Xenotransplantation of microencapsulated canine islets into diabetic rats.

Islets of Langerhans were isolated in high yields from canine pancreata. In the procedure, the pancreata were perfused and digested with collagenase, and the islets were then purified on histopaque density gradients. As many as 60,000 islets were isolated from a single pancreas. Islets were encapsulated in alginate-polylysine-alginate membranes with the aid of an air-jet droplet generator. In vitro studies demonstrated that the isolated and encapsulated islets secreted insulin in response to glucose and IBMX challenge for at least 9 weeks. In in vivo studies 6 diabetic Wistar rats were transplanted with 5,000 to 8,000 encapsulated islets each. The diabetic condition was reversed in all recipients for up to 112 days. In control animals, which received free, unencapsulated islets, the xenografts remained functional for fewer than 21 days. Microcapsules retrieved from normoglycemic transplant recipients 1 and 2 months posttransplantation were shown to contain viable islet tissue, and no cellular overgrowth was observed on capsular surfaces. The results of the study indicate a considerable clinical potential of microencapsulated canine islet xenografts.

Effects of insulin on the cardiovascular integrating mechanisms of brain stem in cats.

In 65 cats anesthetized with alpha-chloralose and urethane, the effects of insulin on cardiovascular responses to stimulation of various structures in the brain stem were studied. The threshold dose of insulin injected intravenously that produced systemic hypoglycemia was 5-10 U/kg. Subthreshold hypoglycemic doses of insulin were used intracerebroventricularly (0.25 U/kg) or intracerebrally (2 mU in 200 nl). Sixty minutes after intravenous insulin, when serum glucose concentrations decreased from 158 to 43 mg/100 ml, pressor responses to stimulation of the periaqueductal gray of midbrain (PAG), locus coeruleus (LC), dorsal medulla (DM), ventrolateral medulla (VLM), and parvocellular reticular nucleus (PVC) decreased significantly. Depressor and bradycardiac response to stimulation of paramedian reticular nucleus or dorsal motor nucleus of vagus (DMV) decreased significantly as well. Thirty minutes after intracerebroventricular insulin, pressor responses of PAG, DM, and the bradycardiac response of DMV decreased significantly. Thirty minutes after intracerebral insulin, pressor responses and renal nerve activities of LC (but not PAG), VLM, DM, and PVC decreased significantly. A similar but faster onset (5 min) of depression of cardiovascular responses on stimulating the LC, VLM, DM, and PVC was observed in another six acutely midcollicular-decerebrate cats recovered from halothane anesthesia. These findings suggest that insulin directly inhibits the vasomotor structures of the brain stem and decreases the pressor responses to stimulation.

Efficacy of thyroxine-suppressive therapy and its relation to serum thyroglobulin levels in solitary nontoxic thyroid nodules.

Patients with nontoxic thyroid nodules are often treated with thyroxine (T4) in order to reduce the size of the nodule, but the efficacy of thyrotropin-suppressive therapy with thyroxine remains uncertain. In this study, 35 patients with a solitary thyroid nodule were given thyroxine (0.1 mg/day) for three months. High resolution (7.5 MHz) sonography was used to measure the size of the nodules before and after thyroxine therapy. The volume of the nodules in 12 patients (34.3%, responders) decreased by more than 50% after thyroxine therapy. In this group, the mean serum thyroglobulin (Tg) level decreased significantly (from 424 to 107 ng/mL, p < 0.001). In the nonresponders, the mean serum Tg level did not change significantly after thyroxine therapy (291 vs 261 ng/mL, p > 0.05). The mean serum total T4 and free T4 concentrations increased significantly in both groups after thyroxine therapy (p < 0.01), the serum total triiodothyronine (T3) level did not change (p > 0.05), and the serum thyrotropin level (TSH) and T3/T4 ratio decreased markedly (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Microencapsulated pancreatic islets: a pathologic study.

