Subcellular distribution of I131-insulin in striated muscle

Subcellular Distribution of P 31 -Insulin in Striated Muscle P . M . EDELMAN, M .D . and I . L . SCHWARTZ, M .D .

Cincinnati, Ohio,

New York, New York

the past five years we have been studying the distribution of I'll-insulin in D rat striated muscle . Methods for fractionation of

intracellular organelles (Fig . 3) . Over 50 per cent of the tubules consisted of a four component sarcolemma with varying numbers of associated mitochondria . (Fig . 4-9 .) The sarcolemma forms the boundary of the muscle cell and appears in the light microscope as a homogeneous membrane, 1 .0 µ thick . In the electron microscope the sarcolemma is found to consist of four components . Visualized from outside to inside, (1) the outermost component consists of a meshwork of fine filaments and is approximately 100 A in diameter ; (2) next to the outer layer is a component of approximately 300 A in diameter consisting of striated collagen filaments ; (3) the next inward component consists of a structureless layer measuring 300 to 500 A in width known as the amorphous zone ; and (4) the innermost component, adjacent to the cytoplasm, consists of a plasma membrane measuring 100 A in thickness and thought to represent a bimolecular leaflet of lipid bounded on either side by protein monolayers [23] . Along the inner margin of the plasma membrane vesicles may be observed in electron micrographs of both the intact muscle cell and in the sarcolemmal preparations . (Fig . 8 and 9 .) These vesicular structures are interpreted by Porter and Palade [7] and Peachey [/7] to be remnants of the triads (T tubules and terminal cysternae) of the sarcotubular system . Apparently, therefore, our method yields the sarcolemma and the T tubular extensions of its innermost component, the plasma membrane . It was also noted that exposure of freshly isolated sarcolemmal membranes to ATP resulted in their prompt shrinkage suggesting that the sarcolemma may contain a contractile protein .

URING

muscle tissue into mitochondrial, microsomal and soluble fractions were modified to our requirements, and technics for preparation of sarcolemmal membranes and muscle cell nuclei were devised . This report summarizes several aspects of these studies ; the findings are discussed in relation to recent advances in our understanding of muscle physiology [1-6] and ultrastructure [7--14] which may be pertinent to the action of insulin in this tissue . THE SARCOLEMMA

The accumulating evidence for the importance of the cell membrane as a major site of insulin action in striated muscle [75-21] led us to study the binding of insulin to the sarcolenima . In the course of these studies it was necessary to prepare sarcolemmal membranes in high yield and with minimal intracellular contamination . We developed a procedure for harvesting a relatively pure preparation of muscle cell "ghosts" [22] . In brief, this procedure involves shredding and shearing of muscle tissue into cell segments, filtration to remove connective tissue, attenuation of the attachments of the sarcolemma to the sarcoplasmic reticulum by incubation in dilute salt solutions, ejection of the cytoplasmic contents from the sarcolemmal envelope by centrifugation, dispersion of the actomyosin gel which traps the sarcolemmal membranes by treatment with adenosine triphosphate (ATP), and finally concentration by flotation-sedimentation procedures . The state of the final product obtained ranged from incompletely evacuated tubules (Fig . 1 and 2) to those totally devoid of

* From the Departments of Physiology and Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio ; The Medical Research Center, Brookhaven National Laboratory, Upton, Long Island, New York, and 'rhe Mount Sinai Medical and Graduate Schools, New York, New York . This study was supported by U . S . Public Ilealth Service Grant No . AM-10080-01 of the National Institute of Arthritis and Metabolic Diseases and by the U . S . Atomic Energy Commission.

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FIG. 1-3. Phase contrast photomicrographs of muscle cells in progressive stages of intracellular evacuation . Note myofibrillae escaping from the ends . Figure 3 is that of the sarcolemmal tubule . Original magnification X 425 . FIG . 4 .

Myofibrillae found in the supernate following settling of the sarcolemmae . Original magnification X 1,062.

