Protein synthesis in pancreatomizad rats

Protein Synthesis in Wound alter Tooth Extraction in Pancreatectomized Diabetic Rats

Sylvestre Arnaldo GRANDINI¹ Luiz Guilherme BRENTEGANI¹ Arthur Belem NOVAES¹ Renato Hélios MIGLIORINI²

¹Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo Ribeirão Preto, SP, Brasil ²Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo Ribeirão Preto, SP, Brasil

Braz Dent J (1990) 1(1): 25-30 ISSN 0103-6440

The incorporation of alanine C14 in protein synthesis was analyzed in recently formed alveolar tissue after tooth extraction in partially-pancreatectomized diabetic rats. The incorporation of alanine C'4 was higher in diabetic animals than in treated diabetic and control groups. The results can be explained by a .delay in bone tissue repair.

Key words: protein synthesis, wound healing, pancreatectomy, diabetes, alanine C14.

Introduction

In wound healing, protein metabolism is fundamental for the repair of collagen, a tissue which is dependent on the synthesis of a large quantity of a special protein. Any factor that can affect either the intracellular mechanism of tropocollagen synthesis or the extracellular mechanism of precipitation and organization of collagen fibers will alter the evolution of the repair process. The importance of carbohydrate and protein metabolism is emphasized when we consider some systemic situations which affect the metabolism of such substances. In this respect, diabetes deserves special attention because of the generalized metabolic changes it causes. Insulin deficiency provokes clear alterations of carbohydrate, protein and lipid metabolism. It has been reported that insulin deficiency in diabetics can restrict physiological stimuli to osteoblasts (Hernberg, .1952) and osteoclasts (Pinheiro, 1966) with a consequent reduction in their number and activity. Grandini et al. (1970) observed histologically that the process of alveolar repair in alloxan diabetic rats is clearly retarded and that insulin administration to the animals leads to a more favorable sequence of healing evolution. Wettenhall et al. (1969) studied the action of insulin and growth hormone on collagen synthesis in vitro and reported that connective tissue is sensitive to insulin but not to growth hormone.

Since it has been well established that insulin acts favorably on the processes of protein synthesis in several tissues (Krahl, 1%2), we undertook the present study to determine whether the delay in alveolar healing reported previously was simply dependent on the activity of protein synthesis. Thus, the objective of the present investigation was to evaluate in vitro the activity of protein synthesis using labelled amino acid alanine C14, in the tissue formed following dental extraction in pancreatectomized diabetic rats.

Material and Methods

Two hundred and forty male rats (Rattus norvegicus albinus, Wistar) weighing 140 to 220 grams were used. The animals were fed a balanced diet before and after the experiment and were divided at random into three groups: 1) control, 2) pancreatectomized diabetic animals and 3) pancreatectomized diabetic animals treated with insulin (NPH, Lilly).

Diabetes was induced by partial pancreatectomy. About 20'11v of the animal's pancreas was maintained after performing the first step of the Migliorini (1970) technique. To check the diabetic condition, the King and Gardner (1947) method was used to determine blood glucose concentration in three stages, I.e., 7 days after pancreatectomy, at extraction time, and on the day of sacrifice. Only the animals which presented blood glucose levels equal to or higher than 250 mg per 100 ml throughout the experiment were utilized. The group of treated diabetic animals started to be treated with 1 unit insulin two days after partial-pancreatectomy and the dose was gradually increased up to 3 units per day and maintained at that level until the day of sacrifice.

The extractions were performed at least 10 days after partial pancreatectomy. Following general anesthesia by inhalation of sulfuric ether, the upper right incisors were extracted using a specially adapted instrument, by the method of Okamoto and Russo (1973). After surgery the animals were given water ad libitum and only soft food in order to maintain the alveolus free of food particles. As prevention against infection, the animals received three daily intramuscular injections of 100 units of penicillin (Despacillin R 400, Squibb).

Based on previous histological findings (Grandini, 1978), it was determined that sacrifice time should be at the following periods after surgery for the control group: 3, 4, 5, 7 and 10 days; and for the other 2 groups: 5, 7, 10, 12 and 14 days. Repair tissue was removed from the alveolus at these times. The animals were sacrificed by decapitation and the right maxillary alveolar processes were immediately removed and carefully dissected. The distal bone wall of the alveolus was then removed for access to, and collection of healing repair tissue. At sacrifice, material was also collected from other animals kept at the same experimental conditions for histological preparation in order to verify microscopically which kind of tissue had been incubated.

Healing repair tissue fragments were incubated in buffered Krebs-Henseleit (1932) medium which was balanced by a gaseous phase containing 95% 02 and 5'11v C02, with final pH between 7.3 and 7.5. To each incubation bottle containing 2 ml medium 5 ml alanine labelled with C14 ( = 0.5 mc) was added. The incubation bottles containing the material collected were exposed one minute to 95% 02 and 5% CO2 and shaken for 2 hours at a constant temperature of 37°c. After incubation, the material was pooled in 15-ml centrifugation tubes. one drop of material and of saturated Na2SO4 was added and then the material was mixed well. The quantity of c14 protein was determined as reported by Migliorini and Manchester (1971).