Dog pancreatic islets isolated by an enzymatic digestion method were encapsulated in an alginate-poly L-lysine-alginate membrane. These microencapsulated pancreatic islets were cultured in vitro to study their ability of insulin secretion. Portions of these in vitro-cultured microencapsulated pancreatic islets were taken out for a viability dye exclusion study as well as for pathologic studies to correlate them with insulin secretion ability. We found that there was a strong correlation between them. Good insulin-secreting microcapsules showed well-preserved cell membranes and beta-cell granules. An in vitro culture for one to two days in RPMI-1640 made the islets more stable, the cellular surface became smoother and the beta-granules were in better shape. The microencapsulated pancreatic islets were also injected into the peritoneum of streptozotocin-induced diabetic CDF1 mice. Blood glucose levels dropped and stayed low for up to 60 days. But, when non-encapsulated dog pancreatic islets were used, the blood glucose levels remained low for only about 14 days. A small portion of the injected microcapsules were washed out at specific times for pathologic study. Up to 28 days after injection, only a few of the injected microcapsules showed pericapsular cellular infiltrate. However, after 56 days, most of the microcapsules showed dense pericapsular cellular infiltrate. Immunohistochemical analysis of these infiltrates showed that the majority of cells were fibroblasts and macrophages. Most of the cells located in the inner portion of the infiltrate were fibroblasts, while the macrophages were located mainly on the outer portion. Both scanning and transmission electron microscopy showed that the surface of the microcapsule outer wall was much smoother than the inner wall. The size of the microcapsules was approximately 0.6-0.8 mm and the thickness of the wall measured around 10 nm. The smaller the microcapsule is, the less chance there is of rupture with release of the xenographic islets. Once the wall of the transplanted microcapsules was ruptured, the inner surface showed more increased inflammatory cell and fibroblast infiltration than the outer surface.

Insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia in the rat.

Microinjection of insulin (0.04-0.12 IU/microliter) into the anterior hypothalamus or the lateral hypothalamus, but not the ventromedial hypothalamus of the rat brain, caused a dose-dependent rise in blood glucose and in serum insulin. The majority (71.5%) of the glucose-facilitated neurons recorded in the lateral hypothalamic area were excited by intracerebral injection of insulin. The data indicate that insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia. It is unknown whether insulin normally reaches the hypothalamic area, or how it might do so.

Stimulation of 5-hydroxytryptamine nerve cells in dorsal and median raphe nuclei elevates blood glucose in rats.

The role played by dorsal or median raphe nuclei in glucoregulation was investigated by stimulating these nuclei in normal rats and in rats with chemical ablation of the hydroxytryptamine (5-HT) nerve cells in these nuclei. Electrical stimulation of either dorsal or median raphe nuclei increased blood glucose or the in vivo voltammetric signal of hypothalamic 5-OH-indole in normal rats; the increase in blood glucose level or the hypothalamic 5-OH-indole release was proportional to the intensity of stimulation. Microinjection of kainic acid or L-glutamate at the same sites also produced hyperglycemia or stimulated the hypothalamic 5-OH-indole release. This stimulation-induced hyperglycemia was significantly reduced by pretreatment of animals with spinal transection or adrenalectomy. In addition, selective destruction of the hypothalamic 5-HT nerve fibers, produced by administration of 5,7-di-hydroxytryptamine (a 5-HT nerve depletor) into both dorsal and median raphe regions, reduced the magnitude of the hyperglycemic responses to electrical stimulation of either dorsal or median raphe nuclei. The data indicate that stimulation of ascending 5-HT pathways in the rat's brain increases the adrenal-sympathetic efferent activity and leads to hyperglycemia.

Further studies on the long-term treatment of Graves' hyperthyroidism with ipodate: assessment of a minimal effective dose.