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Fic. 5 . Phase contrast photomicrograph of the sarcolemma . The black spots observed are mitochondria . Original magnification X 1 .062 . MUSCLE CELL NUCLEI

After the preparation of sarcolemmal membranes was found to be satisfactory for studies of the intracellular distribution of insulin, we developed a method for isolating muscle cell nuclei in aqueous media [31] . This involves mincing and homogenization of muscle tissue,* filtration through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and centrifugation in a 2 .14 M sucrose-salt solution containing 0 .7 mM ATP . The purity and integrity of the nuclei obtained were evaluated by (1) phase contract microscopy, (2) electron microscopy, (3) chemical methods, and (4) enzyme assays . Phase contrast microscopic examination of the nuclei show them to be intact, elongated and frequently multinucleolated . (Fig . 10 .) Electron microscopic examination revealed that the nuclear membranes were generally intact, doublelayered, minimally contaminated with mitochondria and free of other cytoplasmic constituents . The chromatin appeared less dense than that found in the nucleus of an intact cell . (Fig . 11 .) The ratios of DNA : protein and RNA : DNA * The use of a Potter-Elvehjem type homogenizer with a Delrina (acetal resin) pestle and carefully controlled pestle-to-glass clearance proved to be critical in this procedure. vol. . 40,

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found in skeletal muscle nuclei did not appear to differ significantly from those observed in nuclei obtained from other tissues 121-30] . These ratios as well as the low specific activity obtained for cytochrome C oxidase and rnyofibrillar ATPase indicated that, although there was a small amount of mitochondrial and myofibrillar contamination, the nuclear preparation had a relatively high degree of purity . To our knowledge this is the first isolation of skeletal muscle nuclei [31] . DISTRIBUTION OF 1131 INSULIN IN STRIATED MUSCLE

Rat femoral musculature was incubated in a Krebs-Ringer bicarbonate solution containing I 131 -labeled insulin .* The muscle was then removed from the medium and washed repeatedly with 0 .15 M saline solution in order to remove all loosely adsorbed radioactivity . t The tissue was then exposed to 10 -3 M N-ethylmaleimide,I homogenized and fractioneatd into mitochondrial, Inicrosomal, soluble, nuclear and sarcolemmal constituents . The microsomal and *The I 131-insulin employed was chromatographically pure and was employed within two to four days of purchase from the Abbott Co ., Oak Ridge, Tennessee . f The incubation medium and washing procedures are described in greater detail elsewhere [32] . 1 The tissue was bathed in N-ethylmaleimide to bind free SH,groups and to inhibit insulinase activity [38,39] .

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Electron micrograph of a portion of sarcolemmal tubule after the evacuation of myofibrillae . Mitochondria (M) and possibly triadic constituents (er) are observed within the intracellular matrix immediately below the plasma membrane (pm) . Electron micrographs of sarcolemmae illustrated were fixed in KMnO 4 or OsO4 and embedded for sectioning in Araldite® . Original magnification X 32,000 . FIG . 6 .

Electron micrograph of a section of sarcolemma . Components of the sarcolemma such as the plasma membrane (pm), amorphous zone (az), and filamentous strands occurring either singly (c) or in cable forms (cc) may be observed . Original magnification X 38,000 .

FIG . 7 .

8 . Electron micrograph of a section of sarcolemma found in the purest fraction of the preparation . Below the plasma membrane, vesicles (v) are frequently observed . Original magnification X 38,000 .

FIG .

Electron photomicrograph of the sarcolemma . The vesicular structures (V) probably represent elements of the triads (T tubule and terminal cysternae) which assume this appearance following fixation . pm = plasma membrane. Original magnification X 25,000 . FIG . 9 .

