Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

Gross, David J.; Halban, Philippe A.; Kahn, C. Ronald; Weir, Gordon C.; Villa‐Komaroff, Lydia

doi:10.1073/pnas.86.11.4107

Cited by 52 publications

(35 citation statements)

References 30 publications

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“…Wild-type human insulin shows a sorting index of 0.66 (Powell et al, 1988). Thus, the dimeric insulin B10Asp shows a slightly reduced efficiency of sorting, consistent with earlier published results (Carroll et al, 1988;Gross et al, 1989). Monomeric insulin B9Asp shows a sorting index of 0.67 + 0.02 and therefore is sorted as efficiently as wild-type insulin.…”

Section: Table L Comparison Of the Density Of (Pro)insulin Immunolabesupporting

confidence: 80%

“…Taken together, the data showed that both mutated proinsulins entered the regulated secretory pathway where they are proteolytically processed and localized to densecore secretory granules. The B10Asp data are consistent with those of Carroll et al (1988) and Gross et al (1989) who showed that a significant fraction of the B10Asp was processed into mature insulin within the cells, presumably in dense-core secretory granules.…”

Section: Immunolocalization Of the Proinsulin Variantssupporting

confidence: 79%

“…The histidine residue at position B10 is involved in coordinating zinc ions (Blundell et al, 1972), and in the mutated form insulin dimers are formed instead of hexamers (Brange et al, 1988). Therefore, an intriguing possibility is that the aggregation state of insulin is important for its correct sorting into the regulated secretory pathway (Carroll et al, 1988;Gross et al, 1989). In this report, we set out to examine whether the intracellular transport and sorting of insulin is affected by its quaternary structure.…”

mentioning

confidence: 99%

“…In a kindred with this disorder (Gruppuso et al, 1984), the hyperproinsulinemia was found to be associated with a point mutation in the B-chain coding region of the insulin gene (Chan et al, 1987). Carroll et al (1988) and Gross et al (1989) have examined the cellular basis for the defects of this mutant in transgenic mice and in transfected AtT-20 cells, respectively. In both cases, the defects appear to result from a decrease in the efficiency of sorting of the mutant proinsulin, leading to heightened secretion of the unprocessed prohormone in an unregulated fashion.…”

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confidence: 99%

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Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Quinn

Orci

Ravazzola

et al. 1991

The Journal of Cell Biology

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Abstract. Human proinsulin and insulin oligomerize to form dimers and hemmers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. E Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, E Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight.1988. Nature [Lond.]. 333:679-682). One mutant (B10His--Asp) allows formation of dimers but not hexamers and the other (B9 s~r-'ap) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His-~p is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R.

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Section: Table L Comparison Of the Density Of (Pro)insulin Immunolabesupporting

confidence: 80%

Section: Immunolocalization Of the Proinsulin Variantssupporting

confidence: 79%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Quinn

Orci

Ravazzola

et al. 1991

The Journal of Cell Biology

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“…Early experiments of this type were carried out in the neuroendocrine cell line AtT-20ins, which is derived from ACTH-secreting corticotrophs of the anterior pituitary. These cells normally do not express the insulin gene, but upon transfection with a plasmid containing the human proinsulin cDNA, are shown to secrete the mature insulin polypeptide [6,7]. Insulin secretion from AtT-20ins cells can be stimulated by agents such as forskolin or isobutyl methylxanthine (IBMX) [6,8], but not by glucose [8].…”

Section: : S 42-s 47]mentioning

confidence: 99%

Engineered cell lines for insulin replacement in diabetes: current status and future prospects

