On the mechanism of impaired insulin secretion in chronic renal failure.

It has been suggested that a sustained rise in resting levels of cytosolic calcium [Ca2+]i of pancreatic islets is responsible for impaired insulin secretion in chronic renal failure (CRF). Evidence for such an event is lacking and the mechanisms through which it may affect insulin secretion are not known. Studies were conducted in normal, CRF, and normocalcemic, parathyroidectomized (PTX) CRF rats to answer these questions. Resting levels of [Ca2+]i of islets from CRF rats were higher (P less than 0.01) than in control of CRF-PTX rats. [3H]2-deoxyglucose uptake and cAMP production by islets were not different in the three groups. Insulin content of, and glucose-induced insulin secretion by islets from CRF rats was lower (P less than 0.01) than in control and CRF-PTX rats. In contrast, glyceraldehyde-induced insulin release by CRF islets was normal. Basal ATP content, both glucose-stimulated ATP content and ATP/ADP ratio, net lactic acid output, Vmax of phosphofructokinase-1, and Ca2+ ATPase of islets from CRF rats were lower (P less than 0.02-less than 0.01) than in normal or CRF-PTX animals. Data show that: (a) Glucose but not glyceraldehyde-induced insulin secretion is impaired in CRF; (b) the impairment in glucose-induced insulin release in CRF is due to a defect in the metabolism of glucose; (c) this latter defect is due to reduced ATP content induced partly by high [Ca2+]i of islets; and (d) the high [Ca2+]i in islets of CRF rats is due to augmented PTH-induced calcium entry into cells and decreased calcium extrusion from the islets secondary to reduced activity of the Ca2+ ATPase.

Estrone treatment dissociates primary versus secondary consequences of "diabetes" (db) gene expression in mice.

Feeding 0.001% estrone in a diet to C57BL/KsJ mice homozygous for the recessive obesity gene "diabetes" (db) permitted dissociation of the primary consequences of obesity gene expression from the secondary consequences of diabetes effected through interaction between the db gene and other diabetogenic genes in the inbred background. Estrone-treated db/db mice were similar to untreated mutants in exhibiting hyperphagia and marked obesity. However, estrone-treated mutants did not develop the hyperinsulinemia, hyperglycemia, and islet atrophy characteristic of untreated db/db mice. Thus, expression of the primary defect could be studied in the absence of the myriad secondary sequelae elicited by chronic hyperinsulinemia and hyperglycemia. Reduced numbers of hepatocyte plasma membrane insulin receptors (50% of normal) persisted in the estrone-treated mice in the absence of hyperinsulinemia, indicating that this deficiency was a consequence of the primary genetic defect and not merely a downregulation phenomenon secondary to hyperinsulinemia. Comparison of insulin secretion from comparably sized +/+ islets versus islets from estrone-treated db/db mice showed no intrinsic defects in beta-cell sensitivity to glucose. In conclusion, db-induced obesity can be dissociated from hyperinsulinemia, hyperglycemia, beta-cell dysfunction, and hyperphagia but is associated with a generalized membrane defect reflected in part by the persistent deficiency of plasma membrane insulin receptors.

The dynamic insulin secretory response of isolated pancreatic islets of the diabetic mouse. Evidence for a gene dosage effect on insulin secretion.