soluble fractions were obtained by differential centrifugation [33] ; the mitochondrial fraction was obtained by Bellamy's modification [34] of the method of Schneider and Hogeboom [33] . The purity of the cell fractions was evaluated by phase and electron microscopy and, in addition, by assay for cytochrome oxidase activity, succinic acid dehydrogenase activity, ATPase activity, DNA and RNA . The mitochondrial content of the nuclear fraction was /2000th of that of the whole homogenate as judged by the observed ratio of cytochrome C oxidase activity to DNA content . Similarly the myofibrillar contamination of the nuclear fraction amounted to only Y600th of the myofibrillar content of the whole homogenate as judged by the ratio of myofibrillar ATPase activity to DNA content. (Table i .) The sarcolemmal fraction consisted largely of the previously described four layered component AMERICAN JOURNAL O F MEDICINE

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Fro . 10 . Field of isolated intact nuclei (Ni) and partially dama,sed nuclei (N,) of striated muscle . 7 he majorits oI nuclei appear to be i atact, elongated and frequently rnultinucleolated . Original maguiheation X 2 .00(1, Fic . 11 . Electron micrograph of an isolated nucleus obtained from striated muscle . The nuclear membrane appears to be intact and double-layered . A nuclear bleb (R) may be observed at one pole . The nucleolus (NU) is clearly v°isihle . Mitochondria (M) may be seen at each pole . Fixed in osmium tetroxide and embedded in Araldite . Original magnifiratio-a X 12,000 .

with attached T tubules and varying degrees of mitochondrial contamination . A fifteenfold purification of the mitochondrial fraction was achieved as judged by measurements of the specific activity of succinic dehydrogenase ; however, the relative concentrations of RNA in the different fractions suggested that the mitochondrial fraction was somewhat contaminated by microsomes . The "inicrosomal' fraction was contaminated to a slightly higher degree by mitochondria on the basis of the same criteria . (Tables ii and in .) Electron microscopic examination also showed that there was less microsoinal contamination of the mitochondrial fraction than mitochondrial contamination of the inicrosomal fraction . Most of the radioactivity was found in two locations : (1) bound to the sarcolemma and (2) in the soluble fraction of the cell . (Table iv and vot .

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Fig . 12 .) The sarcolemina, which comprises less than 2 per cent of the cell mass, binds 40 per cent of the radioactivity ; the soluble fraction contains 50 per cent of the radioactivity . The combined mitochondrial and microsomal fractions, which comprise approximately 10 per cent of the muscle cell mass, contain less than 2 per cent of the radioactivity . The nuclear fraction, which also comprises less than 2 per cent of the cell mass, contains approximately I per cent of the radioactivity . The myofibrillar fraction, which constitutes over 60 per cent of the cell mass, contains only 4 to 5 per cent of the radioactivity . When the specific radioactivity of each fraction was compared to that of the whole homogenate the following ratios were calculated as an index of labeling : sarcolernma :whole homogenate, 4 to 8 ; soluble fraction : whole homogenate, 2 to 3 ; nuclei :whole homogenate, 0 .5 to



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TABLE I ENZYMATIC ASSAY OF RAT SKELETAL MUSCLE HOMOGENATES AND NUCLEI

Cytochrome C Oxidase * units/mg . proteint units/µg. DNA

Fraction Homogenate

13 .4 f 2 .1

(HI)

Nuclei

ATPase* units/mg. protein t

6 .2 f 1 . 3

0 .431 * 0 .095

units/µg . DNA

289 (147-366)$ 28 .9

0 .00289 + 0 .00096

125 (43-191)t

* Cytochrome c oxidase values are the average of five experiments ; ATPase values are the average of four experiments . t Units for cytochrome oxidase and ATPase and method of DNA determination have been described previously [31] . Protein determined by the Lowry method [35] . I Values in parentheses indicate the range .

TABLE II SUCCINIC DEHYDROGENASE ACTIVITY (SDH) OF RAT SKELETAL MUSCLE FRACTIONS *

SDH (units/mg . protein ± S .D .)t

Fractiont Homogenate Nuclear and myofibrillar Mitochondrial Microsomal

0 .187 f 0 .192 f 2 .55 ± 0 .994 ±

0 .076 0 .053 0 .99 0 .312

* Based on ten to twelve observations . t Rat femoral musculature was fractionated by differential centrifugation into mitochondrial, nuclear and myofibrillar fractions by the method of Bellamy [34] . The mitochondrial supernate was further fractionated into microsomal and soluble fractions by centrifugation at 105,000 g for 1 hour . t SDH was determined by a method previously described by us [36] However, in these experiments the reaction was started with reduced cytochrome c .

