Transmural differences in respiratory capacity across the rat left ventricle in health, aging, and streptozotocin-induced diabetes mellitus: evidence that mitochondrial dysfunction begins in the subepicardium.

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.

Copper(II)-selective chelation improves function and antioxidant defences in cardiovascular tissues of rats as a model of diabetes: comparisons between triethylenetetramine and three less copper-selective transition-metal-targeted treatments.

AIMS/HYPOTHESIS: Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress. METHODS: Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. RESULTS: Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations. CONCLUSIONS/INTERPRETATION: TETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of 'loosely-bound'/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.

A copper(II)-selective chelator ameliorates left-ventricular hypertrophy in type 2 diabetic patients: a randomised placebo-controlled study.

AIMS/HYPOTHESIS: Cu(II)-selective chelation with trientine ameliorates cardiovascular and renal disease in a model of diabetes in rats. Here, we tested the hypothesis that Cu(II)-selective chelation might improve left ventricular hypertrophy (LVH) in type 2 diabetic patients. METHODS: We performed a 12 month randomised placebo-controlled study of the effects of treatment with the Cu(II)-selective chelator trientine (triethylenetetramine dihydrochloride, 600 mg given orally twice daily) on LVH in diabetic patients (n = 15/group at baseline) in an outpatient setting wherein participants, caregivers and those assessing outcomes were blinded to group assignment. Using MRI, we measured left ventricular variables at baseline, and at months 6 and 12. The change from baseline in left ventricular mass indexed to body surface area (LVM(bsa)) was the primary endpoint variable. RESULTS: Diabetic patients had LVH with preserved ejection fraction at baseline. Trientine treatment decreased LVM(bsa) by 5.0 +/- 7.2 g/m(2) (mean +/- SD) at month 6 (when 14 trientine-treated and 14 placebo-treated participants were analysed; p = 0.0056 compared with placebo) and by 10.6 +/- 7.6 g/m(2) at month 12 (when nine trientine-treated and 13 placebo-treated participants were analysed; p = 0.0088), whereas LVM(bsa) was unchanged by placebo treatment. In a multiple-regression model that explained ~75% of variation (R (2) = 0.748, p = 0.001), cumulative urinary Cu excretion over 12 months was positively associated with trientine-evoked decreases in LVM(bsa). CONCLUSIONS/INTERPRETATION: Cu(II)-selective chelation merits further exploration as a potential pharmacotherapy for diabetic heart disease. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN 12609000053224 FUNDING: The Endocore Research Trust; Lottery Health New Zealand; the Maurice and Phyllis Paykel Trust; the Foundation of Research, Science and Technology (New Zealand); the Health Research Council of New Zealand; the Ministry of Education (New Zealand) through the Maurice Wilkins Centre for Molecular Biodiscovery; and the Protemix Corporation.

A novel dephosphorylation-activated conductance in a mouse renal collecting duct cell line.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. It is associated with the progressive development of renal tubular cysts, which may subsequently lead to renal failure. Studies into the genetic basis of ADPKD have identified two genes, PKD1 and PKD2, that are mutated in ADPKD patients. The PKD1 and PKD2 genes encode for two different proteins, TRPP1 and TRPP2. Previous studies have demonstrated the presence of both TRPP1 and TRPP2 in the renal collecting duct cell line M8. The aim of the following study was to investigate the functional properties of cation currents in these cells and to examine the effect of overexpression of TRPP1 using a transgenic cell model (M7). In M8 cells, initial whole cell currents were low. However, over time there was activation of a flow-sensitive current, which was inhibited by gadolinium (I(Gd)). The I(Gd) was more selective for cations over anions, but did not discriminate between monovalent cations and was Ca2+ permeable. Activation of I(Gd) was dependent on the presence of Ca2+ and also required dephosphorylation. The protein phosphatase 2A inhibitor okadaic acid prevented activation of I(Gd), suggesting that protein phosphatase 2A plays an important role in channel activation. The properties and magnitude of I(Gd) were unaffected in M7 cells, suggesting that overexpression of TRPP1 was without effect. I(Gd) was selectively inhibited by an antibody raised against the C-terminus of TRPP2. However, its selectivity profile was different to TRPP2, suggesting that it is attributable to a TRPP2-like channel or a TRPP2-containing heteromeric channel. In conclusion, these data describe the functional identification of a novel dephosphorylation- and flow-activated TRPP2-related channel in mouse collecting duct cells.