Expression of the autosomal recessive (db) gene in homozygous (db/db) C57BL/KsJ mice results in a severe and eventually fatal diabetic syndrome. Many studies of the diabetic mouse have used lean littermates (+/?) as controls despite evidence suggesting a gene dosage effect in heterozygous animals. In order to study the gene dosage effect of the diabetes (db) gene on insulin release in the heterozygote, perifusion experiments were performed on isolated islets of Langerhans of diabetic (db/db), heterozygous (+/db), and normal (+/+) control mice. Islets of normal controls exhibited a fivefold greater increase in insulin release than did those of diabetics in response to 16.7 mM D-glucose. The insulin secretory response of islets of heterozygotes to glucose was intermediate, being two-fold greater than that of diabetics but only about half of that of normal controls. Biphasic insulin release in response to glucose was observed only in islets of normal controls. Islets of all three genotypes exhibited biphasic insulin release in response to 10 mM D-glyceraldehyde; however, overall insulin release in both heterozygotes and diabetics remained diminished as compared with the response of normal controls. This is in contrast to the situation we have previously reported in islets of fasted or aging rats in which, though manifesting defects in glucose-stimulated insulin release, the islets are able to respond normally to 10 mM D-glyceraldehyde in respect to both the dynamic secretory pattern and quantity of insulin released. Our data suggest a gene dosage effect of the (db) gene on glucose-stimulated insulin release in heterozygous (+/db) C57BL/KsJ mice.

Calcium inhibition of parathyroid adenylate cyclase.

Parathyroid adenylate cyclase activity has previously been reported to be inhibited by calcium. However, the concentrations of calcium required to inhibit this enzyme were, in most instances, several orders of magnitude higher than cytoplasmic calcium concentrations. We undertook this study to determine the effects of calcium at submicromolar, as well as higher concentrations on parathyroid adenylate cyclase activity. A partially purified membrane fraction was obtained from normal porcine parathyroid glands using buffers free of divalent cation chelators. Relatively high concentrations of EGTA were then used in the incubation mixture to achieve the desired low ionic calcium concentrations. Addition of 0.5 mM EGTA to the reaction mixture resulted in an approximate 30% increase in basal adenylate cyclase activity and a similar percentage increase in the activity measured in the presence of guanosine triphosphate or NaF, known activators of parathyroid adenylate cyclase. EGTA also stimulated the activity measured in the presence of forskolin, which itself markedly stimulated this enzyme in a concentration-dependent manner. The effects of calcium added in amounts calculated to achieve ionic concentrations of 0.5 X 10(-7) M to 1 X 10(-3) M on the forskolin-activated enzyme activity was investigated. The resultant calcium inhibition curve was found to be stepwise in appearance. Analysis of the data using a mathematical model which assumes multiple, independent calcium-binding sites indicated the presence of two inhibitory sites. One had a high affinity for calcium, Kdiss 0.4-1.5 X 10(-6) M. This site accounted for 50-70% of the calcium-inhibitable activity. The second had a considerably lower affinity, Kdiss = 1.0-5.0 X 10(-4) M. Similar affinities were obtained from experiments in which guanosine triphosphate was used to activate the enzyme instead of forskolin. It is suggested that the high affinity calcium-binding site may be involved in the physiological regulation of parathyroid adenylate cyclase activity and that the previously reported estimates of the calcium sensitivity of parathyroid adenylate cyclase represent composites of the high and low affinity calcium-inhibitable processes.

Insulin release in aging: dynamic response of isolated islets of Langerhans of the rat to D-glucose and D-glyceraldehyde.

Glucose-stimulated insulin release is diminished in islets of Langerhans from older rats compared to that in islets from young controls. The causes of this age-related decrease in hormone release and its relationship to the hyperglycemia seen in aging populations have not been fully elucidated. In attempts to define this secretory defect, we demonstrated in static studies that the insulin secretion to D-glyceraldehyde is not diminished in aging. To gain further insight into the effects of D-glyceraldehyde vs. D-glucose in aging and to understand the dynamics of insulin release from islets of older rats, dynamic insulin release from isolated islets of 2.5- and 13-month-old rats was studied by the technique of perifusion to 2.8 mM and 16.7 mM D-glucose or 2.8 mM D-glucose with 5, 10, or 14 mM D-glyceraldehyde. Insulin secretion at nonstimulatory glucose concentrations (2.8 mM) was similar in the two groups of islets. Insulin release was reduced by 36% from islets of older rats incubated in the presence of 16.7 mM D-glucose, and the first phase of insulin release was largely blunted compared with that in islets from young controls. In the presence of 5.0, 10.0, or 14.0 mM D-glyceraldehyde (plus 2.8 mM D-glucose), total insulin secretion was similar from islets of older and young rats, and normal biphasic release was restored to islets from older rats. Response to the secretagogues was delayed by 1 min in studies on islets from older rats. These findings demonstrate that while the aging process leads to a profound defect in glucose-stimulated insulin release from the pancreatic beta-cell, this defect is not present with every secretagogue, since the normal secretory response is restored in the presence of D-glyceraldehyde. The differences in the insulin secretory responses to D-glucose and D-glyceraldehyde in islets from older rats support the hypothesis that the major rate-limiting step in stimulus-secretion coupling in aging is before the metabolism of the trioses.

