Subcellular Localization of Proinsulin to Insulin Conversion in Isolated Rat Islets

Sorenson, Robert L.; Steffes, Michael W.; Lindall, Arnold W.

doi:10.1210/endo-86-1-88

Cited by 58 publications

(32 citation statements)

References 25 publications

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“…Although radioautographic studies on the biosynthesis and transfer of secretory products in a wide variety of cells strongly support the hypothesis that the secretory products are confined within the cell to the rough endoplasmic reticulum, Golgi apparatus and, finally, to the secretion granules (6,7,(20)(21)(22), even more definitive proof of this strict compartmentation has been obtained in some instances by histochemical localization techniques (23). On the other hand, fractionation studies with disrupted cells have never succeeded in demonstrating this degree of sequestration (4,24), presumably due to the fragility of some of the subcellular membranous structures, which release their contents during homogenization of the tissue. We might then first inquire as to whether evidence was obtained in these experiments for the existence of alternate or nongranule routes of insulin secretion from the islets.…”

Section: Methodsmentioning

confidence: 99%

“…Studies on the biosynthesis of insulin in intact islets of Langerhans isolated from rat pancreas have shown that single chain proinsulin is synthesized in the rough endoplasmic reticulum, presumably on membrane-bound polyribosomes, and is subsequently transformed within the islet cells to insulin, the principal storage product of the beta granules (1)(2)(3)(4). The subcellular site of transformation of proinsulin to insulin has been localized tentatively to the Golgi apparatus and/or the newly formed secretory granules (3,5).…”

Section: Introductionmentioning

confidence: 99%

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Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of langerhans

Sando¹,

Borg²,

Steiner³

1972

J. Clin. Invest.

106

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A B S T R A C T Islets of Langerhans isolated from rat pancreas were incubated at 370C(95% 02/5% C02) in buffered medium containing 1.0 mg/ml glucose and leucine 8H for 1 hr (1st hr), washed, and incubated for an additional hr (2nd hr) in low glucose medium (0.5-1.0 mg/ml) containing unlabeled leucine. A portion of the islets was then extracted with acid-ethanol and the remainder were transferred to medium containing 3.0 mg/ml glucose and incubated for 2 hr (3rd and 4th hr) at 370C. The medium was exchanged at 30-min intervals and portions of the islets were extracted at the 3rd and 4th hr. The total amounts and specific activities of the proinsulin and insulin in the islet extracts and medium samples were determined after fractionation on Biogel P-30 columns in 3 M acetic acid.Maximal release of newly synthesized insulin occurred between the 3rd and 4th hr of incubation, confirming the results of Howell and Taylor (Biochem. J. 102: 922. 1967). The high glucose medium increased the secretion of insulin approximately three to fourfold. The ratio of the specific activities of the insulin in the medium to that in the islets was about 1/1 during incubation in low glucose, but it increased to 2.5/1 during incubation with high glucose. The peak occurred at the 3rd hr, i.e., 1 hr after exposure to high glucose. The ratio of labeled proinsulin to insulin was slightly lower in the medium than in the islets. Addition of sufficient cycloheximide after the 1st hr to inhibit protein synthesis did not inhibit these responses. The specific activity of the proinsulin in the medium was about the same as that in the islets, and both were about 10-fold higher than the specific activity of the insulin. High glucose did not alter the proinsulin specific activity, which tended to decline throughout the period of observation. With cycloheximide present, the concentration of proinsulin in the islets steadily declined while the specific activity of proinsulin remained high, indicating that the proinsulin pool is small and is turning over rapidly. In terms both of amount and radioactivity proinsulin amounted to 6-7% on a molar basis of the insulin in both the medium and the islets. Addition of dibutyryl cyclic 3',5'-adenosine monophosphate (DBCAMP) (0.002 M) with high glucose during the postlabeling period slightly increased the rate of insulin secretion (133% of control) but did not significantly alter the other parameters.The results suggest that while newly synthesized insulin and proinsulin may be preferentially secreted to a slight degree, about 90% of the insulin released during 3 hr in response to glucose, or to glucose and DBCAMP, is derived from pre-existing granule stores. There were no indications of the existence of independent or nongranule pathways of insulin or proinsulin secretion.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of langerhans

Sando¹,

Borg²,

Steiner³

1972

J. Clin. Invest.

106

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“…A 1-ml aliquot of the homogenate was removed; the remaining suspension was carried through differential centrifugation to obtain subcellular fractions. The speeds and times for centrifugation were identical to those we have used with islet tissue to obtain a secretion granule fraction (2). The conditions are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Preliminary Evidence for a Microsomal Precursor to Human Parathyroid Hormone

Wong

Lindall

1973

Proc. Natl. Acad. Sci. U.S.A.

