Summary
The purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned 32P-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000–1500 μg in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10–15 pg/ μg. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 1∶50 those in normal islets. Samples of RNA (20–50 μg) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 μg samples of RNA from brain or in 20 μg samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas was added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50–75 pg proinsulin mRNA/50 μg sample assayed if present. Because none was found, brain must have a concentration <1:6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.
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References
Rehfeld J (1978) Immunologie studies on cholecystokinin II. Distribution and molecular heterogeneity in the central nervous system and small intestine of man and hog. J Biol Chem 253: 4022–4030
Nakata Y, Kasaka Y, Segawa T, Yajimi H, Kitagawa K (1978) Substance P: regional distribution and specific binding to synaptic membranes in rat central nervous system. Life Sci 22: 259–268
Vale W, Ling N, Rivier J, Villarreal J, Rivier C, Douglas C, Brown M (1976) Anatomic and phylogenetic distribution of somatostatin. Metabolism 25: 1491–1494
Havrankova J, Schmechel D, Roth J, Brownstein M (1978) Identification of insulin in rat brain. Proc Natl. Acad Sci 75: 5737–5741
Le Roith D, Hendricks SA, Lesniak MA, Rishi S, Becker KL, Havrankova J, Rozensweig JL, Brownstein MJ, Roth J (1983) Insulin in brain and other extrapancreatic tissues of vertebrates and nonvertebrates. Adv Metab Disorders 10: 303–340
Eng J, Yalow RS (1980) Insulin recoverable from tissue. Diabetes 29: 105–109
Howell SL, Taylor KW (1967) The secretion of newly synthesized insulin in vitro. Biochem J 102: 922–927
Cordell B, Bell G, Tsicher E, DeNoto FM, Ullrich A, Pictet R, Rutter WJ, Goodman HM (1980) Isolation and characterization of a cloned rat insulin gene. Cell 18: 533–543
Kakita K, Giddings SJ, Rotwein PS, Permutt MA (1983) Insulin gene expression in the developing rat pancreas. Diabetes 32: 691–696
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Extraction of RNA from tissues enriched in ribonuclease. Biochemistry 24: 5294–5299
Aviv H, Leder P (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci 69: 1408–1412
Lacy PE, Kostianovsky M (1967) Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35–39
Giddings SJ, Chirgwin J, Permutt MA (1981) The effects of fasting and feeding on pre-proinsulin messenger RNA in rats. J Clin Invest 67: 952–960
McMaster GK, Carmichael GG (1977) Analysis of single- and double-stranded-nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci 74: 4836–4838
Seed B (1982) Diazotisable arylamine cellulose papers for the coupling and hybridization of nucleic acids. Nucleic Acids Res 10: 1799–1810
Wahl GM, Stern M, Stark R (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci 76: 3683–3687
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237–251
Chick WL, Shields W, Chute RN, Lauris U (1976) A transplantable rat insulinoma. Diabetes 25: 344
Villa-Komaroff L, Efstratiadis A, Broome J, Lomedico P, Tizard R, Naber S, Chick W, Gilbert W (1978) A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci 75: 3727–3731
Gazdar AF, Chick WL, Oie HK, Sims HL, King DL, Weir GC, Lauris V (1980) Continuous, clonal, insulin- and somatostatinsecreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci 77: 3519–3523
Rozensweig JL, Havrankova J, Lesniak MA, Brownstein M, Roth J (1980) Insulin is ubiquitous in extrapancreatic tissues in rats and humans. Proc Natl Acad Sci 77: 572–576
Steiner DF (1976) Proteins. In: Fasman GD (ed) Handbook of biochemistry and molecular biology, Vol.3. CRC Press, Cleveland, Ohio, pp 378–381
Chan SJ, Episkopou V, Zeitlin S, Karathanasis SK, MacKrell A, Steiner DF, Efstratiadis A (1984) Guinea pig pre-proinsulin gene: an evolutionary compromise? Proc Natl Acad Sci 81: 5046–5050
Smith JE, Summers MD (1980) The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem 109: 123–129
Alwine JC, Kemp DJ, Parker BA, Reiser J, Renart J, Stark GR, Wahl GM (1979) Detection of specific RNA's or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol 68: 220–242
Giddings S, Chirgwin J, Permutt MA (1982) Effects of glucose on insulin mRNA in rats in vivo. Diabetes 31: 624
Guyette WA, Mutusik RJ, Rosen JM (1979) Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17: 1013–1023
McKnight GS, Palmiter RD (1979) Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J Biol Chem 254: 9050–9058
McKnight GS, Lee DC, Hammarplardh D, Finch CA, Palmiter RD (1980) Transferrin gene expression: Effects of nutritional iron deficiency. J Biol Chem 255: 144–147
Peavy DE, Taylor JM, Jefferson LS (1978) Correlation of albumin production rates and albumin mRNA levels in livers of normal, diabetic, and insulintreated diabetic rats. Proc Natl Acad Sci 75: 5879–5883
Reaven EP, Gold J, Reaven GM (1979) Effect of age on glucosestimulated insulin release by the β-cell of the rat. J Clin Invest 64: 591–599
Giddings SJ, Orland MJ, Weir GC, Bonner-Weir S, Permutt MA (1985) Impaired insulin biosynthetic capacity in a rat model for non-insulin dependent diabetes: studies with dexamethasone. Diabetes 34: 235–240
McConaughy BL, McCarthy BJ (1967) The interaction of oligodeoxynucleotides with denatured DNA. Biochim Biophys Acta 149: 180–189
Bell GI, Merryweather JP, Sanchez-Pescador R, Stempien MM, Priestley L, Scott J, Rall LB (1984) Sequence of a cDNA clone encoding human pre-proinsulin-like growth factor II. Nature 310: 775–777
Dull TJ, Gray A, Hayflick JS, Ullrich A (1984) Insulin-like growth factor II precursor gene organization in relation to insulin gene family. Nature 310: 777–781
Hudson P, Haley J, Cronk M, Shine J, Niall H (1981) Molecular cloning and characterization of cDNA sequences coding for rat relaxin. Nature 291: 127–131
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Giddings, S.J., Chirgwin, J. & Permutt, M.A. Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues. Diabetologia 28, 343–347 (1985). https://doi.org/10.1007/BF00283141
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DOI: https://doi.org/10.1007/BF00283141