1 .0 ; mitochondria : whole homogenate, 0 .25 to 0 .5 ; microsomes :whole homogenate, 0 .25 to 0 .5 . Electrophoresis of the soluble fraction [62] showed that it contained free and intact I'll-insulin . However, when this fraction was incubated for 30 minutes at 37'c . the I'll-insulin was split into peptide components with the mobility of the A and B chains . (Fig. 13 .) A similar phenomenon has been described in liver and muscle extracts by Mirsky [38,39] and more recently by Tomizawa [40] in liver extracts . When muscle tissue is labeled with I'll-insulin and then treated with N-ethylmaleimide prior to homogenization, the soluble fraction no longer retains the ability to degrade insulin even after prolonged incubation . In order to determine the extent to which the insulin found in the soluble fraction of the muscle homogenate could represent an artifact of extracellular contamination, the entire procedure

TABLE III RNA DETERMINATION ON CELL FRACTIONS OF RAT STRIATED MUSCLE *

Fractionst

RNAt (pg ./mg . protein)

RNA (µg ./100 mg. tissue)

% Distribution

Homogenate Nuclei and myofibrils Mitochondria Microsomes Soluble

20 .3 17 .5 59 .4 139 .3 71 .8

256 70 8 .4 19 180

(100) 27 .4 3 .3 7 .4 70 .0

* Results are those of a typical experiment . t Refer to note on fractionation in Table II. t Fractions for RNA determination were precipitated with an equal volume of cold 20 per cent TCA, and the precipitate washed three times in 10 per cent TCA . RNA was then determined on the suspended precipitate by the method of Dische and Borenfreund [371 . AMERICAN

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'FABLE IV CHEMICAL PURITY AND RADIOACTIVITY OF RAT SKELETAL MUSCLE NU( ;LEI ISOLATED FROM I 131-INSULIN-INCUBATED 'TISSUE *

DNA µg . Tissue Fraction

mg. protein

c .p.m ./µg . DNA

c .p .m. /mg. protein

Distribution

Homogenate

5 .53 (3 .6-6 .8)f 27 .0 (16 .1-32 .6) 309 (246-385)

105 .3 (74-160) 10 .5 (6 .7-14 .3) 1 .01 (0 .6-1 .4)

563 (363-812) 285 (142-527) 306 (212-440)

(100) 10 .6 (8 .6---11 .6) 0 .992 (0 .6-1 .7)

(:rude nuclei Purified nuclei

* Average of five experiments . f Figures in parentheses represent the range .

was repeated employing inulin as a marker for extracellular fluid . The results of these experiments indicate that not more than 20 per cent of the insulin found in the soluble fraction could be accounted for in terms of extracellular contamination . BINDING OF 1

131 -INSULIN TO THE

SARCOLEMMAL PREPARATION

Fresh rat femoral muscle tissue was incubated for 30 minutes in bicarbonate-buffered media containing biologically active, chromatographically homogeneous I 131 -insulin (0 .5 to 2 .0 atoms I per mole of insulin) . The tissue was then washed three times in normal saline solution, exposed to 10 -3 M N-ethylmaleimide for 3 minutes, washed three times in 0 .01 N HCl and six to eight times in 8 M. urea-0 .015 N HC1 in order to remove electrovalently bound (salt-linked and

hydrogen bonded) radioactivity . When the radioactivity of the supernatant fluid was at background levels, the sarcolemmal membranes were isolated by the procedure described . Procedures for removal of electrovalently bound radioactivity were then repeated with the sarcolemmal membrane preparation, and the radioactivity of this preparation was assayed . About 25 per cent of the original radioactivity remained associated with the sarcolemmal membranes . This residual radioactivity was then released by exposing the sarcolemmal membranes to mlild conditions which split disulfide bonds but do not hydrolyze peptide bonds (0 .1 M cysteine in 8 M urea, pH 8 ; 0 .1 M 13-mercaptoethylamine in 8 M urea, pH 8 ; 0 .05 M sulfite in 6 M urea, pH 7 ; 0 .05 M sulfite in saturated phenyl mercuric hydroxide, pH 9) . The residual labeling of the membranes now amounted to from 2 to 8 per

+(AChain)

-(B Chain)

1000 volts Microsomes Mitochondria Origin AFTER INCUBATION FOR 30 MIN .