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Human parathyroid glands from a patient with chronic renal failure were used to study in vitro biosynthesis of immunoreactive parathyroid hormone. Parathyroid glands were incubated in the presence of [3Hlleu-cine, and classical subcellular fractions were prepared. Peptides from acetic acid extractions of the fractions were separated on Sephadex G-75 Superfine. Eluates were assayed for immunoreactive parathyroid hormone by a double-antibody technique. Leucine was incorporated into several large immunoreactive peptides of parathyroid hormone having estimated molecular weights of >25,000, 18,000, and 13,000. The large forms of immunoreactive parathyroid hormone were prominant in the microsomes. The secretion granule fraction, as well as the patient's serum, contained principally immunoreactive parathyroid hormone with an apparent molecular weight of 10,000.We have previously demonstrated the existence of immunoreactive peptides of human parathyroid hormone (PTH) that have apparent molecular weights larger than that of highly purified bovine PTH, which has a molecular weight of 9500 (1). Furthermore, we have found in studying the subcellular fractions of human parathyroid tissue that the immunoreactive peptides larger than purified bovine PTH can be localized to the microsome fraction of the gland, which contains protein synthetic components of the cell. From this information we have postulated that human PTH is synthesized by way of a large precursor molecule that is subsequently cleaved to a peptide with molecular weight of about 10,000 and which may be the principal form of PTH stored in the parathyroid's secretory granules. To test this hypothesis, we have incubated human parathyroid tissue with [8H]-leucine to study the in vitro synthesis of this possible precursor to human PTH. METHODSIn Vitro Incorporation of [8HjLeucine. Human parathyroid tissue was obtained fresh at the time of subtotal parathyroidectomy on a patient with chronic renal failure. The tissue was kept briefly on ice until dissected free of connective tissue and minced into pieces of about 1 mm. The tissue was incubated in 5 ml of incubation medium which was Kreb's bicarbonate buffer (gas phase 95% 02-5% C02), containing 1.3 mM calcium, 1.1 mM magnesium, 15 mg/liter of each of 16 amino acids without leucine, 100 ,Ci of [3H]leucine and 1% bovine-serum albumin. The incubation vessel was gassed with 95% 02-5% CO2 and then placed into a shaking water bath at 37°. After 30 min, the vessel was again gassed and Abbreviation: PTH, parathyroid hormone. Pellet from 10,000 X g for 20 min and mitochondrial fraction) F III (microsome fraction) Pellet from 100,000 X g for 60 min F IV Residual soluble fraction incubated for an additional 30 min. After 60 min of incubation, the incubation medium was removed and the tissue was washed twice briefly with ice-cold 0.25 M sucrose. The tissue was homogenized in 5 ml of ice-cold 0.25 M sucrose. A 1-ml aliquot of the homogenate was removed; the remaining suspension was carried through differential centrifugatio...

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“…Sorensen et al 27 have recently presented evidence supporting this hypothesis based on the distribution of labeled proinsulin and insulin in islet subcellular fractions. Such a mechanism would account for the retention of all of the proteolytic conversion products.…”

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confidence: 90%

Identification of Proinsulin and C-Peptide in Human Serum by a Specific Immunoassay

Melani

Rubenstein

Oyer

et al. 1970

Proc. Natl. Acad. Sci. U.S.A.

154

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Abstract. An immunoassay for the measurement of human proinsulin and C-peptide is described. By a combination of this system and an insulin immunoassay, proinsulin, insulin, and C-peptide were identified in acid-ethanol extracts of serum after gel filtration. In keeping with the results of biosynthetic studies, insulin and C-peptide were found to be present in equimolar amounts. The absolute values of serum proinsulin determined independently in the two assays corresponded closely.

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Subcellular Localization of Proinsulin to Insulin Conversion in Isolated Rat Islets

Cited by 58 publications

References 25 publications

Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of langerhans

Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of langerhans

Preliminary Evidence for a Microsomal Precursor to Human Parathyroid Hormone

Identification of Proinsulin and C-Peptide in Human Serum by a Specific Immunoassay

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