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.

MicroRNA Expression Analysis of In Vitro Dedifferentiated Human Pancreatic Islet Cells Reveals the Activation of the Pluripotency-Related MicroRNA Cluster miR-302s.

ß-cell dedifferentiation has been recently suggested as an additional mechanism contributing to type-1 and to type-2 diabetes pathogenesis. Moreover, several studies demonstrated that in vitro culture of native human pancreatic islets derived from non-diabetic donors resulted in the generation of an undifferentiated cell population. Additional evidence from in vitro human ß-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from ß-cells, thus representing a potential in vitro model of ß-cell dedifferentiation. Here, we report the microRNA expression profiles analysis of in vitro dedifferentiated islet cells in comparison to mature human native pancreatic islets. We identified 13 microRNAs upregulated and 110 downregulated in islet cells upon in vitro dedifferentiation. Interestingly, among upregulated microRNAs, we observed the activation of microRNA miR-302s cluster, previously defined as pluripotency-associated. Bioinformatic analysis indicated that miR-302s are predicted to target several genes involved in the control of ß-cell/epithelial phenotype maintenance; accordingly, such genes were downregulated upon human islet in vitro dedifferentiation. Moreover, we uncovered that cell-cell contacts are needed to maintain low/null expression levels of miR-302. In conclusion, we showed that miR-302 microRNA cluster genes are involved in in vitro dedifferentiation of human pancreatic islet cells and inhibits the expression of multiple genes involved in the maintenance of ß-cell mature phenotype.

HDAC7 is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells.

AIMS/HYPOTHESIS: Pancreatic beta cell dysfunction is a prerequisite for the development of type 2 diabetes. Histone deacetylases (HDACs) may affect pancreatic endocrine function and glucose homeostasis through alterations in gene regulation. Our aim was to investigate the role of HDAC7 in human and rat pancreatic islets and clonal INS-1 beta cells (INS-1 832/13). METHODS: To explore the role of HDAC7 in pancreatic islets and clonal beta cells, we used RNA sequencing, mitochondrial functional analyses, microarray techniques, and HDAC inhibitors MC1568 and trichostatin A. RESULTS: Using RNA sequencing, we found increased HDAC7 expression in human pancreatic islets from type 2 diabetic compared with non-diabetic donors. HDAC7 expression correlated negatively with insulin secretion in human islets. To mimic the situation in type 2 diabetic islets, we overexpressed Hdac7 in rat islets and clonal beta cells. In both, Hdac7 overexpression resulted in impaired glucose-stimulated insulin secretion. Furthermore, it reduced insulin content, mitochondrial respiration and cellular ATP levels in clonal beta cells. Overexpression of Hdac7 also led to changes in the genome-wide gene expression pattern, including increased expression of Tcf7l2 and decreased expression of gene sets regulating DNA replication and repair as well as nucleotide metabolism. In accordance, Hdac7 overexpression reduced the number of beta cells owing to enhanced apoptosis. Finally, we found that inhibiting HDAC7 activity with pharmacological inhibitors or small interfering RNA-mediated knockdown restored glucose-stimulated insulin secretion in beta cells that were overexpressing Hdac7. CONCLUSIONS/INTERPRETATION: Taken together, these results indicate that increased HDAC7 levels caused beta cell dysfunction and may thereby contribute to defects seen in type 2 diabetic islets. Our study supports HDAC7 inhibitors as a therapeutic option for the treatment of type 2 diabetes.

Ultrastructural alterations of pancreatic beta cells in human diabetes mellitus.

BACKGROUND: Both types of diabetes are characterized by beta-cell failure and death, leading to insulin insufficiency. Very limited information is currently available about the ultrastructural alterations of beta cells in human diabetes. Our aim was to provide a comprehensive ultrastructural analysis of human pancreatic islets in type 1 (T1D) and type 2 (T2D) diabetic patients. METHODS: We performed a morphometric electron microscopy evaluation of beta cells obtained from the pancreas of 8 nondiabetic (ND), 5 T1D, and 8 T2D organ donors. RESULTS: A lower amount of beta cells was found in both T1D and T2D than in ND islets, whereas alpha cells were increased only in T2D. An increased number of bi-hormonal cells (showing both insulin and glucagon granules in their cytoplasm) were found in T1D. Insulin granules were less represented in T2D than in ND beta cells, whereas no significant changes were found in T1D. Volume density of the endoplasmic reticulum was increased in T2D and unchanged in T1D; mitochondria number and volume were significantly higher in T2D than in ND beta cells, whereas no significant differences were found in T1D. In both T1D and T2D, more beta cells showed signs of apoptosis than in ND. CONCLUSIONS: Our results show that in each type of diabetes, beta cells exhibit specific ultrastructural alterations, whose better understanding might improve therapeutic strategies.

Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion.

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION.

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075-11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

Infection of human islets of Langerhans with two strains of Coxsackie B virus serotype 1: assessment of virus replication, degree of cell death and induction of genes involved in the innate immunity pathway.

Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1.Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFNß, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFNß, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential ß-cell tropism of the virus.

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37 °C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25 mL of cold (4 °C) human serum albumin and centrifuge twice at 150 g for 3 min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30 min. Digested pancreatic tissue was centrifuged at 2200 rpm for 5 min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating ß-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2 weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses.

Pancreatic α-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human α-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality α-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live α-cells from dissociated human islet cells with ~95% purity. The α-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form α-pseudoislets. The α-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key α-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in α-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary α-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate.

Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures. Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested. Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Sex Differences in the Molecular Programs of Pancreatic Cells Contribute to the Differential Risks of Type 2 Diabetes.

Epidemiology studies demonstrate that women are at a significantly lower risk of developing type 2 diabetes (T2D) compared to men. However, the molecular basis of this risk difference is not well understood. In this study, we examined the sex differences in the genetic programs of pancreatic endocrine cells. We combined pancreas perifusion data and single-cell genomic data from our laboratory and from publicly available data sets to investigate multiple axes of the sex differences in the human pancreas at the single-cell type and single-cell level. We systematically compared female and male islet secretion function, gene expression program, and regulatory principles of pancreatic endocrine cells. The perifusion data indicate that female endocrine cells have a higher secretion capacity than male endocrine cells. Single-cell RNA-sequencing analysis suggests that endocrine cells in male controls have molecular signatures that resemble T2D. In addition, we identified genomic elements associated with genome-wide association study T2D loci to have differential accessibility between female and male delta cells. These genomic elements may play a sex-specific causal role in the pathogenesis of T2D. We provide molecular mechanisms that explain the differential risk of T2D between women and men. Knowledge gained from our study will accelerate the development of diagnostics and therapeutics in sex-aware precision medicine for diabetes.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

The potential of the incorporated collagen microspheres in alginate hydrogel as an engineered three-dimensional microenvironment to attenuate apoptosis in human pancreatic islets.

BACKGROUND: Tissue engineering is considered as a promising tool for remodeling the native cells microenvironment. In the present study, the effect of alginate hydrogel and collagen microspheres integrated with extracellular matrix components were evaluated in the decrement of apoptosis in human pancreatic islets. MATERIALS/METHODS: For three-dimensional culture, the islets were encapsulated in collagen microspheres, containing laminin and collagen IV and embedded in alginate scaffold for one week. After that the islets were examined in terms of viability, apoptosis, genes and proteins expression including BAX, BCL2, active caspase-3, and insulin. Moreover, the islets function was evaluated through glucose-induced insulin and C-peptide secretion assay. In order to evaluate the structure of the scaffolds and the morphology of the pancreatic islets in three-dimensional microenvironments, we performed scanning electron microscopy. RESULTS: Our findings showed that the designed hydrogel scaffolds significantly improved the islets viability using the reduction of activated caspase-3 and TUNEL positive cells. CONCLUSIONS: The reconstruction of the destructed matrix with alginate hydrogels and collagen microspheres might be an effective step to promote the culture of the islets.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

Circadian Lipidomics: Analysis of Lipid Metabolites Around the Clock.

Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.

Cathepsin C Regulates Cytokine-Induced Apoptosis in ß-Cell Model Systems.

Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic ß cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates ß-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in ß-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E ß-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of ß-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated ß-cell destruction in T1D.