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Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

ABSTRACT: Ex vivo expansion and differentiation of human pancreatic ?-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which ?-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates with tailored roughness to mimic the nanotopography of the extracellular matrix. We demonstrate that ?-cells can perceive nanoscale features of the substrate and can convert these stimuli into mechanotransductive processes which promote long-term in vitro human islet culture, thus preserving ?-cell differentiation and function. Proteomic and quantitative immunofluorescence analyses demonstrate that the process is driven by nanoscale topography, via remodelling of the actin cytoskeleton and nuclear architecture. These modifications activate a transcriptional program which stimulates an adaptive metabolic glucose response. Engineered cluster-assembled substrates coupled with proteomic approaches may provide a useful strategy for identifying novel molecular targets for treating diabetes mellitus and for enhancing tissue engineering in order to improve the efficacy of islet cell transplantation therapies.

SUBMITTER: Galli A

PROVIDER: S-EPMC6028636 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Galli Alessandra A Maffioli Elisa E Sogne Elisa E Moretti Stefania S Di Cairano Eliana Sara ES Negri Armando A Nonnis Simona S Norata Giuseppe Danilo GD Bonacina Fabrizia F Borghi Francesca F Podestà Alessandro A Bertuzzi Federico F Milani Paolo P Lenardi Cristina C Tedeschi Gabriella G Perego Carla C

Scientific reports 20180702 1

Ex vivo expansion and differentiation of human pancreatic β-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which β-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates ...[more]

PMID: 29967323

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Project description:To investigate substrates for cyclic AMP-dependent protein kinase in intact islets of Langerhans, batches of islets were incubated with [32P]Pi for 1 h in the presence of 10 mM-glucose; the adenylate cyclase activator forskolin, which in parallel experiments was shown to increase islet cyclic AMP content and insulin release, was then added. Islets were homogenized and subcellular fractions prepared by differential centrifugation. Phosphopeptides were electrophoresed on sodium dodecyl sulphate/polyacrylamide gels and quantified by autoradiography and densitometry. Within 5 min forskolin caused increased labelling of Mr-25 000 and -30 000 cytosolic and Mr-23 000 and -32 000 particulate peptides; a rapid decrease in phosphorylation of Mr-18 000 and -34 000 cytosolic peptides was also observed. In addition, rather slower phosphorylation occurred of the Mr-15 000 peptide previously identified as histone H3 [Christie & Ashcroft (1984) Biochem. J. 218, 87-99]. When similar subcellular fractions were incubated with [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase, peptides phosphorylated included cytosolic species of Mr 25 000 and 30 000 and particulate species of Mr 23 000 and 32 000. The distribution of RNA in the subcellular fractions suggested that the Mr-32 000 species could be a ribosomal protein. The 24 000 g pellet was heterogeneous, as judged by marker assays, and was therefore fractionated further by Percoll-density-gradient centrifugation. The peak containing the Mr-23 000 peptide was resolved from marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum and coincided with a peak for insulin: hence the Mr-23 000 peptide is likely to be a secretory-granule component. The study demonstrates that the potentiation of insulin release that occurs when islet cyclic AMP is increased is accompanied by rapid phosphorylation of specific islet substrates for cyclic AMP-dependent protein kinase. The data are consistent with the hypothesis that protein phosphorylation is involved in the regulation of insulin secretion.

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Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Publications

Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

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