180 minutes 8M Urea Pyridine Acetic and

13

12

CELL CONTENT (% of Homogenate ) Mitochondria 5-10 Sarcolemma < 2 Nucleus <2% Myofibrils > 60 Microsomes 2-4 % Soluble <30%

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Specific Radioactivity (c .o.m .l/mg . Prot. ) of Fractions Compared to Homogenate I. 2. 3. 4.

Sarcolemma 4-8x Hom . Soluble Fr. 2-3x Hom. Nuclei z-1 of Hom . Mitochondria and Microsomes

a-2 Hom .

FIG . 12 . Distribution of 1 13'-insulin in

subcellular fractions of striated muscle . Fro . 13 . Electrophoretic pattern of soluble fraction (schematic) .



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J3 mercapto- added at ethanol time-0 - - - Cysteine J3 mercapto-' added at arrow ethanol (time-210min .) Cysteine

40-

U

r=ci

d

a Incubation Time in Minutes

Fio . 14. Specific radioactivity of preparations of 1131insulin-labeled sarcolemmal membranes at various times after incubation in media containing thiol reagents and in control media. The curves show data averaged from ten experiments . Note the fall in the specific activity of the sarcolemmae after addition (arrow) of the thiol reagents . The specific activity has been normalized to a zero time pre-incubation value of 100 c .p.m. per mg, tissue protein . The difference between the labeling of the membranes in thiol-containing and control media is a minimum measure of radioactivity bound to the sarcolemma through disulfide linkage .

cent of the original level . The same quantitative release of radioactivity was observed when membranes were incubated with thiol compounds (0 .1 M cysteine in 8 M urea, pH 8 or 0 .1 M 0-mercaptoethylamine in 8 M urea, pH 8) from the beginning of the experiment . (Fig. 14 .) These findings suggest that insulin is attached to the sarcolemma through electrovalent and covalent (disulfide) linkages . Control studies, in which kidney slices were labeled with I' 31 -insulin and subjected successively to procedures which disrupt ionic, hydrogen and disulfide bonds, showed that no significant radioactivity had become attached to kidney tissue through disulfide linkage . (Fig . 15 .) This finding suggests the value of further exploring the specificity and significance of hormone-tissue disulfide bonding in the over-all mechanism of insulin action, but it does not imply that a thiol-disulfide interchange is one of the reactions which comprises intrinsic hormonal activity .

0 90 210 330 Incubation time ( minutes )

Specific radioactivity of F$'-insulin-labeled kidney slices at various times after incubation in media containing 0 .1 M cysteine and in control media. Data averaged from twelve experiments . Note that the curves relating specific activity of the tissue to incubation time continue to be identical in both control and experimental media . No significant release of radioactivity was observed after the addition of cysteine (arrow) to the control media . The specific activity has been normalized to zero time pre-incubation value of 100 c .p .m, per mg. tissue protein. FIG . 15 .

