Ruolo del sistema ubiquitina-proteasoma (UPS) nella disfunzione beta cellulare del diabete di tipo 2 e possibili opzioni terapeutiche.

Ruolo del sistema ubiquitina-proteasoma (UPS) nella disfunzione beta cellulare del diabete di tipo 2 e possibili opzioni terapeutiche. Sebbene la disfunzione beta cellulare rivesta un ruolo di primaria importanza nel diabete di tipo 2, le informazioni ad oggi disponibili riguardanti il loro fenotipo molecolare in tale tipo di diabete, sono ancora piuttosto limitate. In questo lavoro, dapprima sono state valutate le differenze di espressione tra il trascrittoma di isole purificate da donatori affetti da diabete di tipo 2 (T2D) e quello di isole di donatori non diabetici (ND) mentre in seguito l’attenzione è stata focalizzata sul sistema ubiquitina-proteasoma (UPS), il principale pathway intracellulare deputato alla degradazione delle proteine. L’analisi microarray mostrava che 1230 geni risultavano differenzialmente espressi nelle isole diabetiche e che questi influenzavano sia la struttura che la funzione di tale tessuto. In seguito a questa ed ad altre analisi emergeva decisamente una riduzione dell’UPS nel T2D. Gli esperimenti di RT-PCR quantitativa mostravano una riduzione dell’espressione di UBE2K, PSMB7 e UCHL1 (geni dell’UPS). Studi di immunoistochimica rivelavano un maggior accumulo di ubiquitina accompagnato ad una riduzione dell’attività del proteasoma in T2D. L’inibizione diretta del proteasoma e l’esposizione ad acido palmitico riducevano l’ attività del proteasoma e a questo si accompagnava una riduzione della secrezione insulinica indotta da glucosio. La metformina e il GLP-1, due molecole comunemente utilizzate nel trattamento del diabete di tipo 2 in grado di ripristinare un corretto rilascio insulinico in risposta a glucosio associato ad un aumento dell’attività del proteasoma. In questo studio si mostra la presenza di numerose alterazioni nel trascrittoma delle isole diabetiche e si sottolinea l’importanza delle alterazioni a carico del sistema UPS come meccanismo associato alla disfunzione beta cellulare nel diabete di tipo 2

EFFETTI DEGLI INIBITORI DELLA CALCINEURINA SULLE β-CELLULE UMANE

Un corretto uso dei farmaci immunosoppressori è cruciale per la riuscita di un trapianto di pancreas o isole. Tuttavia, gli agenti immunosoppressori possono avere effetti collaterali deleteri sulla beta cellula pancreatica. Gli studi eseguiti finora, hanno utilizzato per la maggior parte farmaci ormai obsoleti o dosi a concentrazioni farmacologiche più che terapeutiche. Per tale motivo abbiamo osservato gli effetti di una prolungata esposizione di isole pancreatiche umane a 10 ng/ml di Tacrolimo (Tac) o 150 ng/ml di Ciclosporina A (CsA). Per questo studio le isole sono state purificate da pancreas di 14 donatori multi-organo non diabetici (Età: 64±14 anni; Genere: 9M/5F; IMC: 25,9±2,9 kg/m2) tramite un protocollo di digestione con collagenasi e purificazione in gradiente di densità. Gli studi morfologici, funzionali e di sopravvivenza sono stati eseguiti dopo 96 ore di incubazione con Tac o CsA. La secrezione insulinica in risposta a glucosio è risultata significativamente ridotta nelle isole esposte a Tac (p<0.05), ma non con a CsA (Indice di stimolazione, ovvero rilascio in risposta a glucosio 16,7 mM rispetto a glucosio 3,3 mM: Isole di controllo Ctrl, 2.3±0.5; Tac 1.6±0.2; CsA, 2.0±0.4). Questi dati non erano associati a variazioni nell’espressione di mRNA di insulina o glut2, ma ad un significativo aumento nell’espressione della glucochinasi nelle cellule esposte a Tac. La microscopia elettronica (ME), inoltre, mostrava una significativa (p<0.05) riduzione nel numero di granuli di insulina mature nelle β-cellule esposte a Tac (densità di volume, ml%: 1,2±0,5), ma non in quelle esposte a CsA (2,6±0,3), quando comparate a Ctrl (3,1±0,6). Infine, mediante ME si è evidenziato che la percentuale di β-cellule apoptotiche era più alta con Tac (15±6) che con Ctrl (4±4) (il rispettivo valore con CsA era 9±5). In conclusione, il Tac ha effetti deleteri più pronunciati sulla funzionalità e sopravvivenza delle beta-cellule umane rispetto alla CsA, quando entrambe vengono usate a concentrazioni terapeutiche

