Effects of hagfish insulin in the Atlantic hagfish, Myxine glutinosa the in vivo metabolism of [14C]glucose and [14C]leucine and studies on starvation and glucose-loading

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

47,414-425

(1982)

Effects of Hagfish Insulin in the Atlantic Hagfish, Myxine glutinosal The in Vivo Metabolism of [14C]Glucose and [l%]Leucine and Studies on Starvation and Glucose-Loading STEFANO.EMDIN Department

of Pathology,

of UmeB,

University

S-901 87 UmeB,

Sweden

Accepted July 29, 1981 In hagfish, starved for 1 month at 4-6”, blood glucose decreased (1.9 to 0.8 mM) and serum insulin values diminished (2.2 to 1.1 nM). More than 90% of the glycogen in the liver and skeletal muscle was consumed, whereas protein and triglyceride contents were far more stable. The serum levels of amino nitrogen, triglycerides, and free fatty acids were all decreased after starvation. The results indicate that skeletal muscle glycogen was the prime source of energy. In starved hagtish, hag&h insulin (0.1 /&g body weight) induced an approximate twofold stimulation of the synthesis of glycogen, protein, and neutral lipids from [W]glucose after 33 hr at 4-6”. Most of the incorporation was detected in muscle glycogen. No insulin effects were seen in the liver. In analogous studies with [W]leucine, hagfish insulin likewise stimulated the synthesis of glycogen and protein in muscle and protein synthesis in the liver. Despite the evidence of insulin-stimulated syntheses, the total glycogen and protein contents in muscle and liver were unaltered after 33 hr. Likewise, no insulin effects were seen on blood glucose, amino nitrogen, triglycerides, or free fatty acids. Only about 10% of the radioactive dose was incorporated into the muscle and liver, and the metabolic effects of insulin contributed to only about half of this fraction. Glucose-loading increased the serum insulin value from 0.7 to 1.9 t&f. Pretreatment of hagtish with a mixture of glucose and amino acids for 3 days before and after injection of the above isotopes resulted in an increase of the serum insulin values. These endogenously elevated insulin levels were sufftcient to stimulate the incorporation of the label into muscle glycogen and protein. The stimulations were similar to those obtained in experiments with exogenous insulin. It was concluded that the physiological role of insulin in skeletal muscle was similar to what has been observed in higher animals although the quantitative effects of insulin in the hag&h appeared smaller than in higher vertebrates.

In comparison with the extensive information available on insulin physiology in mammals, the corresponding knowledge in lower vertebrates is limited. Most of the work concerning insulin physiology in such animals deal with the effects of an alien insulin on changes in blood metabolites and tissue constituents. Results of such investigations have been reviewed by Epple (1969), Falkmer and Patent (1972), Epple and Lewis (1973), Leibson et al. (1976), and

Thorpe (1976). Tashima and Cahill (1968) were the first who, in a teleost bony fish, used a somewhat different technique. They studied the metabolism of radioactive metabolic precursors in the toadfish, Opsunus tau, in the presence of insulin, and showed that the hormone i.a. accelerated the incorporation of labeled amino acids into skeletal muscle protein. Later, Ince and Thorpe (1976a) and Inui et ul. (1978) reported similar results in the Northern pike, Esox lucius, and in the Pacific hagfish, Eptut* Preliminary reports of parts of this study were retus stouti, respectively, indicating that given at the 2nd International Insulin Symposium at stimulation of muscle protein synthesis is Aachen, Federal Republic of Germany, September 4-7, 1979 (Emdin and Falkmer, 1980) and in a recent among the most original actions of insulin. review (Falkmer and Emdin, 1981). The Myxinoid cyclostomes probably rep414

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@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

PHYSIOLOGY

OF HAGFISH

INSULIN

415

resent the most original vertebrates (cf. (van Handel, 1%5). The colorless acid hydrolysates