Coordination of mammary metabolism and blood flow after refeeding in rats.

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 mumol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.

The role of amylin in the insulin resistance of non-insulin-dependent diabetes mellitus.

Decreased responsiveness of glucose metabolism to insulin in skeletal muscle and the liver (insulin resistance or insensitivity) is characteristic of many conditions, including non-insulin-dependent (type II) diabetes mellitus. Most current work in this area centres on the hypothesis that the primary defect is an impairment of insulin binding and/or transduction of the insulin signal in affected tissues. However, studies imply that defects in the post-insulin receptor signaling pathways are of primary importance in the causation of insulin resistance. Amylin, a novel pancreatic hormone, secreted along with insulin from the pancreatic beta-cells, can modulate insulin effects, to produce insulin resistance in skeletal muscle and liver.

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Effect of high-fat meals and fatty acid saturation on postprandial levels of the hormones ghrelin and leptin in healthy men.

OBJECTIVE: Ghrelin and leptin play a role in control of food intake and adiposity but mechanisms regulating these hormones in man are poorly defined and evidence that dietary fats may have adverse effects is inconclusive. We investigated whether high-fat meals, which differed in saturated fatty acid (SFA) content acutely modified these hormones. DESIGN: Randomised, double-blind, crossover trial. A high-fat (HF) test meal (59 +/- 4 g fat; 71% of energy as fat) was given for breakfast on two occasions. Meals comprised either high (approximately 70:30) or low (approximately 55:45) saturated:unsaturated fatty acid (SFA:USFA) ratio. Fasting and postprandial measurements of serum total ghrelin (RIA), leptin (enzyme-linked immunosorbent assay (ELISA)) and insulin (RIA) were made over 6 h. Postprandial measurements were also made at 10 and 24 h following a fat-exclusion lunch, snack and dinner. SUBJECTS: A total of 18 lean, healthy men. RESULTS: There was no significant effect of the fatty meal (time, P > 0.05), nor a differential effect of SFA:USFA ratio (treatment*time, P > 0.05) on ghrelin over 6h. Leptin decreased in response to both HF treatments (time, P < 0.001) but increased SFA content did not further inhibit hormone secretion (treatment*time, P > 0.05). There was no significant correlation between ghrelin or leptin and circulating insulin (P>0.05). CONCLUSION: We conclude that HF diets may adversely effect serum leptin, although the circadian decrease may account in part for this response. Increasing dietary SFAs had no deleterious effects on leptin or total ghrelin.

Nucleotide sequence of a cDNA for canine amylin.

Canine amylin cDNA was cloned and sequenced from a dog pancreas library using oligonucleotide probes corresponding to portions of human amylin. The sequence reported here is significantly different from an incomplete sequence previously reported. The non-amyloidogenic canine mature peptide is 89% and 95% identical to its amyloidogenic human and cat homologues, respectively. Both the dog and cat peptides are identical over the proposed amyloidogenic region, amino acids 20-29. Hydropathy plots comparing these three peptides are nearly identical suggesting that the primary sequence of amylin is in itself insufficient to explain pancreatic amyloid formation.

Amylin and the amylin gene: structure, function and relationship to islet amyloid and to diabetes mellitus.