It is noteworthy that the radioactivity released from the sarcolemmal membranes in all phases of these experiments proved to be precipitable by trichloroacetic acid (TCA) and associated with intact insulin, large peptide subunits and possibly insulin polymers. No I131 was found to be released as inorganic iodide . Since insulin possesses two disulfide links between the A and B chains, it was possible that these interchain disulfide bonds were being cleaved by the thiol !or sulfite) reagents . However, when a sample of the I131-insulin employed in our experiments was oxidized with performic acid [41] and the chains isolated by countercurrent distribution [42], 56 per cent of the radioactivity was associated with the A chain and 44 per cent with the B chain . Therefore, if our final (thiol or sulfite) treatment of the labeled sarcolemmal membranes resulted only in cleavage of the interchain disulfide bonds, and the B chain was thereby split off from the A chain, the amount of radioactivity released AMERICAN JOURNAL O F MEDICINE



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I 13 I-Insulin Distribution in Striated Muscle-Edelman, Schwartz could amount to 44 per cent at most . Since the observed release actually amounted to 80 per c ent . i t would appear that the hormone-tissue reaction involved formation of a disulfide bond between the membrane and the half-cystine moiety in position 6 and/or position 11 of the A chain of the hormone . However, it should be emphasized again that although a thiol-disulfide interchange involving hormone and target tissue appears to take place in the case of insulin as well as in the case of nevi ohypophyseal peptides, our recent experience with oxytocin and vasopressin analogs lacking a disulfide bond [43a] indicates that this reaction is not essential for intrinsic activity although it may control hormonal potency by orienting and appropriately fixing the hormone to the receptor [4.3h ] . INSULIN-LIKE EFFECTS OF CYCLIC AMP

Evidence that adenosine 3',5'-monophosphate (cyclic AMP) is involved in the action of a number of hormones [44-48] led us to study the effect of this nucleotide on the glucose uptake of the rat diaphragm . In preliminary studies [49] we found that rat hetnidiaphragms exposed to 10' M cyclic AMP in vitro take up significantly more glucose from a glucose-containing medium than do paired untreated (control) hemidiaphragms, whereas, adenosine-5'-monophosphate (5'-AMP) does not significantly increase glucose uptake by this tissue . (Fig . 16 .) Similar experiments conducted with insulin show that the glucose uptake of hemidiaphragms treated with insulin is significantly greater than the glucose uptake of hemidiaphragms treated with cyclic AMP even when the concentration of cyclic AMP in the experimental medium is elevated to maximal levels . The quantitative difference between the effect of insulin and that of cyclic AMP on the diaphragm may in part be due to the relatively low permeability of the tissue to cyclic AMP [50] . We have also observed that the extracellular (inulin) space of rat femoral muscles treated with cyclic AMP is significantly less than that of paired control femoral muscles [49] . Creese and Northover [51], Fritz and Knobil [52] and Randle and Smith [.53,54] have shown that insulin decreases the inulin space in the rat diaphragm and also that this effect may be correlated with other parameters of insulin action . Before considering the possibility that the insulin-induced glucose transport in muscle invot . . 40,

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Paired

Control Insulin No . of Expts . 9-9

Paired

Control C-AMP 9 - 9

Paired

Control 5-AMP 4-4

Effect of insulin and nucleotides on glucose uptake in rat diaphragm .

Fie. 16 .

volves the mediation of cyclic AMP, further studies are necessary : effects on other parameters of insulin action must be tested, the relationship of insulin, epinephrine and cyclic AMP in muscle must be clarified, direct tissue analyses for cyclic AMP must be carried out, and the effects of several other nucleotides must be evaluated . COMMENTS

Almost ten years ago Krahl [55] proposed that an insulin-receptor interaction initiated a disturbance at the cell surface which was propagated throughout the cell . It was suggested that this propagated disturbance produced widespread structural changes leading not only to increased penetration of glucose and other substrates into the cell interior but also to modified substrate and cofactor availability and accordingly, to altered enzymatic activity . Hechter and Halkerston [56] proposed that the macromolecular assembly within the cell which could best serve as the primary network for Krahl's propagated disturbance was the membrane system of the endoplasmic reticulum . In support of this contention the latter investigators cited the demonstration by Barrnett and Ball [57] of dilated endoplasmic reticular cysternae in the cells of insulin-treated epidydimal fat pads and the similar observations of Luft and Hechter [56,58] on adrenal cortical cells perfused with ACTH . The localization of insulin at the sarcolemma is in accord with the idea that the hormone may be acting on the cell membrane and its T tubular invaginations (Fig . 17) which