Falkmer et al., 1974; &berg, 1976). In the were neutralized with NaOH, and samples were taken for glucose determination and scintillation counting. Atlantic haglish, Myxine glutinosa, it has The recovery of added glycogen was 90.6 * 14.2 (SD, been found that glucose loading resulted in n = 5), taking into account that the molecular weight of hyperglycemia lasting l-2 days (Falkmer free glucose is about 11% higher than the molecular weight of a glucose unit in glycogen. The contaminaand Matty, 1966). A crude hag&h insulin preparation evoked a long-lasting but tran- tion of free tracer carried over in the final glycogen precipitate when [i4C]glucose and [r4C]leucine was sient hypoglycemia 2-3 days after the in- added to, and extracted with nonradioactive muscle jection (Falkmer and Matty, 1966), whereas pieces was 0.2 and 0.05%, respectively. higher doses were needed to obtain a simiDetermination of triglycerides and free fatty acids (WA). Duplicate lipid extracts from both liver and lar effect with bovine insulin. In contrast, skeletal muscle tissue from each hagtish were prepared the metabolic sensitivity of Eptalretus to Dole and Meinertz (l%O). The spillover stouti to bovine insulin was found to be at according of added [W]glucose or [‘*C]leucine into the organic least as great as that in most higher verte- phase was less than 0.03%. The recovery of [3H]tribrates (Inui and Gorbman, 1977a). High olein extracted in the presence of nonradioactive tissue doses of alloxan have been shown to cause by this procedure was 97.5 2 0.5% (SD, n = 5), islet cell necrosis and hyperglycemia in whereas only 6.9 f 1.4% (SD, n = 4) of [3H]glycerol extracted. In experiments with animals injected Myxine (Falkmer and Winbladh, 1964). De- was with labeled tracers, the extracted radioactive organic spite these findings hyperglycemia was not phase was counted. Lipid extracts from fed and evoked by isletectomy (Falkmer and Matty, starved animals that were not injected were dried under a stream of nitrogen. Each residue was mixed 1966) or by administration of anti-insulin with 0.5 ml buffer (50 n&’ sodium phosphate, 4 mM serum (Falkmer and Wilson, 1967). magnesium sulfate, and 0.35 mM sodium dodeSince the results of these early studies cylsulfate; pH 7.0) and incubated for 1 hr at 25”. Rewith hag&h were different from the situa- covery of [3H]triolein dried with nonradioactive lipid tion in mammals, this study was performed extracts from tissues and suspended in this buffer was in order to further investigate possible ef- 78.4 2 6.2% (SD, n = 3). Tissue triglycerides were assayed in the fed and starved animals by means of an fects of insulin in the Atlantic hagfish. MATERIAL

AND METHODS

Hagtish insulin was purified and quantitated as previously described (Emdin et al., 1977). Uniformly labeled D-[W]gJucose and L-[“Clleucine (both 330 mCi/mmol), as well as 63Ni (as chloride; 10 mCi/mg Ni), were from the Radiochemical Centre, Amersham, England. Other chemicals were of analytical grade. Substances to be injected were dissolved in a physiological buffer (Emdin, 1981) with 0.25% bovine serum albumin (Fraction V, Sigma Chemicals, St. Louis, MO). Solutions were adjusted so that the injected volume was around 0.1 to 0.2 ml. Buffer alone was given to the control animals. Determination of glucose and glycogen. Glucose was determined by means of the glucose-oxidaseperoxidase procedure, using a commercially available kit (Glox, Kabi, Stockholm, Sweden). Serum and standards were deproteinized with alkaline zinc sulfate (c$ Havu, 1969). Glycogen from duplicate liver and muscle samples was extracted for 15 min with hot 30% (w/v) KOH, precipitated with ethanol, and hydrolyzed with 0.6 N HCl at 100” for 2 hr, essentially according to Good et al. (1933). Saturated sodium sulfate was added to minimize losses when precipitating with ethanol Chemicals.

enzymatic kit (Boehringer, Mannheim, FRG). Duplicate 50-~1 serum samples were assayed enzymatically for triglyceride levels with another enzymatic kit (Calbiochem, San Diego, Calif.) Triolein was used as a standard. Duplicate 10-/d samples of haglish serum were assayed for FFA with 63Ni according to Ho (1970). Palmitic acid in heptane was used as a standard, and the results were expressed as microequivalents palmitic acid per liter serum. The recovery of [Wlpalmitic acid extracted with serum was 72.9 ? 3.2% (SD, n = 4).

Determination of a-amino nitrogen ((Y-NHJV) and protein. Duplicate lo-p1 hagtish serum samples in

small capped polypropylene tubes were deproteinized by treatment with 240 ~1 3% (w/v) sulfosalicylic acid for 2 min at 100”. After centrifugation, 50 ~1 of the supematant was combined with 400 ~1 of 0.2 M sodium borate buffer, pH 9.0 (fmal pH 8.5), whereafter 100 ~1 fluorescamine (Hoffmann La Roche, Nutley, N.J.), 0.25 mg/ml in acetone, was added. Fluorescence was measured in an Aminco-Bowman spectrophotofluorometer (390/480 nm). The fluorescence obtained was taken as a measure of free amino acids in serum. As a standard, glycine, 5.36 mg/ml, was used, corresponding to 1 mg/ml of (Y-NH2N. This method is adapted from Bdhlen et al. (1973). Fluorescamine,