The effects of magnesium on calcium inhibition of parathyroid adenylate cyclase.

cAMP has been shown to mediate the response of the parathyroid gland to a number of agonists and appears to take part in the regulation of this gland by divalent cations as well. We have studied the effects of the concentrations of free magnesium (Mg+2) and ionic calcium (Ca+2) on the kinetic properties of normal porcine parathyroid adenylate cyclase. In a previous study we obtained evidence for two calcium inhibition sites in this enzyme complex. In the present study we observed that the Mg+2 concentration influences the relative contribution of these sites to the overall calcium inhibition. At a high Mg+2 concentration (10 mM), the high affinity site contributes less than 50% of the total calcium inhibitable activity, whereas at a Mg+2 concentration in the low physiological range (0.5 mM), the high affinity site accounts for all the calcium inhibitable activity. Mg+2 was found to be a potent activator of porcine parathyroid adenylate cyclase, with a Ka of Mg of 0.8-2 mM. Ionic calcium at low concentrations (0.2-5 microM) acts as a competitive inhibitor with respect to Mg+2 activation. The calcium inhibition constant was estimated to be 2-3 microM. The Km for ATPMg-2 was 0.3 mM, which is similar to that found in other studies of adenylate cyclase activity in parathyroid tissue. The effects of Ca+2 on enzymatic activity with respect to the ATPMg-2 concentration showed noncompetitive inhibition. The calcium inhibition constant with respect to its effect on Vmax (KIv) was 3 microM; the calcium inhibition constant with respect to its effect on the binding of ATPMg-2 (KIs) was 10 microM. It is concluded from these results that the concentrations of intracellular Ca+2 reported to be present in parathyroid cells could inhibit adenylate cyclase activity. The mode of calcium inhibition that involves competition with magnesium would be particularly significant at low intracellular Mg+2 concentrations, and this phenomenon may account for the parathyroid secretory defect which is a characteristic feature of the magnesium-deficient state.

Immunoreactive luteinizing hormone, follicle stimulating hormone and their subunits in tissue culture media from normal and adenomatous, human pituitary fragments.

Fragments from 4 human pituitaries removed at surgery from one gonadectomized man and three women (one with Nelson's syndrome, one with Forbes Albright syndrome and one with chromophobe adenoma) were grown in tissue culture. The tissue culture medium was changed at weekly intervals, pooled and applied to a Sephadex G-100 column for gel filtration. In each eluate luteinizing hormone (LH), follicle stimulating hormone (FSH), alpha subunit and the beta subunits of LH and FSH were measured by specific radioimmunoassays. For comparison the pituitary standard LER 907 was similarly studied. LH and FSH were measurable in all studies. LH eluted over a broad area whereas FSH eluted as a much sharper peak. In all culture media and in LER 907 large quantities of alpha subunit were detected. The beta subunits of LH and FSH were not present in the LER 907 standard. LHbeta subunit was present in the culture medium of the pituitary fragments from the castrated man and from the women with Nelson's syndrome and Forbes Albright syndrome but not in that of the woman with chromophobe adenoma. FSHbeta subunit was detectable only in the latter case.