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I 131-Insulin Distribution in Striated Muscle-Edelman, Schwartz

FIG. 17 . Transverse section of a glutaraldehyde-fixed white fiber to show the form and disposition of the T system . The section cuts across the Z line (Z) and so includes the middle of the T system cisterna . Openings or orifices of the T system (T) to the outside are shown at the arrow. Strands of sarcoplasm (asterisk) which penetrate into the T system from neighboring fibers may be o bserved . S R = sarcoplasmic reticulum . From Franzini-Armstrong, C. and Porter, K. R . J. Cell . Biol ., 22 : 675, 1964, [8] . Original magnification X 38,000 .

FIG . 18 . A three-dimensional schematic diagram illustrating the sarcoplasmic reticulum and its relationship to the myofibrils. The myofibrils show the light band (I) with the dense Z line (Z) at its center. The A bands (A) have a central light H zone (H) and denser outer regions where primary and secondary filaments overlap. At the center of each A band is a light zone (L) with a dark line (M) in its center . Triads consisting of two terminal cisternae (tc) and a centrally located transverse tubule (tt) are seen adjacent to the Z lines of the myofibrils . Connecting to the terminal cysternae are the intermediate cysternae (ic), and adjacent to the A bands they form the longitudinal tubules (It) . In the vicinity of the H zone in the center of A band, the longitudinal tubules join the fenestrated collar (fc) . glc = glycogen granules . From Peachey, L. D. J. Cell Biol ., 25 : 209, 1965 [11] .

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Fro . 19 . Longitudinal sections through triads and intermediate cisternac of frog sartorius muscle . A, the most common triad morphology : a single transverse tubule (tt) between the flat faces of opposing terminal cysternae (tc) . The intermediate cysternae (ic) connect to the terminal cisternae at points which are sometimes near the centers of the triads (arrow), and pass alongside the terminal cisternae and glycogen masses (gly) toward the A bands, where they join the longitudinal tubules (It) . The granular material within the terminal cysternae is not present in the intermediate cysternae . B, a pair of transverse tubules (tt) in a single "triad," an image encountered only infrequently . A longitudinal tubule is marked (It) . C, a "pentad" consisting of two terminal cisternae (tc), two transverse tubules (tt), and an additional central cysterna (cc) also encountered infrequently . From Peachey, L . D . J. Cell Biol ., 25 : 209, 1965 [11] .

may be regarded as the gateway to the sarcoplasmic reticulum [8-14] . (Fig . 18 and 19 .) Cellular ultrastructural components thought to be involved in extra- to intracellular solute translocations have been the subject of recent investigations [8-14] . Electron microscopic studies of striated muscle reveal the transverse tubules (central element of the triad) as an invagination VOL .

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of the sarcolemma [8,17] ; the lumen of this tubule, therefore, is continuous with the extracellular space [8,10] . (Fig . 17 .) There is evidence that the transverse tubule of the triad functions as a direct channel for the internal passage of electrolytes and other substances [12-74] . This transverse tubular system, which appears to be similar to the "special region" of



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I 131-Insulin Distribution in Striated Muscle-Edelman, Schwartz

Hodgkin and Horowicz [3] and the "interinediary space" of Adrian and Freygang [5], possesses the faculty of altering its capacity by dilation [11-14] . It has been shown that the transverse tubules (T tubules) swell when the muscle cell membrane is hyperpolarized by chloride ion withdrawal or by electrical stimulation [12-14] . If swelling of the T tubules is generally associated with hyperpolarization, the finding of Zierler [59,60] that insulin hyperpolarizes the muscle cell membrane would lead us to expect that insulin also dilates the T tubules . Such an effect on the T tubules of the triadic system could play a role in the extra to intracellular translocation of metabolic substrates such as glucose . The finding that both cyclic AMP and insulin diminish the inulin space of muscle without changing the total water content of the tissue would be expected if the triadic system and the muscle cell as a whole were swelling at the expense of the extracellular space . Our observation of a contractile element in the isolated sarcolemma [22] is in accord with Hechter's suggestion [56] that the membrane systems of the cell have components which function "peristaltically" as a mixing mechanism resulting in facilitated translocations of metabolic intermediaries and mobile macromolecules and, accordingly, in greater opportunities for interactions resulting in increased biosynthetic activity . The appearance of substantial amounts of free insulin in the soluble fraction of the muscle homogenate could be due to liberation during the fractionation procedure of hormone that may have been bound to extracellular constituents . Alternatively, this finding may mean that prior to homogenization of the tissue the hormone had gained access to and had been accumulated on or in the T-tubular system, sarcoplasmic reticulum or cell sap for ultimate release to functional and/or degradative sites . SUMMARY

