Metformin restores prohormone processing enzymes and normalizes aberrations in secretion of proinsulin and insulin in palmitate-exposed human islets.

AIM: To elucidate how proinsulin synthesis and insulin was affected by metformin under conditions of nutrient overstimulation. MATERIALS AND METHODS: Isolated human pancreatic islets from seven donors were cultured at 5.5 mmol/L glucose and 0.5 mmol/L palmitate for 12, 24 or 72 h. Metformin (25 µmol/L) was introduced after initial 12 h with palmitate. Proinsulin and insulin were measured. Expression of prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE), was determined by western blot. Adolescents with obesity, treated with metformin and with normal glucose tolerance (n = 5), prediabetes (n = 14), or type 2 diabetes (T2DM; n = 7) were included. Fasting proinsulin, insulin, glucose, 2-h glucose and glycated haemoglobin were measured. Proinsulin/insulin ratio (PI/I) was calculated. RESULTS: In human islets, palmitate treatment for 12 and 24 h increased proinsulin and insulin proportionally. After 72 h, proinsulin but not insulin continued to increase which was coupled with reduced expression of PC1/3 and CPE. Metformin normalized expression of PC1/3 and CPE, and proinsulin and insulin secretion. In adolescents with obesity, before treatment, fasting proinsulin and insulin concentrations were higher in subjects with T2DM than with normal glucose tolerance. PI/I was reduced after metformin treatment in subjects with T2DM as well as in subjects with prediabetes, coupled with reduced 2-h glucose and glycated haemoglobin. CONCLUSIONS: Metformin normalized proinsulin and insulin secretion after prolonged nutrient-overstimulation, coupled with normalization of the converting enzymes, in isolated islets. In adolescents with obesity, metformin treatment was associated with improved PI/I, which was coupled with improved glycaemic control.

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia.

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1-4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

Palmitate-Induced Insulin Hypersecretion and Later Secretory Decline Associated with Changes in Protein Expression Patterns in Human Pancreatic Islets.

In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.

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.

Inhibiting the endoplasmic reticulum stress response enhances the effect of doxorubicin by altering the lipid metabolism of liver cancer cells.

Hepatocellular carcinoma (HCC) is characterized by a low and variable response to chemotherapeutic treatments. One contributing factor to the overall pharmacodynamics is the activation of endoplasmic reticulum (ER) stress pathways. This is a cellular stress mechanism that becomes activated when the cell's need for protein synthesis surpasses the ER's capacity to maintain accurate protein folding, and has been implicated in creating drug-resistance in several solid tumors. OBJECTIVE: To identify the role of ER-stress and lipid metabolism in mediating drug response in HCC. METHODS: By using a chemically-induced mouse model for HCC, we administered the ER-stress inhibitor 4µ8C and/or doxorubicin (DOX) twice weekly for three weeks post-tumor initiation. Histological analyses were performed alongside comprehensive molecular biology and lipidomics assessments of isolated liver samples. In vitro models, including HCC cells, spheroids, and patient-derived liver organoids were subjected to 4µ8C and/or DOX, enabling us to assess their synergistic effects on cellular viability, lipid metabolism, and oxygen consumption rate. RESULTS: We reveal a pivotal synergy between ER-stress modulation and drug response in HCC. The inhibition of ER-stress using 4µ8C not only enhances the cytotoxic effect of DOX, but also significantly reduces cellular lipid metabolism. This intricate interplay culminates in the deprivation of energy reserves essential for the sustenance of tumor cells. CONCLUSIONS: This study elucidates the interplay between lipid metabolism and ER-stress modulation in enhancing doxorubicin efficacy in HCC. This novel approach not only deepens our understanding of the disease, but also uncovers a promising avenue for therapeutic innovation. The long-term impact of our study could open the possibility of ER-stress inhibitors and/or lipase inhibitors as adjuvant treatments for HCC-patients.

Signaling in insulin-secreting MIN6 pseudoislets and monolayer cells.

Cell-cell interactions are of fundamental importance for cellular function. In islets of Langerhans, which control blood glucose levels by secreting insulin in response to the blood glucose concentration, the secretory response of intact islets is higher than that of insulin-producing beta-cells not arranged in the islet architecture. The objective was to define mechanisms by which cellular performance is enhanced when cells are arranged in three-dimensional space. The task was addressed by making a comprehensive analysis based on protein expression patterns generated from insulin-secreting MIN6 cells grown as islet-like clusters, so-called pseudoislets, and in monolayers. After culture, glucose-stimulated insulin secretion (GSIS) was measured from monolayers and pseudoislets. GSIS rose 6-fold in pseudoislets but only 3-fold in monolayers when the glucose concentration was increased from 2 to 20 mmol/L. Proteins from pseudoislets and monolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins were mapped onto KEGG pathways. Protein profiling identified 1576 proteins, which were common to pseudoislets and monolayers. When mapped onto KEGG pathways, 11 highly enriched pathways were identified. On the basis of differences in expression of proteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation were performed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathways regulating glucose metabolism, cell interaction, and translational regulation.