Diabetes in the elderly: diagnosis, pathogenesis, and therapy.

The prevalence of diabetes mellitus increases with age; it occurs in approximately 10 percent of Americans 60 years of age and in 16 to 20 percent of those 80 years old. Type II diabetes mellitus is primarily found in the elderly, and it is estimated that an additional 20 percent of the elderly population has age-associated hyperglycemia, which may be part of a spectrum between normality and type II diabetes. The diabetic group is at risk for both microvascular and macrovascular complications of diabetes, whereas the group with the hyperglycemia of aging may be at risk for macrovascular-type complications. Thus, 40 percent of our senior population has abnormal carbohydrate tolerance and is at risk for diabetic-type chronic complications. The basis for both the diabetic state as well as for the hyperglycemia of aging is probably multifactorial--involving both altered insulin secretion and altered insulin action. Unique problems arise in treating older diabetic patients. Physiologic changes occurring during normal aging, age-associated pathologic processes, the increased prevalence of other chronic diseases, and polypharmacy must all be considered in selecting appropriate therapy for these patients. A rational approach for the maintenance of glucose homeostasis is presented for older patients with diabetes.

Treatment of hypertension in diabetic men: problems with sexual dysfunction.

Hypertension occurs 2 to 3 times more frequently in diabetic persons than in nondiabetic persons. Care must be taken in treating hypertension in diabetic patients because the choice of antihypertensive agent may worsen the diabetic state or its complications or cause additional health problems for the patient. Sexual dysfunction is a common problem in diabetic patients; however, diabetes need not be the cause. Diabetic men with hypertension have an increased sensitivity to the side effect of sexual dysfunction, which occurs from the use of centrally acting antihypertensive agents. By using prazosin, an alpha 1-adrenergic blocking agent, this cause of sexual dysfunction was eliminated. The reasons for the increased prevalence of hypertension in diabetic patients are discussed and a rational approach is given for the treatment of elevated blood pressure in these individuals.

Detecting diabetes in an elderly dental patient population.

The prevalence of both diagnosed and undiagnosed diabetes, a disease which has a significant impact on dental care delivery, increases with age. Because of these two factors, the dental practitioner must pay special attention to the detection of diabetes in this age group. This paper describes a study that validates the use of a simple blood test in conjunction with a questionnaire as a method to detect undiagnosed diabetes in an elderly dental patient population.

The high affinity calcium inhibition of parathyroid adenylate cyclase is not calmodulin dependent.

To investigate the possible role of calmodulin in the calcium sensitivity of parathyroid adenylate cyclase (AC), the effect of the calmodulin inhibitor trifluoperazine hydrochloride (TFP) on the calcium sensitivity of forskolin-stimulated AC activity was investigated in membranes prepared from normal porcine parathyroid glands. TFP inhibited AC in a concentration-dependent manner, the IC50 being approximately 100 microM. The inhibition of the enzyme occurred at roughly the same concentration of TFP in the presence and absence of calcium. Another calmodulin inhibitor, N-(6-aminohexyl)-chloro-1-naphthalenesulfonamide (W-7), also inhibited AC in a calcium-independent manner with a IC50 of approximately 200 microM. The pattern of calcium inhibition of AC was compared in membranes prewashed with either EGTA or 2 microM ionic calcium plus 100 microM TFP in an attempt to remove endogenous calmodulin. Neither treatment significantly altered the apparent affinities of the two previously reported calcium inhibition sites, nor did they alter the relative contribution of the individual calcium inhibition sites to the overall calcium inhibition. Inclusion of 100 microM TFP in the incubation mixture resulted in no change in the apparent affinities of the calcium inhibition site although it did result in a significant decrease in the relative contribution of the high affinity site (P less than 0.05). Addition of exogenous calmodulin (5-50 micrograms/ml) had no significant effect on AC. We conclude from these studies that the inhibition of parathyroid AC by calcium is independent of calmodulin and that this enzyme has intrinsic high sensitivity to calcium.