Methods have been developed for isolating muscle cell nuclei and sarcolemmal tubules . With these methods it has been found that I131_ insulin is associated primarily with the sarcolemmal and soluble fractions of the muscle cell . I'll-insulin binds to the sarcolemma (and possibly its T tubular extensions) by electrovalent and covalent (disulfide) bonds, the latter involving the 6 and/or 11 positions on the A

chain . Cyclic AMP increases the glucose uptake of rat diaphragm and decreases the extracellular space of rat femoral muscle . It is suggested that insulin may act upon the T tubular extensions of the sarcolemmal membrane, swelling the transverse tubular system, increasing the intracellular compartment and decreasing the extracellular compartment of muscle tissue . These effects would stretch the sarcolemma and expand pathways by which water, glucose and amino acids enter the cell . A hormone-sensitive, possibly contractile system of this type involving the membrane systems of the cell as a whole could also explain effects of insulin on protein synthesis by facilitating translocations and interactions of substrates, cofactors, enzymes and mobile templates within the cell, This proposal is an extension of the cytoskeletal hypothesis of Peters [67], as are the related ideas of Krahl [55] and Hechter [56] which have been advanced to account for the secondary reactions of hormone action . REFERENCES 1 . HUXLEY, A. F . and TAYLOR, R . E . Local activation of striated muscle fibers . J. Physiol ., 144 : 426

1958 . 2 . ANDERSSON-CEDERGREN, E . Ultrastructure of motor end plate and sarcoplasmic components of mouse skeletal muscle fiber as revealed by three-dimensional reconstructions from serial sections . J.

Ultrastruc . Res ., 3 (supp. 1) : 1, 1959 . 3 . HODGKIN, A . L . and HoROwICZ, P. The effect of sudden changes in ionic concentrations on the membrane potential of single muscle fibers . J.

Physiol ., 153 : 370, 1960. 4 . FAWCETT, D. W. and REVEL, J . P. The sarcoplasmic reticulum of a fast-acting fish muscle . J. Biophys. & Biochem . Cytol., 10 : 89, 1961 . 5 . ADRIAN, R . H. and FREYGANG, W. H . The potassium and chloride conductance of frog muscle membrane . J. Physiol ., 163 : 61, 1962 . 6 . FALK, G . and FATT, P . Linear electrical properties of striated muscle fibres observed with intracellular electrodes . Proc. Royal Soc. London, s.B.,

160 : 69, 1964 . 7 . PORTER, K . R. and PALADE, G . E. Studies on the endoplasmic reticulum, III . Its form and distribution in striated muscle cells . J. Biophys. & Biochem .

Cytol ., 3 : 269, 1957 . 8 . FRANZINI-ARMSTRONG, C. and PORTER, K . R . Sarcolemmal invaginations constituting the T system in fish muscle fibers. J. Cell Biol ., 22 : 675, 1964. 9 . HUXLEY, H . E. Evidence for continuity between the central elements of the triads and extracellular space in frog sartorius muscle . Nature, 202 : 1067,

1964. 10 . ENDO, M. Entry of a dye into the sarcotubular system of muscle . Nature, 202 : 1115, 1964 . 11, PEACHEY, L . D . The sarcoplasmic reticulum and transverse tubules of the frog's sartorius . J. Cell

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