GENERAL AND COMPARATIVE ENDOCRINOLOGY 50, 374-317 (1983)

Insulin-like Peptides in the Lobster Homarus americanus II. Insulin-like Biological Activity

BRENDA SANDERS’

College of Marine Studies, University of Delaware, Lewes, Delaware 19958

Accepted June 16, 1982

The in vitro incorporation of [“Clglucose into glycogen in lobster muscle was used to measure insulin-like biological activity. Glycogenesis was significantly increased by the same hepatopancreas eluate which was previously found to have the greatest insulin im- munoreactivity. Hemolymph but not gut extract also increased the rate of glycogenesis.

Most reports of invertebrate insulin-like activity have involved the use of mamma- lian bioassay systems to measure ILA in invertebrate tissue extracts (Wilson and Falkmer, 1965; Davidson et al., 1971). Un- fortunately, these studies offer no informa- tion about possible invertebrate target tis- sues. De Martinez et al. (1973) were the first to use crude extracts from the oyster Ostrea edulis to enhance glycogenesis in oyster gill tissue demonstrating that mol- luscan gill tissue has insulin receptors, and that insulin can increase the activity of its glycogen synthetic system. Later it was found that insulin also increased glucose uptake in molluscan muscle (Plisetskaya et al., 1978a).

In this bioassay lobster muscle was used as the target tissue for two reasons. Muscle is considered a primary target for the action of insulin in other animals (Harper et al., 1977). Also, since crustacean muscle stores substantial amounts of glycogen (Van Weel, 1970), there is a good possibility that the muscle is a target for insulin in vivo in Homarus americanus.

The highest insulin immunoreactivity of comparable molecular weight to bovine in- sulin is present in hepatopancreas eluate (Sanders, 1983). The present study was

1 Present address: Duke University Marine Labo- ratory, Beaufort, N.C. 28516.

undertaken to determine if this im- munoreactive insulin (IRI) had insulin-like biological activity (ILA). Specifically, I measured the biological activity of hepato- pancreas eluate from chromatographic frac- tions that had previously been radioim- munoassayed (Sanders, 1983). ILA was then compared with IRI. Gut and hemo- lymph extracts were also assayed for the presence of biologically active insulin.

MATERIALS AND METHODS Animal care, tissue procurement, and insulin ex-

traction techniques have been previously described (Sanders, 1983). Insulin-like activity (ILA) was as- sayed by the in vitro incorporation of [14C]glucose into glycogen (de Martinez et al., 1973). ILA was measured in bovine insulin, tissue extracts, and controls. Lob- ster perfusion fluid (Cole, 1941), gelatin (2 mg ml-‘), and glucose (4 mg ml-‘) were combined and used as the incubation medium. Each assay tube contained 0.5 ml incubation fluid, [Wlglucose (0.05 &i), and ap- prox 30 mg of freshly dissected lobster abdominal muscle. All tubes were incubated and shaken for 20 hr at room temperature under constant gassing (5% CO, in 0,).

The glycogen extraction procedure of Good et al. (1933) was modified. First, the assay tissue was di- gested with an equal volume of 60% KOH and heated in a boiling water bath for 0.5 hr. Oyster glycogen (0.5 mg) was added as a carrier, and each assay tube was mixed on a vortex shaker. Glycogen was then precipi- tated by adding 1.25 vol of 96% ethanol, shaking, and leaving overnight at 4”. After all tubes were centri- fuged at 1200g for 30 min and the supernatant fluid decanted, the precipitate was dissolved in 1 ml of water and transferred to a scintillation vial containing 10 ml Aquasol (New England Nuclear). Each vial was

374 0016~6480/83 $1.50 Copyright @ 1983 by Academic Press, Inc. AlI rigbts of reproduction in any form reserved.

LOBSTER INSULIN-LIKE ACTIVITY 375

shaken and counted in a Beckman scintillation counter for 10 min.

Data from the assay were analyzed using the Stu- dent t distribution (Sokal and Rolf, 1969). Significance levels were set at the 95% probability level unless otherwise specified.

RESULTS

Bovine insulin increased [14C]glucose in- corporation into [14C]glycogen in lobster muscle tissue (Fig. 1). An increase in rate of glycogenesis was observed as bovine insu- lin was increased from 0.05 to 2.5 pg ml-‘. Glycogenesis was not significantly en- hanced with 0.05 pg ml-’ of bovine insulin (t = 1.78, P > 0.10). However, an increase in [14C]glycogen biosynthesis with bovine insulin concentrations of 0.21, 0.63, and 2.50 pg ml-’ were significant at the 9% confidence interval (t = 3.42, P < 0.01; c = 3.09, P < 0.01; I = 5.00, P < 0.001, respec- tively) .

The biological activity of H. americanus tissue extracts differed (Table 1). The high- est levels of activity per weight of extract were measured in hepatopancreas extract. Gut extract did not increase the incorpora- tion of [14C]glucose into glycogen. Several attempts to use higher concentrations of gut extract in this bioassay resulted only in greatly increasing the variability of the rep- licates. A significant increase in the rate of

0 0.1 1.0 10.0 BOVINE INSULIN (99)

FIG. 1. Mean [Wlglycogen deposition in lobster muscle tissue as a function of concentration of bovine insulin. Each mean is calculated from 8 to 10 repli- cates; bars represent one standard deviation.

glycogenesis was observed when hemo- lymph extract was added to the incubation medium. These data suggest that there is a protein with insulin-like biological activity in both the hepatopancreas and the hemo- lymph.