Presence of calmodulin in parathyroid adenomas.

A significant percent of parathyroid adenomas demonstrate decreased sensitivity to the suppressive effects of calcium on parathyroid hormone secretion. The recognition that calmodulin mediates a large number of calcium-dependent cellular events suggests that calmodulin may play a key role in the normal regulation by calcium of parathyroid activity. We therefore undertook to determine if parathyroid adenomas contained calmodulin and if so, if the calmodulin content of adenomas could be correlated with the serum concentrations of calcium and immunoreactive parathyroid hormone (IPTH) in hyperparathyroid patients prior to surgery. Calmodulin was assayed by activation of a calmodulin-stimulatable phosphodiesterase (PDE) obtained from rat brain. We found PDE-stimulating activity in each of six adenoma extracts examined. The stimulation had the following properties, which are characteristic of calmodulin-mediated processes: (1) it required the presence of calcium; (2) the dose response to adenoma extract and human red blood cell calmodulin were parallel, and (3) the stimulation was phenothiazine-inhibitable. The amount of calmodulin present in the adenoma extracts ranged from 0.4 to 1.25 units/micrograms protein. No correlation was found between the calmodulin content of the adenomas and the presurgical levels of either serum calcium or serum IPTH.

Impaired potassium-induced insulin secretion in chronic renal failure.

Extrarenal disposal of potassium load is impaired in chronic renal failure (CRF). This has been attributed to excess PTH since extrarenal disposition of potassium is normal in CRF-PTX animals. Insulin augments potassium entry into cells and hyperkalemia stimulates insulin secretion. Since glucose-induced insulin secretion is impaired in CRF and normal in CRF-PTX, it is possible that K(+)-induced insulin secretion is also impaired in CRF due to excess PTH. Such a defect would contribute to the abnormality in extrarenal disposal of potassium in CRF. We examined K(+)-induced insulin secretion, cytosolic calcium ([Ca2+]i) and the changes in [Ca2+]i in response to 20 mM KCl of islets from normal, CRF, and CRF-PTX rats; and normal and CRF animals treated with verapamil (normal-V and CRF-V). K(+)-induced insulin secretion by islets isolated from CRF rats was significantly (P less than 0.01) lower than that from normal, CRF-PTX, CRF-V and normal-V rats. Basal level of [Ca2+]i in islets of CRF rats was significantly (P less than 0.01) higher than in islets of the other four groups of animals. The calcium signal (delta [Ca2+]i) and the delta [Ca2+]i/basal [Ca2+]i ratio in response to 20 mM KCl observed in islets from CRF rats were significantly lower than in the other four groups of animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of adrenal glands in the development of abnormal glucose and insulin homeostasis in genetically obese (ob/ob) mice.

This study evaluates the role of adrenal hormones in the development of hyperinsulinaemia and impaired glucose homeostasis in genetically obese hyperglycaemic C57BL/6J ob/ob mice. Lean (+/?) and obese mice were bilaterally adrenalectomised or sham operated at 5 weeks of age, and glucose tolerance was examined after 7 and 14 days. Adrenalectomy temporarily reduced food intake and body weight gain in lean mice, and improved glucose tolerance without a significant change in plasma insulin concentrations at both intervals studied. In obese mice adrenalectomy permanently reduced body weight gain and food intake to values comparable with lean mice. Glucose tolerance was improved in adrenalectomised obese mice at both intervals studied, resulting in plasma glucose concentrations similar to adrenalectomised lean mice. Plasma insulin concentrations during the tolerance tests were reduced in adrenalectomised obese mice, but remained higher than in lean mice. Adrenalectomy did not improve the poor insulin response to parenteral glucose in obese mice. The results indicate that adrenal hormones play an important role in the development of glucose intolerance and contribute to the hyperinsulinaemia in obese (ob/ob) mice, in part by promoting hyperphagia.