Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-ßH1. We demonstrate that PCSK9 is expressed and secreted by EndoC-ßH1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLA-ABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells.

Characterization of the Secretome, Transcriptome, and Proteome of Human ß Cell Line EndoC-ßH1.

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was ∼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

Loss of Human Beta Cell Identity in a Reconstructed Omental Stromal Cell Environment.

In human type 2 diabetes, adipose tissue plays an important role in disturbing glucose homeostasis by secreting factors that affect the function of cells and tissues throughout the body, including insulin-producing pancreatic beta cells. We aimed here at studying the paracrine effect of stromal cells isolated from subcutaneous and omental adipose tissue on human beta cells. We developed an in vitro model wherein the functional human beta cell line EndoC-ßH1 was treated with conditioned media from human adipose tissues. By using RNA-sequencing and western blotting, we determined that a conditioned medium derived from omental stromal cells stimulates several pathways, such as STAT, SMAD and RELA, in EndoC-ßH1 cells. We also observed that upon treatment, the expression of beta cell markers decreased while dedifferentiation markers increased. Loss-of-function experiments that efficiently blocked specific signaling pathways did not reverse dedifferentiation, suggesting the implication of more than one pathway in this regulatory process. Taken together, we demonstrate that soluble factors derived from stromal cells isolated from human omental adipose tissue signal human beta cells and modulate their identity.

Overexpression of Wild-Type Human Alpha-Synuclein Causes Metabolism Abnormalities in Thy1-aSYN Transgenic Mice.

Parkinson's disease is a progressive neurodegenerative disorder characterized by loss of dopaminergic neurons, pathological accumulation of alpha-synuclein and motor symptoms, but also by non-motor symptoms. Metabolic abnormalities including body weight loss have been reported in patients and could precede by several years the emergence of classical motor manifestations. However, our understanding of the pathophysiological mechanisms underlying body weight loss in PD is limited. The present study investigated the links between alpha-synuclein accumulation and energy metabolism in transgenic mice overexpressing Human wild-type (WT) alpha-synuclein under the Thy1 promoter (Thy1-aSYN mice). Results showed that Thy1-aSYN mice gained less body weight throughout life than WT mice, with significant difference observed from 3 months of age. Body composition analysis of 6-month-old transgenic animals showed that body mass loss was due to lower adiposity. Thy1-aSYN mice displayed lower food consumption, increased spontaneous activity, as well as a reduced energy expenditure compared to control mice. While no significant change in glucose or insulin responses were observed, Thy1-aSYN mice had significantly lower plasmatic levels of insulin and leptin than control animals. Moreover, the pathological accumulation of alpha-synuclein in the hypothalamus of 6-month-old Thy1-aSYN mice was associated with a down-regulation of the phosphorylated active form of the signal transducer and activator of transcription 3 (STAT3) and of Rictor (the mTORC2 signaling pathway), known to couple hormonal signals with the maintenance of metabolic and energy homeostasis. Collectively, our results suggest that (i) metabolic alterations are an important phenotype of alpha-synuclein overexpression in mice and that (ii) impaired STAT3 activation and mTORC2 levels in the hypothalamus may underlie the disruption of feeding regulation and energy metabolism in Thy1-aSYN mice.