Pooled chromatographic fractions of hepatopancreas eluate were assayed to de- termine which molecular size fractions were associated with insulin-like activity (Fig. 2). In pooled fractions 1 and 2, [14C]glycogen was 112 + 21 and 118 + 27% of controls. Increased glycogenesis was not significant in either group (t = 0.99, P > 0.2; t = 1.34, P > 0.2). Glycogen deposition was significantly increased in pooled frac- tion 3, which was 194 + 42% of the controls (t = 5.31, P < 0.001). These are the same fractions which had the greatest insulin immunoreactivity (Sanders, 1983). When a bovine insulin standard was passed through the column, it also eluted with these frac- tions. A significant increase in [14C]glyco- gen, 136 + 22% of controls, was also ob- served in pooled fraction 4 (t = 3.03, P < 0.01). Therefore, in the hepatopancreas ILA was found with proteins of comparable size to bovine insulin. Also, this insulin-like activity was observed in the same fractions which Sanders (1983) found contained the largest immunoreactive insulin peak.

DISCUSSION

The repeatable dose-response curve ob- served when bovine insulin was used in this bioassay demonstrated that the amount of [14C]glycogen is a measure of relative hormone concentration. The curve also showed H. americanus muscle has insulin receptors and bovine insulin can stimulate glycogenesis in this tissue. The low potency of bovine insulin in this invertebrate bioas- say suggests evolutionary differences in the insulin receptors (Falkmer and Wilson, 1967; Falkmer and Gstberg, 1976).

This study presents evidence of a protein in H. americanus that stimulates glyco- genesis in a manner similar to bovine in-

376 BRENDA SANDERS

TABLE 1 MEAN [14C]G~~~~~~~ PER MILLIGRAM OF MUSCLE TISSUE FOR EACH LOBSTER EXTRA@

Tissue

Hepatopancreas Gut Hemolymph

Immunoreactive Insulin

equivalent@ (ng)

0.20 1.18

4.72

[W]Glycogen (cpm mg tissue-‘)

320 k 47 226 ” 27 295 2 43

Percentage of controls

150 -c 22 95t 11

136 f 16

t P

4.30* 0.001 0.85 0.300 3.77* 0.010

a Ten replicates are used to calculate each mean and standard deviation. Student t values and probability are also given.

b From Sanders (1983). * Statistically significant at the 99% confidence level.

sulin. Furthermore, greatest relative ILA was found in hepatopancreas extract, the same extract shown by Sanders (1983) to have the greatest insulin immunoreactivity. ILA in the hepatopancreas of a crustacean

5

- 200

s

. 100 3 E

c 1 2 3 4

POOLED FRACTIONS

FIG. 2. Mean [Ylglycogen in glycogenic assay when pooled chromatographic fractions of hepatopan- cress eluate were added to the incubation media. Each bar represents the mean and 1 SD calculated from 8 replicates of the pooled fractions in the brackets di- rectly beneath it. “C” is the control which is the pooled void fractions. The immunoreactive insulin profile presented in Sanders (1983) has been included below the bars to facilitate comparison between IRI and insulin-like biological activity.

has been reported previously; acid -ethanol extracts of the hepatopancreas of Carcinus maenas enhance glycogenesis in mouse hemidiaphragm tissue (Davidson et al., 1971). These results, and histological evi- dence that the hepatopancreas may have insulin producing B cells (Davidson et al., 1971), suggest this organ is a major site of insulin biosynthesis in decapod crusta- ceans.

Insulin-like activity was also found for the first time in crustacean hemolymph. In- sulin immunoreactivity was previously demonstrated in this tissue (Sanders, 1983).

Although gut extract was shown to have low insulin immunoreactivity, no ILA was detected (Sanders, 1983). The crustacean gut may not contain the active form of the hormone or it could contain a biological antagonist to insulin. The increased vari- ability of replicates containing high con- centrations of the extract, and the report of glucagon-like activity and immunoreactiv- ity in the hepatopancreas of another deca- pod, Astacus fluviatilis, support the latter explanation (Maier et al., 1975).

These results indicate that most ILA is found in the lobster hepatopancreas and hemolymph. No measurable insulin-like activity is present in the gut. This distribu- tion of ILA among tissue extracts contrasts with the tissue distribution of insulin-like activity in molluscs. Although the hepato- pancreas of the oyster 0. edulis has in-

LOBSTER INSULIN-LIKE ACTIVITY 377

sulin-like activity (de Martinez et al., 1973), very high ILA is also found in the gut of several mollusc species (Davidson et al., 1971). Also, molluscan crystalline style, hepatopancreas, and small intestine contain high concentrations of immunoreactive in- sulin (Plisetskaya et al., 1978). These ob- servations suggest the gut plays a lesser role in insulin biosynthesis or storage in crustaceans than in molluscs.

Mammalian proinsulin, which is im- munologically reactive to insulin an- tibodies, has only a small fraction of the biological activity of insulin (Berson and Yalow, 1969; Tager and Steiner, 1974). The authors suggest the receptor binding site for the higher molecular weight IRI cannot bind efficiently with insulin receptors. The same explanation may apply to these re- sults, i.e., that only the smaller im- munoreactive insulin molecules have biological activity.

In summary, these results indicate a protein that has insulin biological activity and immunoreactivity is produced in the hepatopancreas of H. americanus. This protein circulates in the hemolymph and has a direct effect on glycogen metabolism in the muscle tissue of the lobster in vitro. However, the physiological significance of these insulin-like peptides in the carbohy- drate metabolism of crustaceans has yet to be elucidated.

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