We have previously described that sodium ipodate (500 mg/day, p.o.) is effective in normalizing serum T3 and T4 levels in most patients with Graves' hyperthyroidism. In this study, we examined serum T3, T4, and rT3 levels in 14 hyperthyroid patients with Graves' disease during treatment with a lower dose (500 mg, every other day, p.o.) of sodium ipodate for a period of 3-30 weeks (mean 15.5 weeks). Three types of responses were observed. In group I (4 patients), both serum T3 and T4 were in the normal range at the end of treatment [baseline: mean +/- SEM T3, 6.8 +/- 0.96 nmol/L (normal 0.92-3.0)] and T4 [256 +/- 44 nmol/L (normal 62-167); post-ipodate: T3, 2.0 +/- 0.46 nmol/L and T4 107 +/- 28 nmol/L]. In group II (n = 5), either serum T3 (3 patients) or serum T4 (2 patients) did not become normal (baseline: T3 7.7 +/- 1.1 and T4 228 +/- 3.9; post-ipodate: T3 2.9 +/- 0.57 and T4 188 +/- 27 nmol/L). In group III (5 patients), neither serum T3 nor serum T4 returned to normal following ipodate treatment (baseline: T3 11.9 +/- 1.8 and T4 260 +/- 23; post-ipodate: T3 7.5 +/- 0.49 and T4 322 +/- 17 nmol/L). The mean serum rT3 concentration increased during ipodate treatment to a peak value of 100% above baseline and remained elevated (20-75% above baseline) throughout the study. Some improvement in hyperthyroidism was suggested by increase in body weight during ipodate treatment in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)

[Microalbuminuria in insulin-dependent diabetes mellitus: a comparison of specimen collection, analytic methods and relationship with glycemic control and blood pressure].

The excretion of small quantities of urinary albumin (microalbuminuria = urinary albumin excretion rate, UAER = 20-200 micrograms/min) may predict renal function in both insulin-dependent and noninsulin-dependent diabetes. We compared radioimmunoassay with the immunoturbidimetric method to detect early increases in urine albumin concentration. More problems have been encountered in deciding which method of collecting urine best differentiate between early onset diabetic nephropathy and normality. Random urine samples collected at clinics are convenient but show wide variations in concentration and the effects of exercise. Such variations may be overcome by using a rest period and correcting for urine creatinine concentration. We studied 21 IDDM patients (12 female, 9 male), aged 13-33 years old (mean 21) and 11 nondiabetics (6 female, 5 male), aged 15-30 years old (mean 23). All gave negative results on testing with Albustix at clinic visits. All subjects passed urine immediately after they got up in the morning. The results disclosed: (1) The correlation coefficient of albumin excretion (micrograms/ml) in the urine collected overnight with that collected over 24 hours was good (r = 0.89, p less than 0.001). (2) When the albumin excretion rate of the urine collected overnight was expressed as microgram albumin/mg creatinine, the correlation was also as good as the 24-hr urine albumin excretion (microgram albumin/mg creatinine) (r = 0.87, p less than 0.001). (3) The results of our study support the use of urine samples collected overnight, corrected for creatinine, to estimate microalbuminuria.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of a bulbospinal 5-HT pathway in the rat brain produces hyperglycaemia.

The glucoregulatory role of spinally projecting serotonin (5-HT) neurones near the ventrolateral surface of the medulla oblongata was investigated by stimulating these nerve cells in normal rats and in rats with selective chemical ablation of 5-HT nerves in the spinal cord. Electrical stimulation of the lateral medulla produced hyperglycaemia in normal rats; the increase in blood glucose was proportional to the intensity and frequency of stimulation. Furthermore, microinjection of kainic acid or L-glutamate at the same sites also produced hyperglycaemia. This stimulation-induced hyperglycaemia was significantly reduced by spinal transection or adrenalectomy. Selective destruction of spinal 5-HT nerves produced by intraspinal injection of 5,7-dihydroxytryptamine also reduced the magnitude of the hyperglycaemia response to electrical stimulation of the lateral medulla. This indicates that stimulation of 5-HT nerve cells adjacent to the ventrolateral surface of the medulla oblongata and projecting to the spinal cord increases the adrenal-sympathetic efferent activity and leads to hyperglycaemia in rats.