416

STEFAN

which reacts only with primary amines, gives in comparison with determinations of (Y-NH2N using ninhydrin practically no interference with ammonia and urea. Tissue protein from duplicate liver and skeletal muscle samples was determined by homogenizing the tissue in 2 X 1 ml 5% (w/v) trichloroacetic acid. The centrifuged precipitate was washed twice with 2 ml trichloroacetic acid and the supernatant was discarded. The samples containing precipitated protein were allowed to dissolve in 1 ml 1 M NaOH at 25” for 16 hr. After centrifugation, an aliquot was taken for protein determination according to Lowry, using bovine serum albumin as a standard, and another fraction was taken for scintillation counting. Less than 0.01% of either [‘Vlglucose or [“‘C]leucine, added to nonradioactive homogenized tissue, were carried over to the washed protein pellet. Insulin assays. Duplicate SO-p1 serum samples were assayed for insulin by radioimmunoassay, and charcoal-treated insulin-free hagfish serum was included in the standards (Emdin and Steiner, 1980). Radioactivity measurements. Radioactive samples were counted in a toluene-based scintillation fluid with 10% (w/v) Triton X-100 in a Packard Tri-Carb or an LKB scintillation counter. The [1251]activity was counted in a Packard 5320 gamma counter. Quenching of the samples was corrected by internal standardization. Animals and experimental design. Hagfish, 20-40 cm long, weighing 20-60 g, were used. They were caught at all seasons except summer at the Kristineberg Marine Biology Station at Fiskebackskil on the Swedish West Coast. Traps, baited with rotten fish, were used as previously described (Gustafson, 1935; Falkmer et a/., 1976). The hagfish were usually kept in large aquaria with running deep-sea water at 4-8” and at about 3.3% salinity. Hagfish with large visible eggs were not used; otherwise no attention was paid to different sexes of the expeirmental animals. Myxine glutinosa shows incomplete sexual differentiation and determination, and a kind of rudimentary hermafroditism exists (cf Falkmer et al., 1974). About 1 to 2 hr after catching the hagfish, some were bled from their large caudal venous sinuses (Falkmer and Matty, 1966) and killed. Pieces of liver and skeletal muscle were excised and weighed. These samples were frozen for later analysis. It was checked by inspection of the guts that these animals had been feeding. Animals that did not appear healthy, as well as those with large liver tumors (Falkmer et al., 1976) were omitted from the study. In the starvation experiments the guts of the animals were manually emptied, and the hagfish were then transferred to smaller aquaria and kept in running sea water at 4-6” in the dark for 30 days when samples from blood and tissues were taken for subsequent analysis. Hagtish used in experiments involving administration of radioactive isotopes. insulin, and glucose were

0. EMDIN transported by railway to the laboratory in Umea. A maximum of 30 fish were kept in 200-liter aquaria with recirculating sea water at about 4-6” and 3.3% salinity. The animals were starved for 10 days before the experiments began. During the experiments with radioactive tracers the hagfish were kept in individual aquaria. Injections were given into the dorsal muscles at different sites for each substance and care was exercised not to enter the peritoneal cavity or the gut. From hagfish injected with insulin and/or radioactive isotopes, six pieces of liver and skeletal muscle, each about 0.2 g, were excised and weighed on pieces of aluminum foil and kept frozen at -20” until duplicate determination of glycogen, lipids, and protein were carried out. To try to synchronize the maximum blood levels of im injected radioactive isotopes and insulin, their rates of resorption into the circulation were measured at 4-6” (Fig. 1). The two isotopes reached their maximum levels within 3 hr. Insulin was much more slowly resorbed and cleared from circulation. This marked difference was paid attention to in the subsequent experiments. Groups of hagtish were injected with hagfish insulin (0.1 pg/g body wt) or buffer. After 9 hr the animals were given either [“C]glucose or [‘YZ]leucine (both usually 0.15 &i/g body wt). The hagtish were then kept for another 24 hr before the experiment was terminated and samples were taken for analyses.

FIG. 1. Rate of resorption of insulin and isotopes in hagfish. Insulin (O), 0.1 &g body wt, was injected intramuscularly. The serum insulin values were then determined after repeated sampling at the times indicated. [‘VZ]Glucose (0) and [‘*C]leucine (A), both at 0.15 &i/g body wt, were injected intramuscularly. The serum radioactivity was then calculated after repeated sampling at the times indicated. For all substances n = 5. Bars indicate SEM.

PHYSIOLOGY

OF

HAGFISH

fish are generally low, and those observed here are in agreement with those reported

Statistics. Differences between the mean experimental and control values wereconsidered significant if P < 0.05, using the two-tailed Student’s t test.

earlier (Falkmer and Matty, 1966). The amount of (u-NH2N, reflecting free amino acids, also decreased during starvation, but to a more moderate extent. Likewise, triglycerides and FFA dropped to about half their initial values. This was true also of the insulin levels which were reduced significantly upon starvation.

RESULTS Liver and Muscle Glycogen, Triglycerides, in Fed and Starved Hagjish

Protein,

and

The levels of glycogen, protein, and triglycerides in hagfish liver and skeletal muscle are given in Table 1. Glycogen was found in liver and muscle at levels below 1%. Upon 1 month’s starvation at 4”, more than 90% of the glycogen was consumed. Protein and triglyceride levels remained far more stable. A slight, but significant, reduction of both protein and triglyceride levels was seen in the liver parenchyma. Surprisingly, a reverse situation was observed in the skeletal musculature.

