences. INTERPRETATION: IPDT was non-inferior to ICBT in terms of change in depression for the treatment of adolescents with MDD. This finding increases the range of accessible and effective treatment alternatives for adolescents with depression. FUNDING: Kavli trust