Amylin, the major peptide component of the islet amyloid commonly found in the pancreases of patients with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM), is a recently discovered islet polypeptide. This peptide has many structural and functional features suggesting that it is a novel hormone, which may control carbohydrate metabolism in partnership with insulin and other glucoregulatory factors. Amylin is synthesised in, and probably secreted from, the beta-cells of the islets of Langerhans, where it has recently been immunolocalised to secretory granules. DNA cloning studies indicate that in the human and the rat, amylin is generated from a precursor, preproamylin, which displays a typical signal peptide followed by a small prohormone-like sequence containing the amylin sequence. The presence of the signal peptide suggests that amylin is secreted and plays a physiological role. Amylin is probably generated by proteolytic processing similar to that for proinsulin and other islet prohormones. The human amylin gene encodes the complete polypeptide precursor in two exons which are separated by an intron of approx. 5 kb, and is located on chromosome 12. Amylin is a potent modulator of glycogen synthesis and glucose uptake in skeletal muscle, and is capable of inducing an insulin-resistant state in this tissue in vitro, and perhaps also in the liver in vivo. In normal metabolism, amylin could act in concert with insulin as a signal for the body to switch the site of carbohydrate disposal from glycogen to longer-term stores in adipose tissue, by making skeletal muscle relatively insulin-resistant, whilst at the same time leaving rates of insulin-stimulated carbohydrate metabolism in adipose tissue unaltered. Several lines of evidence now implicate elevated amylin levels in the pathogenic mechanisms underlying NIDDM, and suggest to us that the obesity which frequently accompanies this syndrome is a result of, rather than a risk factor for, NIDDM. Following the beta-cell destruction which occurs in type 1 (insulin-dependent) diabetes mellitus (IDDM), it is probable that amylin secretion disappears in addition to that of insulin. As patients with insulin-treated IDDM frequently experience problems with hypoglycaemia, and as amylin acts to modulate the action of insulin in various tissues, it is possible that amylin deficiency may contribute to morbidity in insulin-treated IDDM, perhaps through the loss of a natural damping mechanism which guards against hypoglycaemia under conditions of normal physiology.

Induction of insulin resistance in vivo by amylin and calcitonin gene-related peptide.

During hyperinsulinemic glucose-clamp studies, intravenous infusion of calcitonin gene-related peptide (CGRP) in rats antagonized the ability of insulin to stimulate peripheral glucose disposal by 52% (196 +/- 7.2 vs. 105 +/- 10.5 mumol.kg-1.min-1, P less than 0.05) and to inhibit hepatic glucose output by 54% (P less than 0.01). CGRP also inhibited the in vitro effects of insulin to stimulate hexose uptake in cultured BC3H1 myocytes at all insulin concentrations studied. Amylin is a peptide isolated from amyloid deposits in pancreatic islets of type II (non-insulin-dependent) diabetic subjects, is present in normal beta-cells, and bears a striking homology to CGRP. When synthetic human amylin was infused during clamp studies, it inhibited the ability of insulin to stimulate glucose disposal by 56% (96.9 +/- 9.4 vs. 42.4 +/- 5.0 mumol.kg-1.min-1, P less than 0.05) and to suppress hepatic glucose output by 64%. Therefore, amylin and CGRP can cause insulin resistance in vivo and may be implicated in insulin-resistant states such as type II diabetes mellitus.

Alteration in phosphorylation of P20 is associated with insulin resistance.

We have recently identified a small phosphoprotein, P20, as a common intracellular target for insulin and several of its antagonists, including amylin, epinephrine, and calcitonin gene-related peptide. These hormones elicit phosphorylation of P20 at its different sites, producing three phosphorylated isoforms: S1 with an isoelectric point (pI) value of 6.0, S2 with a pI value of 5.9, and S3 with a pI value of 5.6 (FEBS Letters 457:149-152 and 462:25-30, 1999). In the current study, we showed that P20 is one of the most abundant phosphoproteins in rat extensor digitorum longus (EDL) muscle. Insulin and amylin antagonize each other's actions in the phosphorylation of this protein in rat EDL muscle. Insulin inhibits amylin-evoked phosphorylation of S2 and S3, whereas amylin decreases insulin-induced phosphorylation of S1. In rats made insulin resistant by dexamethasone treatment, levels of the phosphoisoforms S2 and S3, which were barely detectable in healthy rats in the absence of hormone stimulation, were significantly increased. Moreover, the ability of insulin to inhibit amylin-evoked phosphorylation of these two isoforms was greatly attenuated. These results suggested that alterations in the phosphorylation of P20 might be associated with insulin resistance and that P20 could serve as a useful marker to dissect the cellular mechanisms of this disease.

Watching amyloid fibrils grow by time-lapse atomic force microscopy.

Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

Insulin resistance in the Zucker diabetic fatty rat: a metabolic characterisation of obese and lean phenotypes.