Metformin Can Attenuate Beta-Cell Hypersecretion-Implications for Treatment of Children with Obesity.

In children with obesity, insulin hypersecretion is proposed to precede insulin resistance. We investigated if metformin could be used to attenuate insulin secretion from palmitate-treated isolated islets and its implication for children with obesity. Human islets were exposed to palmitate for 0.5 or 1 day, when metformin was introduced. After culture, glucose-stimulated insulin secretion (GSIS) was measured. Children with obesity, who had received metformin for over six months (n = 21, age 13.9 ± 1.8), were retrospectively evaluated. Children were classified as either "reducing" or "increasing" based on the difference between AUC0-120 of insulin during OGTT before and after metformin treatment. In human islets, GSIS increased after culture in palmitate for up to 1 day but declined with continued palmitate exposure. Whereas adding metformin after 1 day of palmitate exposure increased GSIS, adding metformin after 0.5 days reduced GSIS. In children with "reducing" insulin AUC0-120 (n = 9), 2 h glucose and triglycerides decreased after metformin treatment, which was not observed in patients with "increasing" insulin AUC0-120 (n = 12). In isolated islets, metformin attenuated insulin hypersecretion if introduced when islet secretory capacity was maintained. In children with obesity, improved glycemic and lipid levels were accompanied by reduced insulin levels during OGTT after metformin treatment.

Low Fasting Concentrations of Glucagon in Patients with Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

(1) Background: Deficiencies of mitochondrial fatty acid oxidation (FAO) define a subgroup of inborn errors of metabolism, with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCAD) being two of the most common. Hypoketotic hypoglycemia is a feared clinical complication and the treatment focuses on avoiding hypoglycemia. In contrast, carnitine uptake deficiency (CUD) is treated as a mild disease without significant effects on FAO. Impaired FAO has experimentally been shown to impair glucagon secretion. Glucagon is an important glucose-mobilizing hormone. If and how glucagon is affected in patients with VLCAD or MCAD remains unknown. (2) Methods: A cross-sectional study was performed with plasma hormone concentrations quantified after four hours of fasting. Patients with VLCAD (n = 10), MCAD (n = 7) and CUD (n = 6) were included. (3) Results: The groups were similar in age, sex, weight, and height. The glucagon and insulin levels were significantly lower in the VLCAD group compared to the CUD group (p < 0.05, respectively). The patients with CUD had glucagon concentrations similar to the normative data. No significant differences were seen in GLP-1, glicentin, glucose, amino acids, or NEFAs. (4) Conclusions: Low fasting concentrations of glucagon are present in patients with VLCAD and cannot be explained by altered stimuli in plasma.

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.

Iron Chelator VLX600 Inhibits Mitochondrial Respiration and Promotes Sensitization of Neuroblastoma Cells in Nutrition-Restricted Conditions.

Neuroblastoma, the most common solid tumor in children, is characterized by amplification of the MYCN proto-oncogene, a high-risk aggressive clinical marker associated with treatment failure. MYCN plays an important role in cell growth, proliferation, metabolism, and chemoresistance. Here, we show for the first time that in neuroblastoma, iron chelator VLX600 inhibits mitochondrial respiration, decreases expression levels of MYCN/LMO1, and induces an efficient cell death regardless of MYCN status in both 2D and 3D culture conditions. Moreover, insufficient induction of autophagy was observed in cells treated with VLX600, which is essential as a protective response in the event of ATP synthesis disruption. Further inhibition of glucose uptake using DRB18, a pan-GLUT (glucose transporter) inhibitor, synergized the effect of VLX600 and no significant cell death was found in immortalized epithelial cells under this combination treatment. Our results demonstrate that inhibition of mitochondrial respiration by iron chelator VLX600 accompanied by autophagy deficiency promotes sensitivity of neuroblastoma cells in a nutrition-restricted microenvironment regardless of MYCN status, indicating that MYCN expression level is an essential clinical marker but might not be a necessary target for the treatment of neuroblastoma which warrants further investigation. VLX600 has been studied in Phase I clinical trials; combining VLX600 with conventional chemotherapy could be an innovative therapeutic strategy for neuroblastoma.