Pancreatic Beta Cell Identity in Humans and the Role of Type 2 Diabetes

Pancreatic beta cells uniquely synthetize and release insulin. Specific molecular, functional as well as ultrastructural traits characterize their insulin secretion properties and survival phenotype. In this review we focus on human islet/beta cells, and describe the changes that occur in type 2 diabetes and could play roles in the disease as well as represent possible targets for therapeutical interventions. These include transcription factors, molecules involved in glucose metabolism and insulin granule handling. Quantitative and qualitative insulin release patterns and their changes in type 2 diabetes are also associated with ultrastructural features involving the insulin granules, the mitochondria, and the endoplasmic reticulum

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

A role for autophagy in β-cell life and death.

Autophagy is a vacuolar, self-digesting mechanism responsible for the removal of organelles and defined regions of the cytoplams. This process has, in general, a beneficial role for the cell, since it regulates the turnover of aged proteins and eliminates damaged structures. However, cells that undergo altered autophagy may be triggered to die in a non-apoptotic manner. As a matter of fact, in recent years it has become clear that dysregulated autophagy may be implicated in several disorders, such as cancer, neurodegenerative diseases and hepatic encephalopathy. We have recently shown that β-cells of type 2 diabetic subjects show signs of autophagy associated death, which may contribute to the overall loss of β-cell mass in type 2 diabetes. In addition, studies with cell lines and rodent models have demonstrated the importance of autophagy in β-cell function and survival. Altogether, the available evidence supports the view that autophagy is implicated in β-cell pathophysiology, and suggests that addressing the molecular mechanisms involved in autophagic regulation might provide clues for preventing or treating β-cell damage in diabetes

FGF-2b and h-PL transform duct and non-endocrine human pancreatic cells into endocrine insulin secreting cells by modulating differentiating genes

Background: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes

Ubiquitin D regulates IRE1 α/c-Jun N-terminal kinase (JNK) protein-dependent apoptosis in pancreatic beta cells

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1 alpha is critical for this transition. IRE1 alpha activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1 alpha-interacting protein ubiquitin D (UBD) on the regulation of IRE1 alpha and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1 alpha interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown ofUBDin human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1 alpha activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1 beta and interferon (IFN)-gamma in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1 alpha in human and rodent beta cells, modulating IRE1 alpha-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1 alpha/JNK pro-apoptotic pathway in cytokine-exposed beta cells

Histopathology and ex vivo insulin secretion of pancreatic islets in gestational diabetes: A case report

Gestational diabetes (GD) results from insufficient endogenous insulin supply. No information is available on features of islet cells in human GD. Herein, we describe several properties of islets from a woman with GD. Immunohistochemical stainings and EM analyses were performed on pancreatic samples. Islet isolation was achieved by enzymatic dissociation and density gradient centrifugation. Ex vivo insulin secretion was studied in response to fuel secretagogues. Control islets were obtained from matched non-pregnant, non-diabetic women. Total insulin positive area was lower in GD, mainly due to the presence of smaller islets. β-cell apoptosis and the presence of Ki67 positive islet cells were similar in GD and controls, whereas the amount of insulin positive cells in or close to the ducts was decreased in GD. Ex vivo insulin secretion did not differ between GD and non-pregnant, non-diabetic islets. These findings suggest that in this case of human GD there might mainly be a defect of β-cell amount, not due to increased apoptosis, but possibly to insufficient regeneration

Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition