Anna Sirek

The effect of sex hormones on glycosaminoglycan content of canine aorta and coronary arteries.

Hyaluronate (HA), heparan sulfate (HS), dermatan sulfate (DS) and isomeric chondroitin sulfates (CS) were measured in vascular walls of 9-10 months old normal and hypophysectomized female beagles treated with sex hormones. Following hypophysectomy the animals were maintained for 8 weeks without any hormonal replacement therapy and then they were exposed for 3 weeks to parenteral treatment with sex hormones. One group received twice weekly 25 mg testosterone, another group was given the same amount of progesterone, and a third group received on day 1 and day 14, estrogens in 2 injections, consisting of a mixture of 10 mg short-acting estradiol-17-phenylpropionate and 2.5 mg long-acting estradiol benzoate. After 11 weeks all animals were sacrificed, coronary arteries and aortas were immediately removed and the latter were divided into three segments: arch, thoracic and abdominal. Removal of the pituitary led to a reduction of the HA content in the aortic arch and thoracic segment, but coronary arteries and abdominal aorta were not affected. The main consequence of hypophysectomy both in the entire aorta and in coronary arteries was a sharp reduction of the sulfated glycosaminoglycan (GAG) content. All three hormones produced a modest rise in the HS content of coronary arteries. A more definite response was seen in the thoracic aorta where each of the three hormones raised the low DS content to normal levels. Concerning the effect of sex hromones on aortic GAG other than DS, TESTOSTERONE RAISED THE CS content towards normal in thoracic and abdominal segments, while estrogen by doubling the normal HA concentration was particularly potent in the abdominal aorta. It is conceivable that the different sensitivity of various segments of aorta and coronary arteries to sex (and other) hormones in terms of regulating GAG metabolism may prove to be of relevance to the uneven distribution of lesions in degenerative vascular disease.

Intermittent hyperinsulinaemia and arterial glycosaminoglycans in dogs.

SummaryThe effect of insulin on the glycosaminoglycan content of the arterial ground substance was compared in age-matched alloxan-diabetic dogs, hypophysectomized dogs and normal controls. Hyaluronic acid and the three sulphated components, heparan, dermatan and isomeric chondroitin sulphates were quantitatively determined in the arch, thoracic and abdominal aorta, and in the carotid, coronary, iliac, renal and mesenteric arteries. Diabetic animals were either well or poorly controlled. Six well controlled dogs with a fasting mean plasma glucose level of 8.5 mmol/l were given around 30 units of porcine insulin/day and showed fluctuations in plasma insulin concentrations between 24 and 175 mU/l. Four poorly controlled dogs with a mean fasting plasma glucose level of 18.5 mmol/l received an average of 14 units of insulin/day, and the fluctuations ranged from 8 to 113 mU/l. Eight normal, untreated controls showed mean fluctuations between 15 and 22 mU/l. Within 14 weeks of insulin treatment, well controlled animals displayed glycosaminoglycan alterations in five arterial segments, usually involving more than one glycosaminoglycan constituent. In poorly controlled animals the number of segments that showed glycosaminoglycan alterations was the same, but abnormalities were limited to one of the sulphated components only. The coronary arteries displayed identical glycosaminoglycan alterations in the two groups of diabetic dogs, namely a significant rise in dermatan sulphate content (p < 0.01, mean±SEM) from the normal value of 1.12±0.03 mg/g dry defatted tissue to 1.31±0.03 mg/g in well controlled animals and to 1.37±0.08 mg/g in poorly controlled animals. In order to ascertain that the abnormalities in the glycosaminoglycan chemistry were related to hyperinsulinaemia rather than hyperglycaemia, six hypophysectomized dogs were treated with 1.5 U/day of porcine protamine zinc insulin, for 3 weeks. Average plasma glucose levels were in the order of 4.3 mmol/l and plasma insulin levels fluctuated between 9 and 27 mU/l; the latter represented twice the peak value seen in five untreated hypophysectomized dogs. The resulting chemical changes in the glycosaminoglycan content were in line with those encountered in diabetic animals, including the rise in dermatan sulphate content of the coronary arteries. These results indicate that: (1) hyperinsulinaemia produced by injections of insulin causes alterations of the arterial glycosaminoglycan content; (2) not all segments of the arterial tree are equally responsive to insulin and (3) the coronary arteries have a particularly insulin-sensitive dermatan sulphate metabolism.

Arterial Glycosaminoglycans in Diabetic Dogs

The glycosaminoglycan (GAG) composition of a number of large and medium-sized arteries was studied in 6 alloxan-diabetic beagles and was compared with 6 normal, age-matched controls. Diabetic animals were maintained on diet and insulin for 100 days. The aortic arch, thoracic and abdominal segments, external iliac, superior mesenteric, renal, common carotid and coronary arteries were analyzed for hyaluronic acid (HA) and for heparan (HS), dermatan (DS), and chondroitin (CS) sulphates. All diabetic dogs displayed significant alterations. The HA content was reduced in iliac arteries, and together with HS, also in the thoracic aorta. HS or CS were increased in carotid, iliac and renal arteries, DS, a GAG constitutent with very high affinity for low density lipoproteins, was significantly increased in coronary arteries alone. 2 additional animals which are excluded from this series did not become diabetic after alloxanization and showed no change in arterial GAG content. Early changes in the chemistry of the arterial ground substance seem to provide a clue to the precocious development of atherosclerotic disease in diabetes.

Action of growth hormone and thyroxine on aortas of hypophysectomized dogs.

Young dogs were surgically hypophysectomized and maintained for eleven weeks postoperatively with age-matched normal controls. For three weeks prior to sacrifice, four hypophysectomized dogs were given daily injections of bovine growth hormone (GH, 0.2 mg./kg.) and another four were given daily injections of thyroxine (T4, 5 μg./ kg.). Aortas were removed, cleaned of adventitia and divided into three segments: arch, thoracic and abdominal. Each portion was analyzed for collagen, elastin, deoxyribonucleic acid (DNA), calcium, total mucopolysaccharides (MPS), hyaluronic acid (HA), heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (GS). The arch and thoracic aortas of normal animals were found to contain more DNA, CS and elastin but less collagen than the abdominal aorta. Removal of the hypophysis resulted in an overall increase in elastin and DNA content and caused a decrease in all sulfated MPS. Administration of either CH or T4 to hypophysectomized dogs had a profound effect on the majority of constituents in all segments of aorta. GH returned the content of elastin, DS and CS toward normal in at least two of the three aortic segments. T4 returned the content of DNA, DS and CS toward normal in all segments. Moreover, T4 treatment caused significant reductions in collagen and HA contents of thoracic and abdominal segments. These results indicate that: (1) the composition of normal aorta varies with the segment studied; (2) the composition of the aorta is markedly affected by hypophysectomy, CH and T4 treatment; and (3) individual aortic segments show differential sensitivity to a given hormone.

Effect of Pancreatectomy, with and without Hypophysectomy, and of Insulin Treatment on the Composition of Canine Aorta

The chemical composition of aorta from normal and endocrine deficient dogs was studied in terms of collagen, elastin, calcium, “water plus lipid” and mucopolysaccharide (MPS) content. The latter component was resolved into four fractions: hyaluronic acid (HA), heparan sulfate (HS), dermatan sulfate (DS), and the isomeric chondroitin sulfates (CS). The results were compared in the following way: (1) normal versus pancreatectomized; (2) hypophysectomized versus hypophysectomized and pancreatectomized (Houssay); and (3) Houssay with and without insulin treatment. Pancreatectomy affected the content of two constituents only: the HS and CS levels were lowered. The low content could be raised by insulin treatment. These results indicate that except for the sulfated MPS, insulin does not appreciably alter the composition of the aortic wall.

Pituitary growth hormone and the question of pancreatic secretion of glucagon.

Experiments are presented in which normal dogs were injected with a highly purified growth hormone preparation. One hour later blood samples were drawn simultaneously from the pancreatico-duodenal and jugular veins. The ‘central’ and ‘peripheral’ blood samples were injected into depancreatized dogs. While the injection of ‘peripheral’ blood produced no appreciable change in the blood sugar level of the diabetic recipient, the ‘central’ blood caused a definite but transient rise in blood sugar. The rise could be prevented by treating the recipient diabetic animal with the adrenergic blocking agent dihydroergotamine. It has been concluded that the hyperglycemic factor present in the blood of the pancreatico-duodenal vein after injection of growth hormone preparations is not identical with glucagon-Lilly, the effect of which is not influenced by dihydroergotamine. In two experiments in which completely depancreatized, instead of intact, dogs were used as donors the administration of growth hormone produced the characteristic hyperglycemic response when ‘central’ blood samples were injected into other depancreatized dogs. These findings cast doubt on the pancreatic origin of the hyperglycemic material and add interest to the search for its source and nature. Further experiments using depancreatized dogs are in progress.

Diurnal Episodic Pattern of Insulin Secretion in the Dog

Sampling portal blood every 15 min by means of indwelling cannulae, we have found evidence that basal insulin secretion in nonanesthetized dogs takes place in six or seven major secretory episodes over a 24-h period. Reproducible patterns were obtained in four experiments conducted on three normal animals. When frequency of peaks was plotted against the log of insulin concentration, a normal distribution was obtained (lognormal distribution). The means and variances were nearly the same in the three animals. Low levels of insulin (10–25 μU/ml) alternated with peaks which were occasionally higher than 200 μU/ml. The average duration of a peak was 30 min and seemed to be independent of ambient glucose levels. It is suggested that these peaks are the result of surges in plasma growth hormone concentrations and/or spontaneous repetitive discharges along neuronal pathways functionally related to insulin secretion.

Inhibition of sulphonylurea-stimulated insulin secretion by beta adrenergic blockade

SummaryNormal dogs were injected i.V. with a single dose of 0.25 mg/kg sodium salt of HB 419 (Glibenclamide). Plasma insulin and glucose concentrations were measured at stated intervals over a period of two hours. The rise in insulin, but not the hypoglycemic response was abolished in peripheral blood when the animals were pretreated with a single i.v. injection of either 0.1 mg/kg or 0.3 mg/kg dl-propranolol, 30 min prior to the administration of HB 419. The d-isomer of propranolol was ineffective in this respect. These results indicate that a) the mechanism by which propranolol inhibits sulphonylurea-stimulated insulin secretion involves beta adrenergic receptors; and b) the hypoglycemia produced by HB 419 in the presence of propranolol could be the result of extrapancreatic effects. Since the possibility of an early rise in the insulin concentration of portal blood was not excluded in our present series of experiments, final proof will have to be provided by studies in totally pancreatectomized dogs.

A New Technique for Hepatic Portal Sampling in the Conscious Dog

Abstract A technique for implantation of a silastic catheter into the portal vein is described. The central end of the catheter is passed through a puncture hole into a tributary of the portal vein. The peripheral free end, occluded by a rubber membrane, is passed through the abdominal wall and buried under the skin. Once the catheter is in place it can be kept patent for several weeks. Injection of a substance and withdrawal of blood from the portal vein are carried out by percutaneous puncture of the rubber membrane, a virtually painless procedure that can be carried out in fully conscious nonrestrained dogs.

Effect of prolonged administration of tolbutamide in depancreatized dogs.

In a preliminary communication we have reported that depancreatized dogs treated with tolbutamide for prolonged periods showed derangements of liver function. The present paper gives a detailed account of our studies on twelve depancreatized dogs treated orally with various doses of the drug. When given relatively large doses, the animals required appreciably smaller amounts of exogenous insulin to maintain a standard degree of control.