Serum Insulin Values after Glucose Loading

In order to further substantiate the findings of different serum insulin concentrations in fed and starved hagfish, and to give additional aspects on some concomitant observations on hagfish insulin secretion in vitro (Emdin, 1982), serum insulin values were measured in glucose-loaded hagfish. The hagfish were fasted for 10 days at 4-6” and then injected with either buffer or 0.5 g/kg glucose im. Serum samples were taken after 3 hr, and the insulin values were assayed. The mean serum level of insulin in fasted buffer injected hagfish was 0.70 + 0.07 n&Y (SEM, n = 17) which is lower (P < 0.01) than the fasting value given in Table 1. It should be emphasized, however, that the

Serum Contents of Glucose, a-NH,N, Triglycerides, FFA, and Insulin in Fed and Starved Hag&h

In serum all substances studied were reduced during starvation (Table 1). It is known that blood glucose levels in the hag-

MEAN

417

INSULIN

TABLE 1 CONTENTS OF GLYCOGEN, PROTEIN, AND TRIGLYCERIDES IN THE LIVER, SKELETAL MUSCLE, SERUM OF FED HAGFISH, AND OF HAGFISH AFTER I MONTH OF STARVATION AT 4-6”

AND

Contents f SEM” Tissue Liver

Substance GWxen

Mhw)b

Protein (&mg) Triglycerides (PELglmg)

Fed 4.38 f 84.93 2 4.15 2

0.52 (43) 3.45 (43) 0.20 (35)

0.33 lr 0.03 (36) 56.55 r 4.22 (44) 3.28 k 0.21 (39)

<0.005 <0.005 <0.005

0.64 (43) 2.95 (43) 0.24 (28)

0.31 + 0.03 (33) 135.63 k 2.81 (41) 2.11 + 0.22 (39)

<0.005 CO.005 CO.005

0.76 56.7 4.08 129.8 1.10

CO.005 CO.05 CO.005 CO.005 CO.005

Muscle

Glycogen b&x4 Protein (&mg) Triglycerides (&mg)

8.04 ” 114.95 t 1.45 k

Serum

Glucose (mM) a-NHoN (mg/liter) Triglycerides (g/liter) FFA (peqiliter) Insulin (WV)

1.89 84.1 8.26 222.0 2.16

a Number of hag&h b Tissue wet weight

in parentheses. throughout.

P

Starved

?I * k -c k

0.12 13.2 0.24 14.2 0.20

(35) (40) (31) (41) (40)

f 2 t k -r-

0.11 4.5 0.34 9.0 0.12

(33) (40) (32) (39) (38)

418

STEFAN

0. EMDIN

lower insulin values were obtained in animals injected with buffer and handled more often than the group left starving for a month. Since catecholamines inhibit insulin release in the hagfish (Emdin, 1981), it is possible that stress reactions could have depressed the insulin values. The mean insulin concentration of glucose-injected animals was 1.90 +- 0.17 n&Y (SEM, n = 18, P < 0.005). Thus, glucose stimulates insulin release in viva, and the insulin concentration matches the concentration seen in newly fed hagtish (Table 1).

14C-label roughly twofold into glycogen, protein, and lipids. In the liver, lipids had the highest specific activity, whereas in muscle the highest specific activity was found in glycogen and lipid counts were low (Table 2). Insulin stimulated the incorporation from [14C]leucine into liver protein, whereas the effect on liver glycogen was more equivocal (Table 2). Somewhat surprisingly, both labeled leucine and glucose were incorporated into liver glycogen and protein with similar preference and efficiency, whereas only negligible incorporation occurred into lipids. The effects exerted by hagfish insulin on the metabolism of [14C]leucine in muscle were similar to the effects seen when labeled glucose was employed (Table 2). Hence, the highest specific activity was found in muscle glycogen. Hardly any measurable incorporation into muscle lipids occurred. Skeletal muscle comprises some 50% of the hagfish body weight (A. Mattison, personal communication) and the liver only

Effects of Hagfish Insulin on [14C]Glucose and [14C]Leucine Incorporation into Liver and Muscle Glycogen, Protein, and Lipids

In the liver, the radioactivity from [i4C]glucose was incorporated into glycogen, protein, and lipids, but no obvious effect of insulin was seen on the specific activity (Table 2). In skeletal muscle, however, insulin stimulated the incorporation of TABLE EFFECTS INCORPORATION