The Zucker diabetic fatty (ZDF) rat is a commonly used animal model of type 2 diabetes yet complete descriptions of insulin resistance in this model are limited. We present a full characterisation of in vivo insulin resistance in obese (fa/fa) animals compared to lean (+/?) littermates. Anaesthetised, ten-week old, obese ZDF rats and their lean littermates underwent a hyperinsulinaemic euglycaemic glucose clamp. Compared with lean littermates, obese ZDF rats required an 89% lower glucose infusion rate to maintain euglycaemia and showed a 35% decrease in peripheral glucose disposal. Insulin-stimulated glucose uptake (R(g')) in obese animals was also significantly less in all skeletal muscles studied. R(g') in cardiac and white adipose tissue was not different between the two groups. Total glycogen content in skeletal and cardiac muscle was significantly less in obese animals, while total glycogen content in the liver was significantly greater than in lean littermates. Glycogen synthesis was also decreased in skeletal muscle of obese animals. Compared with lean animals, total triglyceride content was significantly greater in skeletal muscle, heart and liver of obese ZDF rats. Obese animals also showed significantly increased glucose incorporation into lipid in all of these tissues, indicating an increase in lipogenesis. Collectively, these results provide an integrated characterisation of in vivo insulin resistance in obese ZDF rats and a direct comparison with lean littermates.

HIV-1 western blot: development and assessment of testing to resolve indeterminate reactivity.

OBJECTIVE: To reduce the number of HIV-1 Western blot (WB)-indeterminates requiring follow-up and the time taken to provide a clear positive or negative result. DESIGN: In the first of two stages, a testing and follow-up strategy was developed to resolve anti-HIV-1 status of WB-indeterminates. In the second stage, implementation of this strategy was assessed. METHODS: After dividing indeterminates into four groups according to WB profile, samples were tested for anti-HIV-1, anti-HIV-2, anti-HTLV-I antibodies, and HIV-1 antigen using the most sensitive assays available. When testing failed to clarify anti-HIV-1 status, follow-up samples were taken to monitor changes in antibody status. RESULTS: Samples in two out of the four indeterminate groups were negative for anti-HIV-1. The other two groups required additional testing and/or follow-up to distinguish reactivity caused by anti-HIV-1 from cross-reactivity. CONCLUSION: Grouping HIV-1 WB-indeterminates according to profile allows a significant percentage to be reported as anti-HIV-1-negative, while additional testing may allow others to be reported as anti-HIV-1-positive. The remainder require a maximum of 3 months' follow-up to resolve anti-HIV-1 status.

Comparison of the effects of calcitonin gene-related peptide and amylin on osteoblasts.

Calcitonin gene-related peptide (CGRP) and amylin are homologous 37-amino-acid peptides which have been demonstrated to have anabolic effects on bone. It is not clear whether these effects are mediated by a common receptor, nor is it known which ligand is the more potent. These questions are addressed in the present study using cultures of fetal rat osteoblasts. CGRP increased cell number when present in a concentration >/=10-9 M, but 10-8 M CGRP was required to stimulate thymidine and phenylalanine incorporation. Amylin was effective on these indices at 100-fold lower concentrations, and its maximal effects were about twice as great as those of CGRP. ED50's for the effects of amylin and CGRP on cell number were 10-12 M and 10-10 M, respectively. There was no additivity between maximal doses of the peptides on these indices. The effects of specific receptor blockers on the maximal stimulation of cell number by these peptides were also studied. The CGRP receptor-blocker, CGRP-(8-37), completely blocked the effect of CGRP at blocker concentrations >/=10-9 M. In contrast, the amylin receptor blocker, amylin-(8-37), completely blocked the effects of CGRP when the blocker was present in concentrations as low as 10-11 M. The KI of CGRP-(8-37) was 2 x 10-10 M and that of amylin-(8-37) was 7 x 10-12 M. In converse experiments studying the blockade of maximal doses of amylin, amylin-(8-37) 10-10 M was effective (KI 1 x 10-10 M), whereas a 100-fold greater concentration of CGRP-(8-37) was necessary to achieve the same effect (KI 6 x 10-9 M). It is concluded that amylin and CGRP probably act through a common receptor to stimulate osteoblast growth, and that this receptor has a higher affinity for amylin than for CGRP.

A potential role for adrenomedullin as a local regulator of bone growth.