Effect of fructose-induced hypertension on the renin-angiotensin-aldosterone system and atrial natriuretic factor.

Studies were performed to examine the effects of a high fructose intake on blood pressure, the renin-angiotensin-aldosterone system, and atrial natriuretic factor in normal rats. Sprague-Dawley rats were fed with either normal rat chow or a fructose-enriched diet for four or seven weeks. High dietary fructose consumption induced hyperinsulinemia and hypertriglyceridemia, and increased the systolic blood pressure by 19 +/- 4 mm Hg (four weeks of diet) and 24 +/- 1 mm Hg (seven weeks of diet), respectively. There were no significant changes in the plasma renin activity at either time. However, an increase in plasma atrial natriuretic factor was noted in rats treated with a high fructose diet for four weeks, and a decrease in plasma aldosterone was found in those rats fed with fructose for a longer period. There was no significant difference in body weight gain between rats fed with fructose-enriched diet and those fed with control diet. These data indicate that the increase in blood pressure associated with a high fructose intake is not due to a direct effect or the renin-angiotensin-aldosterone system.

Opsonophagocytosis of Staphylococcus aureus by diabetics' sera and monocytes.

The ingestion and the intracellular killing of Staphylococcus aureus by monocytes from patients with noninsulin-dependent diabetes mellitus (NIDDM) under good control (mean 2-h postprandial blood glucose less than 150 mg/dl in the past 6 months) or under poor control (mean 2-h postprandial blood glucose greater than 200 mg/dl in the past 6 months) were investigated. The ingestion was expressed as a phagocytic index which was a percentage of the total bacteria added in the reaction mixtures, while the intracellular killing was expressed as the percentage of ingested bacteria which were destroyed. The phagocytic index (mean +/- S.E.) of healthy subjects (HS), diabetics under good control (DGC) and diabetics under poor control (DPC) were 45.1 +/- 3.5, 38.2 +/- 2.2, and 32.1 +/- 3.4%, respectively. This showed that monocytes from diabetics ingested S. aureus more poorly than did those from HS (p less than 0.001) and monocytes from DPC ingested more poorly than did those from DGC (p less than 0.001). There was no difference in intracellular killing between HS and DGC, but a marked difference was noted between DGC and DPC. Specific antibodies (IgG) as demonstrated by indirect immunofluorescence assay had little effect on ingestion but could enhance complement activation and then increase ingestion whether the serum was from HS, DGC, or DPC. On the other hand, the significant effect of complement activity in the presence of specific antibodies on intracellular killing was only observed on HS and DGC, suggesting that antibody-dependent complement activation could be a crucial mechanism for efficient intracellular killing.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin secretion and insulin action in Taiwanese with type 2 diabetes.

In contrast to the United State, type 2 diabetes appears to be a common occurrence in non-obese Asians. In order to evaluate the possibility that this epidemiologic difference was indicative of a basic metabolic phenomenon, estimates of insulin secretion and insulin action were generated in 32 Chinese males, 16 with type 2 diabetes and 16 with normal glucose tolerance. Half of the individuals in each diagnostic category were obese (body mass index greater than 28 kg/m2) and half were non-obese (less than 26 kg/m2). Plasma glucose responses to a 75-g oral glucose challenge were significantly higher in patients with type 2 diabetes, but did not vary significantly within either group as a function of obesity. Plasma insulin concentrations were lower than normal when patients with type 2 diabetes were compared to their weight-matched controls. In addition, the absolute insulin values also varied as a function of body weight, with higher plasma insulin concentrations observed in the obese individuals. Insulin action was estimated by determination of the steady-state plasma insulin (SSPI) and glucose (SSPG) concentrations during the last 60 min of a continuous 180-min intravenous infusion of somatostatin, crystalline insulin, and glucose. Under these conditions endogenous insulin secretion is suppressed, SSPI concentrations are similar in all individuals, and SSPG concentrations provide a quantitative estimate of insulin-stimulated glucose disposal. The results of these studies indicated that patients with type 2 diabetes had significantly elevated SSPG concentrations as compared to normals, and this was true whether the diabetic subjects were obese or non-obese.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between plasma cortisol and CSF catecholamines in endogenous depressed dexamethasone nonsuppressors.