Results and Discussion

The results obtained for each group during the postoperative period are presented in Table 1 and Figure i. The activity of protein synthesis in the recently formed alveolar tissue following dental extraction was calculated as the average value obtained from all the incubation tubes, being the estimation done for each 25 mg of tissue for 5W,000 ppm.

The new tissue formed and incubated presented good activity of protein synthesis in the three groups of animals. This activity showed some changes related to chronology, similar to the findings reported by Grandini et al. (1970). In quantitative terms, however, alanine C14 incorporation into protein was higher in the diabetic animals than in the treated diabetic and control groups. Protein synthesis peaked on the fifth day in the control group and on the tenth day in the diabetic group. In contrast, in the treated diabetic group, protein synthesis peaked around the 5th, 7th and 10th postoperative days, with slightly higher levels on the 7th day.

A hypothesis that could be considered to explain the high level of protein synthesis in the diabetic animals is the lack of evolution from repair tissue to bone tissue. Guimarães (1967) reported a longer healing phase in diabetic animals, due to the fact that the fibroblastic phase started later and progressed more slowly than in the control animals. In the control group, starting on the 5th postoperative day, the alveolar apical third was almost completely filled with recently formed osseous trabeculae; the medial third presented mostly new connective tissue, and the cervical part showed only granulation tissue. This picture was not the same in the diabetic group in which the apical and part of the medial third remained filled with newly formed repair tissue that only reached some maturation and characteristics of young connective tissue by the 10th postoperative day.

In the control group, tissue formation occurred throughout the alveolus, whereas in the diabetic group it occurred mostly in the apical third and in the treated diabetic group in the apical and medial thirds of the alveolus.

The animals of the treated diabetic group received insulin throughout the experiment and showed marked improvement in alveolar repair, very close to the healing rate observed in the control group.

The present results do not permit us to state with certainty that the level of diabetes affects the healing process, because the sample studied was obtained from animals with blood glucose levels of 250 to 618 mg/ml (few of the animals used had blood glucose levels below 300 mg/ml). The animals with higher blood glucose levels had frequent infections and showed a quantitative reduction in repair tissue formation. This finding was also reported by Otaka (1962) working with glycemia over 300 mg/ml.

The amino acid of choice for the present study was alanine C14 because it is evenly incorporated into all proteins and therefore can be used to estimate total protein synthesis.

Pancreatectomy was considered to be an excellent model of experimental diabetes for the study of the metabolic effects of the acute absence of insulin in rats and was helpful in the study of alveolar repair subsequent to tooth extraction.

Conclusion

Diabetic subjects showed protein synthesis with more activity and a later peak during healing when compared with those in the control and treated diabetic groups. The timing of the activity of protein synthesis is altered by insulin administration. Partial pancreatectomy proved to be an excellent means of inducing diabetes in subjects for the study of wound healing in dental sockets.

References

Grandini SA: The effect of partial-pancreatectomy induced diabetes on wound healing subsequent to tooth extraction. Oral Surg Med Oral Path 45: 190-199, 1978

Grandini SA, Migliorini RH, Okamoto T: Processo de reparo em ferida de extração dental em rates cam diabete aloxanico (Estudo Histológico). Bol Fac Farm Odont Ribeirão Preto 7: 47-71, 1970

Guimarães SAC: Contribuição ao estudo morfológico - Descrito, histométrico e histoquímico do tecido de granulação cicatricial em rates diabéticos aloxanicos. Doctoral thesis, Faculdade de Odontologia de Bauru 1967

Hemberg CA: The bone structure in alloxan induced diabetes mellitus in rats. Acta Med Scand 142: 274-277, 1952

King EJ, Gardner RJ: Colorimetric determination of glucose. J Clin Pathol 1: 30-33, 1947

Krahl ME: Insulin and protein synthesis in isolated tissue. Diabetes II: 144-146, 1962

Krebs HA, Henseleit K: Untersuchungen über die Hamstoffbildung im Tierkorjer. Ztschr Physiol Chem 210:33-66, 1932

Migliorini RH: Two-stage procedure for total pancreatectomy in the rat. Diabetes 19: 694-697, 1970

Migliorini RH, Manchester KL: Diminution in starvation of capacity of cell rap to support protein synthesis in cell-free systems. Febs Letters 123: 104-144, 1971

Okamoto, T, Russo MC: Wound healing following tooth extraction. Histochemical study in rats. Rev Fac Odont Araçatuba 2: 153-169, 1973

Otaka Y: Histochemical studies on mucopolysaccharides in the field of pathology. In: Biochemistry and medicine of mucopolysaccharides. Egami F, Oshima Y, ed. 305-321, Maruzen, Tokyo 1962

Pinheiro CE: Estudo de alguns aspectos da ossificação em rates diabéticos aloxânicos. Doctoral thesis, Faculdade de Odontologia de Bauru 1966

Wettenhall REH, Schwartz PL, Bomstein J: Actions of insulin and growth hormone on collagen and chondroitin sulfate. Synthesis in bone organ cultures. Diabetes 18: 280-284, 1969

Correspondence: Dr. Sylvestre Arnaldo Grandini, Departamento de Cirurgia, Faculdade de Odontologia de Ribeirão Preto, USP, 14050 Ribeirão Preto, SP, Brasil.

Accepted October 9, 1990
Eletronic publication: august 1997

BACK TO CONTENTS