Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

Levy, James R.; Ullrich, Axel; Olefsky, Jerrold M.

doi:10.1172/jci113465

Cited by 12 publications

(10 citation statements)

References 36 publications

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“…This process involves the rapid exocytosis of intact ligands from neurons, a process that may also be utilized by other cell proteins, peptides or small molecules. Retroendocytosis of insulin has been described in kidney epithelial cells, fibroblasts transfected with insulin receptors, adipocytes and other cells (Dahl et al, 1989;Levy et al, 1988;Marshall, 1985). While largely unexplored in neurons, this pathway may be important in bidirectional signaling.…”

Section: Discussionmentioning

confidence: 99%

Intraganglionic interactions between satellite cells and adult sensory neurons

Christie

Koshy

Cheng

et al. 2015

Molecular and Cellular Neuroscience

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Section: Discussionmentioning

confidence: 99%

Intraganglionic interactions between satellite cells and adult sensory neurons

Christie

Koshy

Cheng

et al. 2015

Molecular and Cellular Neuroscience

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“…Furthermore, after in vivo uptake into the liver, proinsulin is more slowly cleared than insulin, suggesting a slower intracellular processing (15,16). However, although proinsulin has been shown to be less efficiently degraded than insulin by isolated liver subcellular fractions (11,17) and purified liver insulindegrading enzyme (IDE) (18), studies on proinsulin processing in intact cells have been limited to adipocytes (19) and fibroblasts overexpressing the insulin receptor (20). In the latter cells, proinsulin was found to be less efficiently degraded than insulin and, in part, released intact from the cell via a retroendocytotic pathway; this observation was suggested to account for the prolonged biological activity of proinsulin in vivo (20).…”

Section: As [ 125 I]-insulin [ 125 I]-proinsulin Taken Up Into Livermentioning

confidence: 99%

“…Male Sprague Dawley rats, weighing 200 Ϯ 20 g, were obtained from Charles River (St. Aubin les Elbeufs, France) and Elevage Janvier (Le Genest-St-Isle, France) and were fasted for 16 h before killing. When indicated, porcine [ 125 I]-labeled peptides (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) or native human peptides (0.06 -13 nmol), diluted into 0.5 ml 0.15-m NaCl, were rapidly injected into the penis vein under ether or pentobarbital anesthesia. At the time indicated, the liver was rapidly removed, minced in ice-cold 0.25 m sucrose, and immediately homogenized.…”

Section: Animals and Peptide Injectionsmentioning

confidence: 99%

Cell Itinerary and Metabolic Fate of Proinsulin in Rat Liver: In Vivo and in Vitro Studies

et al. 2003

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Proinsulin, the insulin precursor in pancreatic beta-cells, displays a slower hepatic clearance than insulin and exerts a more prolonged metabolic effect on liver in vivo. To elucidate the mechanisms underlying these differences, the cellular itinerary and processing of proinsulin and insulin in rat liver have been comparatively studied using cell fractionation. As [125I]-insulin, [125I]-proinsulin taken up into liver in vivo was internalized and accumulated in endosomes, in which it underwent dissociation from the insulin receptor and degradation in a pH- and ATP-dependent manner. However, relative to [125I]-insulin, [125I]-proinsulin showed a delayed and prolonged in vivo association with endosomes, a slower in vivo and cell-free endosomal processing, and a higher cell-free endosome-lysosome transfer. Endosomal extracts degraded to a lesser extent proinsulin than insulin at acidic pH; so did, and even proportionally less, at neutral pH, plasma membrane and cytosolic fractions. Proinsulin degradation products generated by soluble endosomal extracts were isolated by HPLC and characterized by mass spectrometry. Under conditions resulting in multiple cleavages in insulin, proinsulin was cleaved at eight bonds in the C peptide but only at the Phe24-Phe25 bond in the insulin moiety. As native insulin, native proinsulin induced a dose- and time-dependent endocytosis and tyrosine phosphorylation of the insulin receptor; but at an inframaximal dose, proinsulin effects on these processes were of longer duration. We conclude that a reduced proteolysis of proinsulin in endosomes, and probably also at the plasma membrane, accounts for its slower hepatic clearance and prolonged effects on insulin receptor endocytosis and tyrosine phosphorylation.

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“…In the phosphorylation assay, supernatants of FurHPI-transduced cells displayed a slightly higher activity than expected on a molar base. This is probably due to the presence of variable amounts of proinsulin and des-split insulin forms, which retain a residual activity (Levy et al, 1988). Overall, these results demonstrate that the vector-derived, furin-cleaved human insulin retains full activity in vitro.…”

Section: Functional Properties Of the Vector-derived Human Insulinmentioning

confidence: 75%

Reversal of Diabetes in Mice by Implantation of Human Fibroblasts Genetically Engineered to Release Mature Human Insulin

Falqui

Martinenghi²,

Severini³

et al. 1999

Human Gene Therapy

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Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

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Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

Abstract: The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used.

Cited by 12 publications

References 36 publications

Intraganglionic interactions between satellite cells and adult sensory neurons

Intraganglionic interactions between satellite cells and adult sensory neurons

Cell Itinerary and Metabolic Fate of Proinsulin in Rat Liver: In Vivo and in Vitro Studies

Reversal of Diabetes in Mice by Implantation of Human Fibroblasts Genetically Engineered to Release Mature Human Insulin

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