Nineteen endogenous depressive in-patients (13 with major depression and 6 with bipolar disorder-depressed) and 10 other patients with dysthymic disorder serving as the control group were given the dexamethasone suppression test (DST, 1 mg/subject). The results showed that the DST sensitivity in endogenous depressives was 73.7% and the specificity was 90%. After the patients were treated daily for 6 weeks with 150-200 mg imipramine, 88.9% of those endogenous depressive patients who previously had a positive DST response exhibited a negative response. Moreover, a significantly negative correlation was found between the CSF norepinephrine level and the pre-dexamethasone 4 p.m. plasma cortisol level in those endogenous depressed patients who had a positive DST response. Pre-treatment data also showed that the 4 p.m. plasma cortisol had a significant negative correlation with CSF dopamine. These findings suggest that endogenous depression with positive DST could be related not only to a lower norepinephrine level, but also to a lower dopamine level.

Alterations in physiologic functions and in brain monoamine content in streptozocin-diabetic rats.

In the present study, we used streptozocin (STZ) to induce diabetes in rats and observed alterations in several physiologic functions and in monoamine content of different brain regions. Rats with STZ diabetes displayed a thermoregulatory deficit in the cold. Both the body temperature and metabolic rate of the diabetic animals were reduced at ambient temperatures below 22 degrees C. These diabetic animals had a higher level of the spontaneous pain threshold, but displayed a reduced sensitivity of analgesic responses to morphine injection. In addition, these diabetic animals had a lower level of spontaneous motor activity, but displayed an increased sensitivity of locomotor stimulant responses to amphetamine administration. Biochemical examination revealed that the diabetic animals had a lower serotonin level in both the hypothalamus and the brainstem without changes in the serotonin levels of the corpus striatum. These diabetic animals also had a lower catecholamine level in the hypothalamus, but a higher catecholamine level in the corpus striatum. The alterations in brain monoamine content and in the above-mentioned physiologic parameters were reversed after insulin replacement therapy. The data suggest that alterations in various autonomic, somatosensory, and motor neural functions of untreated STZ-diabetic rats correlated with a reproducible pattern of monoamine content in various brain regions (a pattern that differed from that observed in healthy control rats), and that both the altered neural function and the altered brain monoamine pattern were reversed after insulin therapy.

Thyrotropin-releasing hormone-induced hyperglycemia: possible involvement of cholinergic receptors in the lateral hypothalamus.

The influence of the site of action of thyrotropin-releasing hormone (TRH) on the production of hyperglycemia was studied in rats by comparing the effectiveness of TRH administered by different routes. Administration of TRH (5 micrograms) into the lateral hypothalamus (LH) produced a hyperglycemia with a peak elevation of blood glucose of 140 mg/dl. Injection of 5 micrograms TRH into the ventromedial hypothalamus (VMH) produced a blood glucose elevation of 44 mg/dl, while injection of the same dose of TRH into the anterior hypothalamus (AH) produced a blood glucose elevation of 23 mg/dl. These findings indicate an LH site of action of TRH. Indeed, intra-LH administration of TRH (1-10 micrograms) caused dose-related increases in blood glucose. Administration of acetylcholine into the same site was also shown to induce hyperglycemia. The hyperglycemic effects of TRH and acetylcholine were antagonized by previous treatment of the LH site with atropine, a cholinergic receptor antagonist. Furthermore, the TRH-induced hyperglycemia was not observed or was greatly reduced in spinal rats or in adrenalectomized rats. The results indicate that TRH may act through the cholinergic receptor mechanisms within the LH region to induce hyperglycemia by promoting an increase in the sympathetic-adrenal medullary efferent activity.