Bone remodeling is a complex process of coordinated resorption and formation of bone, which is regulated by systemic hormones and by local factors. We have previously shown that the peptide hormone adrenomedullin is mitogenic to osteoblastic cells in vitro and that it promotes bone growth in vivo. The aim of the present study was to characterize the expression of molecules that may mediate adrenomedullin signaling in osteoblasts and to investigate the expression of adrenomedullin itself in these cells. The first adrenomedullin receptor that was cloned is the seven-transmembrane G protein-coupled receptor, L1. Two additional receptors for adrenomedullin, which arise from interactions between calcitonin receptor-like receptor and receptor activity modifying proteins 2 or 3, have now been described. In the current study, we used RT-PCR and Northern blot analysis to demonstrate that messenger RNA for the three adrenomedullin receptors, as well as for adrenomedullin itself, is expressed in primary rat osteoblasts. Treating primary osteoblasts with transforming growth factor-beta and insulin-like growth factor-I moderately reduced adrenomedullin RNA levels, whereas PTH had no effect. We have shown by immunocytochemistry that adrenomedullin peptide is present in osteoblasts, and by competitive binding assays that (125)I-adrenomedullin binds with high affinity to intact osteoblasts and to osteoblast cell membranes. Coexpression of adrenomedullin and adrenomedullin receptors in osteoblasts, taken together with our previous finding that adrenomedullin is mitogenic to these cells, raises the possibility that this peptide is a local regulator of bone growth.

Hemodynamic, hormonal, and renal effects of intracerebroventricular adrenomedullin in conscious sheep.

Adrenomedullin, the recently described vasodilator that exhibits potent hypotensive actions when administered systemically, is also found in the central nervous system, suggesting a role for adrenomedullin as a neurohormone. However, only a limited number of studies have examined the central effects of adrenomedullin. Therefore, we have examined the integrative hemodynamic, renal, and hormonal effects of intracerebroventricular (I.C.V.) adrenomedullin in conscious sheep. Eight surgically prepared sheep received I.C.V. infusions of adrenomedullin at two doses (2 ng/kg x min followed immediately by 20 ng/kg x min each for 90 min) in a vehicle-controlled study. Water deprivation for 48 h before control infusion resulted in sheep drinking 2617 +/- 583 ml in the 90-min period following reintroduction of water. On the adrenomedullin day, drinking was halved to 1392 +/- 361 ml (P < 0.05). Adrenomedullin had no significant effect on urinary volume and sodium excretion. Plasma adrenomedullin levels remained unchanged during control infusions but were elevated by the end of I.C.V. adrenomedullin infusions (P < 0.001). Plasma ANP levels were also increased approximately 50% (P < 0.05). Plasma levels of both ACTH and cortisol were also increased 3- to 4-fold in response to I.C.V. adrenomedullin (P < 0.05). There was no significant difference in arterial pressure, heart rate, or cardiac output between study days. In conclusion, adrenomedullin within the central nervous system may have at least two roles: modulation of the hypothalamo-pituitary-adrenal axis and protection against fluid overload.

Hyperamylinemia, hyperinsulinemia, and insulin resistance in genetically obese LA/N-cp rats.

The experimental evidence supporting a direct role for hyperinsulinemia as a cause of insulin resistance remains equivocal. Amylin, an islet beta-cell peptide cosecreted with insulin in response to nutrient stimuli, causes insulin resistance when infused into intact animals or applied to isolated skeletal muscles. We compared measures of amylin and insulin gene expression between control and genetically obese, insulin-resistant Lister Albany/NIH-(LA/N-cp) rats. Pancreatic amylin messenger RNA levels were increased 7.8 +/- 0.7-fold (mean +/- SEM), and plasma amylin-like immunoreactive material was increased 10.9 +/- 1.1-fold (LA/N-lean, 14 +/- 4 pM; LA/N-cp, 153 +/- 16 pM; p less than 0.0001) in obese rats. Pancreatic insulin I mRNA levels were increased 7.4 +/- 0.5-fold, and plasma insulin levels 20.0 +/- 5.0-fold, in these rats (LA/N-lean, 308 +/- 84 pM; LA/N-cp 6,120 +/- 1,540 pM; p less than 0.0001). The EC50 for insulin-stimulated incorporation of glucose into glycogen was about fourfold higher in muscles isolated from obese rats. The present results, coupled with previous observations, support the hypothesis that hyperamylinemia, rather than hyperinsulinemia per se, could have directly caused the insulin resistance in the obese LA/N-cp rats. Hyperamylinemia needs to be considered in future experimental studies probing the relation between hyperinsulinemia and insulin resistance.