2

OF HAGFISH INSULIN (0.1 pLg/g BODY WEIGHT im) ON [%]GLUCOSE INTO GLYCOGEN, PROTEIN, AND UPIDS IN LIVER AND SKELETAL

in vivo AT 4-6”,

DURING

33 hr

[L4C]Incorporation W-Labeled Compound

Tissue

Substance

Glucose

Liver

Glycogen Protein Lipid Glycogen Protein Lipid

Muscle

Leucine

Liver

Muscle

Glycogen Protein Lipid Glycogen Protein Lipid

Saline-buffer-injected Hagtish 3.29 0.33 26.52 10.83 0.06 3.76

2 k 2 k 2 2

0.51” o.07b 5.21’ 4.31 0.004 0.21

3.66 2 1.02 0.38 2 0.06 0’ 1.24 + 0.29 0.03 + 0.002 0

a Disintegrations per minute (dpm) per microgram glycogen. * dpmlpg protein. o dpm/mg wet weight of tissue. d ns, no statistically significant difference. ” Almost no incorporation into lipids occurred.

AND [14C]L~~~~~~ MUSCULATURE, STUDIED

+ SEM (n = 10) Hag&h-insulin-injected Hagiish 5.16 0.49 30.38 27.39 0.15 5.76

k f + k it 5

1.40 0.09 7.90 1.11 0.024 0.57

5.58 t 0.91 0.78 -c 0.09

P nsd ns ns

<0.005 <0.005 co.025 ns <0.005

0

14.53 t- 4.21 0.16 2 0.033 0

co.005 <0.005

PHYSIOLOGYOF about 4% (Fange, 1973). Hence, when considering the total incorporation of label from either [‘*C]glucose and [14C]leucine into liver and skeletal muscle, it is found that, regardless of the tracer, 60-70% of the recovered label was found in muscle glycogen and 20-30% was incorporated into muscle protein. The liver, as a whole, incorporates 10% or less. Effects of Hagfish Insulin on Total Glycogen and Protein Contents in Liver and Muscle

HAGFISHINSULIN (?SEM)

a-NH2N

419 levels were 57.4 + 6.7

mg,Mer in the buffer-injectedcontrols and 70.9 5 5.2 mglliter in the insulin-injected hagfish. The corresponding values for triglycerides were 6.51 + 0.72 and 6.00 + 1.90 g/liter, respectively. For the FFA contents the values were 114.6 + 16.2 and 129.4 * 54.4 peq/liter, respectively. All these values were within the range observed in fed and fasted hagfish (Table 1). The disappearance rates of the radioactive tracers from the serum were not significantly affected by insulin under either of these experimental conditions since the mean (?SEM) radioactivities in the serum for [14C]glucose were 27.2 t 3.6 dpm x 10m4/ml in the controls (n = 10) and 29.2 ? 3.5 dpm x 10m4/ml in the insulin-injected hagfish (n = 10). The corresponding values for [14C]leucine were 6.9 t 1.5 dpm X IOe4/ml (n = 10) and 3.4 * 1.8 X 10P4/ml (n = lo), respectively.

In none of the above experiments could any significant insulin effects be seen on total glycogen or protein in liver or muscle. Thus, in the same 20 buffer-injected hagfish the mean liver and muscle glycogen content (?SEM) were 1.75 + 0.32 and 2.75 ? 0.47 pglmg wet wt, respectively, and in the 20 animals receiving an im injection of hagfish insulin the corresponding values were 1.61 + 0.40 and 2.54 ? 0.58, respectively. For total protein, the liver and muscle contents Experiments with Hagfish Pretreated with Glucose and Amino Acids were 114.2 ? 10.7 and 120.1 ? 7.1 pg/mg wet wt, respectively, in the controls, and An experiment was set up to test whether 92.2 ? 7.2 and 111.7 + 3.6 pglmg wet wt, it might be possible to induce a more prorespectively, in the insulin injected hagfish. found insulin effect if the metabolism of the hagfish was kept above “starvation level.” Effects of Hagfish Insulin on Glucose, Hagfish were given injections (im) of glu(Y-NH~N, Triglycerides, FFA, cose (0.5 mg/g body wt) and a mixture of 15 [‘“Cl Glucose, and [‘“Cl Leucine L-amino acids (without leucine, arginine, or in Blood proline; total dosage: 0.25 mg mixture/g The experiments with radioactive tracers body wt) at 8-hr intervals 3 days before and described above gave no indication of sig- during the insulin administration. [14C]Glunificant insulin effects on measured serum case (0.45 &i/g body wt) and [‘4C]leucine constituents. Thus, the mean serum glucose (0.15 @/g body wt) were used as labeled value in the buffer-injected hagfish, previ- precursors. The dose of [‘4C]glucose was ously starved for 10 days at 4-6”, was 1.37 increased in order to compensate for an 2 0.19 mM @EM, n = 20) and that of the otherwise expected lower specific activity insulin-injected hagfish was 1.25 2 0.14 due to the injections of unlabeled glucose. mM (SEM, n = 20) at the end of the ex- Otherwise, the design was identical to that periment. The fasting glucose level was sig- of preceding experiments. nificantly higher than that in the starvation There were no differences between the experiments described in Table 1; a fact controls and the insulin-treated animals, that might be due to stress evoked by han- neither in their specific activities (Table 3) dling the hagfish and to differences in the nor in their absolute contents of glycogen length of the starvation time. The mean and protein in liver and skeletal muscle