Altered mitochondrial metabolism in peripheral blood cells from patients with inborn errors of ß-oxidation.

Inborn errors of mitochondrial fatty acid oxidation (FAO), such as medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCAD) affects cellular function and whole-body metabolism. Carnitine uptake deficiency (CUD) disturbs the transportation of fatty acids into the mitochondria, but when treated is a mild disease without significant effects on FAO. For improved clinical care of VLCAD in particular, estimation of FAO severity could be important. We have investigated whether the oxygen consumption rate (OCR) of peripheral blood mononuclear cells (PBMCs) obtained from patients with MCAD, VLCAD, and CUD can be used to study cellular metabolism in patients with FAO defects and to determine the severity of FAO impairment. PBMCs were isolated from patients with VLCAD (n = 9), MCAD (n = 5-7), and CUD (n = 5). OCR was measured within 6-hours of venous puncture using the Seahorse XFe96. The PBMCs were exposed to glucose alone or with caprylic acid (C8:0) or palmitic acid (C16:0). OCR was significantly lower in cells from patients with ß-oxidation deficiencies (MCAD and VLCAD) compared to CUD at basal conditions. When exposed to C16:0, OCR in VLCAD cells was unchanged, whereas OCR in MCAD cells increased but not to the levels observed in CUD. However, C8:0 did not increase OCR, as would be expected, in VLCAD cells. There was no clear relationship between clinical severity level and OCR. In patients with ß-oxidation deficiencies, changes of mitochondrial respiration in PBMCs are detectable, which indicate that PBMCs have translational potential for studies of ß-oxidation defects. However, further studies are warranted.

Impaired HMG-CoA Reductase Activity Caused by Genetic Variants or Statin Exposure: Impact on Human Adipose Tissue, ß-Cells and Metabolome.

Inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase is associated with an increased risk of new-onset type 2 diabetes. We studied the association of genetic or pharmacological HMG-CoA reductase inhibition with plasma and adipose tissue (AT) metabolome and AT metabolic pathways. We also investigated the effects of statin-mediated pharmacological inhibition of HMG-CoA reductase on systemic insulin sensitivity by measuring the HOMA-IR index in subjects with or without statin therapy. The direct effects of simvastatin (20-250 nM) or its active metabolite simvastatin hydroxy acid (SA) (8-30 nM) were investigated on human adipocyte glucose uptake, lipolysis, and differentiation and pancreatic insulin secretion. We observed that the LDL-lowering HMGCR rs12916-T allele was negatively associated with plasma phosphatidylcholines and sphingomyelins, and HMGCR expression in AT was correlated with various metabolic and mitochondrial pathways. Clinical data showed that statin treatment was associated with HOMA-IR index after adjustment for age, sex, BMI, HbA1c, LDL-c levels, and diabetes status in the subjects. Supra-therapeutic concentrations of simvastatin reduced glucose uptake in adipocytes and normalized fatty acid-induced insulin hypersecretion from ß-cells. Our data suggest that inhibition of HMG-CoA reductase is associated with insulin resistance. However, statins have a very mild direct effect on AT and pancreas, hence, other tissues as the liver or muscle appear to be of greater importance.

GLP-1 analogue recovers impaired insulin secretion from human islets treated with palmitate via down-regulation of SOCS2.

Elevated circulating palmitate levels have been connected with type 2 diabetes mellitus. GLP-1 has favorable effects on beta-cells function. The aim was to identify mechanisms for decreased GSIS after long-term palmitate exposure and restoration by GLP-1 by analyzing changes in G-protein coupled receptor (GPCR) pathway signaling. Insulin secretory response to 20 mM glucose was attenuated after 7 days in islets exposed to palmitate but inclusion of exendin-4 restored secretion. Palmitate treatment altered genes of several GPCR signaling pathways including inflammatory pathways with up-regulated IL-1B, SOCS1 and SOCS2 transcript levels. Protein level of SOCS2 was also up-regulated by palmitate and accompanied by down-regulation of pAkt(T308), which was restored by exendin-4 treatment. When SOCS2 was knocked down, palmitate-induced down-regulation of IRS-1 and pAkt(T308) was prevented and GSIS, proinsulin to insulin ratio and apoptosis was restored. Long-term palmitate treatment up-regulates SOCS2 and reduces PI3K activity, thereby impairing GSIS. GLP-1 reverts the palmitate-induced effects.

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.