Newgard

Clark²,

BeltrandelRio

et al. 1997

Diabetologia

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The recently concluded diabetes control and complications trial (DCCT) [1] has emphatically underscored the potential for improvement in current treatment protocols for diabetes mellitus. 'Conventional' insulin therapy, involving 1-2 injections of the hormone per day and intermittent glucose monitoring is clearly not as effective at slowing the progression of the debilitating and costly secondary complications of the disease as 'tight control', in which insulin pumps or multiple injections are combined with frequent glucose monitoring to limit hyperglycaemic excursions. Unfortunately, improvement in glycaemic control through increased insulin dosing requires a new level of compliance and discipline, and places the patient at significantly enhanced risk for dangerous hypoglycaemic episodes. These concerns have produced a surprising reticence among both physicians and patients towards implementation of tight control therapy. This reaction has served to place a new emphasis on the design of better methods for insulin replacement in insulin-dependent diabetes mellitus (IDDM). A primary consideration in such work is to develop a 'closed loop' system in which the hormone is delivered in response to metabolic demand, thereby eliminating the need for self monitoring by the patient. Three basic approaches to achieving this end have been contemplated, and are being investigated with ever increasing vigour. The first involves development of mechanical devices consisting of a pump or other mechanism for insulin delivery linked to a detector system for blood glucose. The second involves transplantation of insulin-producing cells from human or large mammal donors into IDDM patients. This can be achieved by transplantation of the entire pancreas or isolated pancreatic islets. Because of the difficulty and cost associated with islet isolation and pancreas transplantation, a third approach has emerged, in which the tools of molecular biology are used to fashion insulin-secreting cell lines with fuelmediated insulin secretion responses resembling Diabetologia (1997) Summary The recently completed diabetes complications and control trial has highlighted the need for improvement of insulin delivery systems for treatment of insulin-dependent diabetes mellitus. Despite steady improvement in methods for islet and whole pancreas transplantation over the past three decades, the broad-scale applicability of these approaches remains uncertain due in part to the difficulty and expense associated with procurement of functional tissue. To address this concern, we and others have been using the tools of molecular biology to develop cell lines with regulated insulin secretion that might serve as a surrogate for primary islets or pancreas tissue in transplantation therapy. This article seeks to provide a brief summary of the current status of this growing field, with a particular emphasis on progress in producing cell lines with appropriate glucose-stimulated insulin secretion. [Diabetologia (1997) 40: S 42-S 47]

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Insulin Gene Expression and Biosynthesis

Poitout¹,

Stein²,

Rhodes³

2003

International Textbook of Diabetes Mellitus

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The insulin gene is expressed specifically and at very high levels in pancreatic β‐cells. Most of its tissue‐specific expression and metabolic regulation are conferred by approximately 340 bp upstream of the transcription initiation site. Several glucose‐responsive elements have been identified within this region, and physiological regulation of insulin gene expression relies on the cooperative and synergistic interactions between DNA‐binding factors and coactivators. Glucose is the major regulator of insulin gene expression. It activates transcription and stabilizes insulin mRNA. In addition, insulin gene expression is stimulated by glucagon‐like peptide 1, growth hormone, and lactogenic hormones, and inhibited by epinephrine, somatostatin, glucagon, and leptin. In type 2 diabetes, chronic elevations of blood glucose and fatty acid levels impair insulin gene expression. Under most circumstances, the production of insulin is also highly regulated at the translational level. Indeed, this is the predominant control mechanism in the β‐cell whereby intracellular insulin stores are efficiently maintained in the short term. Essentially, translation of the preproinsulin mRNA template to preproinsulin protein occurs in a fashion typical of most eukaryotic mRNAs. In general, nutrients that stimulate insulin secretion, of which glucose is the most physiologically relevant, also stimulate proinsulin biosynthesis at the translational level. Recently, it has been shown that specific control of glucose‐induced proinsulin biosynthesis in the β‐cell resides in cis ‐elements in the 5′‐ and 3′‐untranslated regions of preproinsulin mRNA itself. After proinsulin is translocated into the lumen of the rough endoplasmic reticulum, correctly folded proinsulin can then be delivered in transport vesicles to the cis ‐Golgi apparatus in an ATP‐dependent process. The major site for processing of the proinsulin to biologically active insulin is the immature granule compartment of the β‐cell. Production of insulin occurs via limited proteolysis of the proinsulin precursor molecule, which is catalyzed by two endopeptidases, proprotein convertase 2 (PC2) and proprotein convertase 3 (PC3) (also known as PC‐1), and an exopeptidase, carboxypeptidase‐H (CP‐H). Similar to their deleterious effects on insulin gene expression, chronic hyperglycemia and hyperlipidiemia impair proinsulin biosynthesis.

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Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

Cited by 52 publications

References 30 publications

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Engineered cell lines for insulin replacement in diabetes: current status and future prospects

Insulin Gene Expression and Biosynthesis

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