420

STEFAN

0. EMDIN

TABLE EFFECTS INCORPORATION in viva

3

OF HAGFISH INSULIN (0.1 &g BODY WEIGHT im) ON [%]GLUCOSE AND [‘TILEUCINE INTO GLYCOGEN, PROTEIN, AND LIPIDS IN LIVER AND SKELETAL MUSCULATURE, STUDIED AT 4-6”, DURING 33 hr,~~ HAGFISH PRETREATED WITH GLUCOSE AND A MIXTUREOF 15 NATURAL AMINO ACIDS

[WlIncorporation W-Labeled Compound Glucose

Substance

Liver

Glycogen Protein Lipid Glycogen Protein Lipid

5.40 0.78 28.3 23.2 0.43 26.3

Glycogen Protein Lipid Glycogen Protein Lipid

5.41 k 1.01 0.72 L 0.09 0 15.93 ? 1.55 0.17 + 0.01 0

Muscle

Leucine

Saline-buffer-injected Hagfish

Tissue

Liver Muscle

k 1.47Q 2 0.15* 2 3.97’ + 4.6 + 0.05 -+-3.9

f SEM (n = IO) Hagfish-insulin-injected Hagfish 6.22 0.84 24.9 22.1 0.34 24.9

2 0.88 2 0.13 -+ 2.12 * 5.43 * 0.06 + 2.1

6.49 k 0.92 0.57 * 0.19 0 13.38 t 1.13 0.16 2 0.01 0

P

nsd ns ns ns ns ns ns ns ns ns

u Disintegrations per minute (dpm) per microgram glycogen. b dpmlpg protein. c dpm/mg wet weight of tissue. d ns, no statistically significant difference.

(data not shown). The data on specific ac- hagfish. In addition, the serum levels of tritivities in liver and muscle show that glycerides and FFA did not change (data [14C]leucine was incorporated into the tis- not shown). The serum radioactivity of sues in a manner similar to the insulin[14C]glucose (73.8 + 9.4 vs 66.9 + 5.1 dpm stimulated animals described in Table 2. x 10M4/ml) were similar in the controls and When comparing the specific activities of the experimental animals. The specific acmetabolized [14C]glucose in the two ex- tivity of [r4C]glucose in blood was considperiments, incorporation into glycogen was erably lower in this experiment when comquantitatively similar, and liver and muscle pared with the previous one. protein, along with muscle lipid, all had inIt is important to note that the serum increased their specific activities, despite the sulin values in both groups of glucose and lower specific activity of the substrate in amino acid injected hagfkh were signitiblood (see below). cantly above the fasting insulin level in the In the blood, the serum insulin level in hagfish, and that the serum insulin value in the 20 saline-buffer-injected control hagfish the control group was roughly equal to the was 1.92 2 0.15 n&f (?SEM) and 31.8 + value seen in newly fed or glucose-loaded 2.40 nM in the same number of insulinanimals. injected hagfish after 33 hr observation time DISCUSSION at 4-6”. In contrast, there were no signifiThe islet organ of the Atlantic hagfish is cant differences in the blood glucose (6.09 -+ 0.62 vs 5.65 -+ 0.73 mM) or cu-NHzN (95.6 the first to appear in evolution. The islet consists of insulin and so? 4.1 vs 82.8 k 5.25 mg/liter) contents be- parenchyma matostatin cells only (cf. Van Noorden tween the controls and the insulin-injected

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and Falkmer, 1980). The primary and ter- the blood glucose for prolonged periods of time since hepatectomized Pacific hagfish tiary structure, as well as several biolgoical keep their glucose and a-NH2N at normal effects of hagfish insulin are known (cf. values for at least 1 month (Inui and GorbFalkmer and Emdin, 1981). The radioimman, 1977b). It is likely that upon starvamunoassay for hagfish insulin (Emdin and Steiner, 1980) opened the possibility to in- tion beyond 1 month other tissue constituents may provide the energy required for vestigate hagfish insulin release (Emdin, 1981) and its physiological role. Studies of survival. Moreover, additional energy may be provided by other tissues, for example, this kind may give an idea of the development of insulin release and peripheral ef- the scattered fat pads in the visceral peritofects of insulin in higher animals. These neum, which undergo atrophy during starprocesses and mechanisms could have been vation (Emdin, unpublished observation). The preferential utilization of carbohypresent in less complicated forms in early drate derived energy during starvation in vertebrates (cf. Van Noorden and Falkmer, 1980). Gorbman (1980), however, pointed the haglish is in accordance with observaout that structural and functional features tions made in some teleosts. Hence, Ince found in the endocrine systems in Agnatha and Thorpe (1976b) found that in starved of today have had as much time to evolve Northern pike both liver and muscle glycoand adapt as the entire vertebrate group has gen decreased, whereas the levels of protaken for its phyletic differentiation. Con- tein and lipid were stable. Similar results sequently, these features cannot a priori be have been reported by Inui and Yokote assumed to represent conditions that (1974) when studying the eel, Anguilfa existed in their Silurian progenitors, the juponica. Others have, however, reported Ostracoderms. Myxine glutinosa and Ep- that either protein or fat may be used as the tatretus stouti are adapted to a life in the prime source of energy during starvation bottom mud, where the temperature is (Kamra, 1966; Butler, 1968; Patent, 1970; around 5” and darkness is almost complete Dave et al., 1975). The fasting insulin values observed here (cf. Falkmer et al., 1974; Inui et al., 1978). Only little is known about their feeding were somewhat higher than those reported habits (Gustafson, 1935), but hagfish can be in 10 different teleost species (Patent and kept unfed in the laboratory for as long as Foa, 1971; Thorpe and Ince, 1976) and a 14 months (Inui et al., 1978). cyclostome, the river lamprey (Plisetskaya et al., 1976), where values around 0.1 to 0.4 Effects of Starvation nM were found. These determinations Starvation drastically reduced the glycowere, however, based on heterologous ingen content in both hagfish liver and sulin radioimmunoassays, with one excepskeletal muscle (Table 1). Hence, muscle tion, viz., cod insulin, which was estimated glycogen is the most important source of to be around 1 nM. It is likely that the insuenergy during 1 month of starvation. After lin values in the other species were underinitial hyperglycemia, blood glucose levels estimated. Thus, the fasting insulin values in are kept quite constant in starving hagfish some teleost fish and cyclostomes appear to in captivity (Falkmer and Matty, 1966). The be higher than in mammals (Goodner and blood glucose is presumably kept constant Porte, 1972). Despite the possibility that the through glycogenolysis rather than glu- catching of hagfish is likely to cause stress coneogenesis in muscle since no decrease and release of catecholamines, thus supof muscle protein or triglycerides was ob- pressing insulin release (Emdin, 1981), the served (Table 1). The hagfish liver appears insulin levels in newly fed, as well as in to be of minor importance in maintaining glucose loaded hagfish were roughly dou-

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bled. Such a low insulin secretory response was also found in some fish, both in vitro (Patent and Foa, 1971) and in vivo (Ince and Thorpe, 1976~). This could indicate a less sensitive control of insulin regulated metabolism in lower vertebrates compared to that in mammals. Muggeo et al. (1979a) have reported that insulin receptors (from blood cells with unknown physiological significance) from a wide range of species, including teleost fish and the Atlantic hagfish (Muggeo et al., 1979b), have the same rank order of preference for species variant insulins in vitro. Hence, the insulin receptor appeared functionally more conserved throughout evolution that insulin, and regardless of the species the most potent insulins have the highest receptor affinities. This concept is in contrast with earlier observations in vivo (Falkmer and Matty, 1966; Falkmer and Wilson, 1967; Ince and Thorpe, 1974) where the species-specific insulins were the most potent. However, it is not possible to extrapolate the observed receptor affinities on blood cells to the complex effects of different insulins on the whole animal. The elevated fasting insulin values seen in marine vertebrates may be a way to compensate for an unfavorable receptor affinity as has been suggested in the guinea pig. (cf. Muggeo et al., 1979a). Compensation may also be brought about by changes in receptor number per unit membrane area (Muggeo et al., 1979a).

0.

EMDIN

studying the effects of codfish insulin (0.1 pg/g) on the metabolism of [14C]glucose and [14C]glycine in the Northern pike, Ince and Thorpe (1976a) found an increased incorporation of radioactivity into muscle protein and lipid, but no effects of insulin were seen on muscle [14C]glycogen synthesis. In their study of the effects of insulin on [14C]glytine metabolism in the Pacific hagfish, Inui et al. (1978) found that bovine insulin (0.02 &g) augmented both skeletal muscle glycogen and protein synthesis. They also observed that gluconeogenesis from [14C]glytine, measured as the appearance of uncharged 14C-label in blood, was inhibited by insulin. In none of the above in vivo studies was there any change of the absolute concentrations of glycogen, lipid or protein, and the effects of insulin on the metabolism of isotopes in the liver were absent or inhibitory. In the Atlantic hagfish, 60-70% of the total label incorporated was found in muscle glycogen, and this seems to meet with the animal’s requirements for energy during fasting, and here the glycogen-promoting ability of insulin appears to be of great quantitative importance. Leucine was chosen as tracer since it was expected to be a source primarily for protein synthesis. In higher animals, alanine is the most important amino acid for gluconeogenesis in the liver (Felig et al., 1970). In the hagfish, label from [14C]leucine was, however, found in the radioactive glycogen fraction. This observation requires further investiExperiments with Radioactive Isotopes gation since leucine is considered a ketoThe ability of hagfish insulin to stimulate genie amino acid in vertebrates. In fact, reskeletal muscle glycogen and protein syn- gardless of which labeled substrate that was thesis in the hagfish (Table 2) is in accor- used, both the liver and the muscle seemed dance with earlier observations. Hence, in to handle the two isotopes quite similarly the toad&h (Tashima and Cahill, 1968) a with regard to their final destinies (Table 2). mixture of bovine and porcine insulins (4 Despite the observed insulin effects on pg/g) caused a twofold increase of the me- muscle, no change of the absolute values of glycogen and protein was observed. This tabolism of [14C]glucose into muscle protein indicates that the amount of synthesized and glycogen and enhanced the disappearance rate of [14C]glucose from the blood products during 24 hr are much smaller than without affecting total blood glucose. When the total amounts, and supports the idea of

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a relatively slow metabolism. It is not glucosefrom the blood into the tissues,at known to what extent the radioactive pre- leastpartly stimulatedby insulin, and a @ucursorswere oxidized or excreted in the case efflux from tissues as a result of urine. Consideration of the given dose in relation to its fractional utilization by the liver and skeletal muscle, shows that only about 10% of the injected isotopes appear to have been recovered in these tissues. Insulin stimulation contributed to about half of this tissue radioactivity. This could possibly explain why no significant difference of serum radioactivity between the insulin treated animals and the controls was found, since a small difference may have escaped detection. Recycling of the label could also have occurred and would complicate the interpretation (Ince and Thorpe, 1976a). The unaltered a-NH2N value is in contrast to the findings in the pike (Thorpe and Ince, 1974), as well as to the findings in the Pacific hagfish (Inui and Gorbman, 1977a). These authors observed a lowering of CYNH,N after insulin administration. Likewise, insulin lowers serum FFA in several teleost fish (Minick and Chavin, 1972; Ince and Thorpe, 1975). In the Atlantic hagfish no such effect could be seen. That the blood glucose values were not lowered by insulin (after 33 hr) is not surprising, since Falkmer and Matty (1966) found that insulin-induced hypoglycemia required at least 48 hr for its onset. Maybe longer observation times are needed to see any effects on a-NH,N and FFA. The time course of metabolic events in the hagfish requires further consideration. The hagfish islets respond to glucose by secreting insulin within 20 min in vitro (Emdin, 1982) and no more than 3 hr in viva. Exogenous insulin reaches its peak serum value within 12 hr, and the stimulatory effects of insulin on muscle metabolism are noted within 24 hr. Yet, hypoglycemia, even after huge doses of exogenous insulin requires 48 hr to develop (Falkmer and Matty, 1966). During starvation glucose homeostasis could be maintained by a combination of movement of

gluconeogenesis. It is further speculated that: If the insulin stimulated transport of glucose is of small magnitude and, in addition, the presumed inhibitory effect of insulin on gluconeogenesis takes days to develop, the time required for development of hypoglycemia in Myxine will be long. If the number of glucose carriers in the muscle membrane is rather low it is reasonable that they cannot rapidly cope with high concentrations of glucose. This could also explain the slow clearance of glucose from the circulation observed in hag&h by Falkmer and Matty (1966). The attempt to induce an enhanced metabolism and an increased response to insulin was largely unsuccessful (Table 3). Certainly, with [14C]leucine there was no enhancement. Due to the different specific activities of [14C]glucose in blood in the experiments described in Tables 2 and 3, comparisons must be made with caution. It is, nevertheless, possible that an enhancement of lipid and protein synthesis occurred in muscle, perhaps due to the increased substrate levels. The endogenous insulin levels in the control animals were increased above fasting levels, due to exogenous glucose administration. This could explain the lack of significant differences with respect to metabolic turnover of 14C-label between insulin-treated animals and their controls. If so, this is taken as evidence favoring the idea that the increase of endogenous insulin by glucose administration or feeding is sufficient to bring about the metabolic effects observed with much higher doses of exogenous insulin in the Atlantic hagtish. ACKNOWLEDGMENTS I am indebted to Dr. S. Falkmer and Dr. A. Thorpe for valuable discussions and suggestions. Thanks are due to the staff at the Kristineberg Marine Biology Station, Fiskeblickskil, Sweden. Technical assistance was provided by Mr. Lars-Ake Damber. This work was supported by grants from the Swedish Medical

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Research Council (Project 12X-718), and the Swedish Diabetes Association, and the Medical Faculty of the University of Umefi.

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