Elevated plasma immunoreactive neuropeptide Y concentrations and its increased urinary excretion in patients with advanced diabetic nephropathy.

Neuropeptide Y (NPY) is a potent vasoconstrictor peptide that is abundant in the brain and the peripheral sympathetic nervous system. In the present study we investigated possible changes in plasma immunoreactive (IR)-NPY concentrations and urinary IR-NPY excretion in patients with non-insulin dependent diabetes mellitus (NIDDM) and the relationship to diabetic complications, such as nephropathy and neuropathy. IR-NPY in plasma and urine was measured by radioimmunoassay in 69 patients with NIDDM. Plasma IR-NPY concentrations in patients with advanced nephropathy (creatinine clearance <30 ml/min) (100.5 +/- 10.3 pmol/l, n=9, mean +/- SEM) were higher than in the control subjects (55.0 +/- 6.8 pmol/l, n=15) (P<0.02). Urinary excretion of IR-NPY and fractional excretion of NPY were also increased in the patients with advanced nephropathy. Sephadex G-50 column chromatography of the urine extracts obtained from healthy subjects, diabetic patients with renal failure and non-diabetic patients with renal failure showed an immunoreactive peak eluting in the NPY position. On the other hand, neither plasma nor urinary IR-NPY was high in patients with retinopathy, or in patients with peripheral neuropathy. The present study has, for the first time, shown high plasma IR-NPY concentrations and urinary IR-NPY excretion in NIDDM patients with advanced nephropathy.

Biotin enhances glucose-stimulated insulin secretion in the isolated perfused pancreas of the rat.

The effects of biotin on insulin secretion in pair-fed control rats and biotin-deficient rats were investigated using the method of isolated pancreas perfusion. Isolated pancreas perfusion was performed using 20 mM glucose, 10 mM arginine, and 20 mM glucose plus various concentrations of biotin (20 mM glucose + biotin solution) as stimulants of insulin secretion. The insulin response to 20 mM glucose in biotin-deficient rats was approximately 22% of that seen in control rats. The level of the insulin response to 10 mM arginine was also significantly lower in biotin-deficient rats than in control rats. These results indicate that insulin release from the pancreas was disturbed in biotin-deficient rats. The insulin responses to 20 mM glucose + 1 mM biotin in biotin-deficient and control rats increased to 165% and 185%, respectively, of that to 20 mM glucose. These biotin-induced increases in glucose-stimulated insulin release were evident within the first few minutes of the infusion. An enhancement of the arginine-induced insulin response in control rats was not found when arginine and biotin was administered. These results suggest that biotin may play an important role in the mechanism by which glucose stimulates insulin secretion from the beta cells of the pancreatic islets.

Role of nitric oxide in obesity-induced beta cell disease.

Here we report that free fatty acid-induced suppression of insulin output in prediabetic Zucker diabetic fatty (ZDF) rats is mediated by NO. When normal islets were cultured in 2 mM FFA, NO production and basal insulin secretion increased slightly. In cultured prediabetic ZDF islets, FFA induced a fourfold greater rise in NO, upregulated mRNA of inducible nitric oxide synthase (iNOS), and reduced insulin output; both nicotinamide and aminoguanidine, which lower NO, prevented the FFA-mediated increase in iNOS mRNA, reduced NO, and minimized the loss of insulin secretion. In vivo nicotinamide or aminoguanidine treatment of prediabetic ZDF rats prevented the iNOS expression in islets and decreased beta cell dysfunction while blocking beta cell destruction and hyperglycemia. We conclude that NO-lowering agents prevent adipogenic diabetes in obese rats.

Cerebellin and cerebellin mRNA in the human brain, adrenal glands and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma.

The expression of cerebellin and cerebellin mRNA was studied by radioimmunoassay and Northern blot analysis in the human brain, adrenal gland and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma. Immunoreactive cerebellin was detected in every region of brain studied, with the highest concentrations found in the hemisphere of the cerebellum (424.2 +/- 12.6 pmol/g wet weight, n = 6, mean +/- S.E.M.) and the vermis of the cerebellum (256.8 +/- 30.5 pmol/g wet weight). Immunoreactive cerebellin was also detected in the pituitary (8.2 +/- 1.8 pmol/g wet weight), the spinal cord (3.3 +/- 0.3 pmol/g wet weight) and the normal parts of adrenal glands (2.98 +/- 0.37 pmol/g wet weight, n = 9) and some tumour tissues, such as phaeochromocytomas, cortisol-producing adrenocortical adenomas, ganglioneuroblastomas and neuroblastomas. Northern blot analysis showed that cerebellin mRNA was highly expressed in the hemisphere and vermis of the cerebellum. Cerebellin mRNA was also expressed in other regions of the brain and the tumour tissues of phaeochromocytoma, cortisol-producing adrenocortical adenoma, ganglioneuroblastoma and neuroblastoma. Immunocytochemistry of the normal adrenal gland showed that immunoreactive cerebellin was localized in the adrenal medulla. The present study has shown the expression of cerebellin and cerebellin mRNA, not only in the cerebellum but also in other regions of the brain and some tumours, such as cortisol-producing adrenocortical adenoma, phaeochromocytoma and neuroblastoma. These findings suggest possible pathophysiological roles of cerebellin peptides, not only in the cerebellum, but also in the extra-cerebellar tissues.

Immunoreactive adrenomedullin in human adrenal glands and adrenal tumors.

Adrenomedullin is a potent vasodilator peptide that was isolated from human pheochromocytoma. We developed a sensitive and specific radioimmunoassay for adrenomedullin and studied the presence of adrenomedullin in human adrenal glands and adrenal tumors, including pheochromocytoma. High concentrations of immunoreactive adrenomedullin were found in normal parts of adrenal glands (cortex and medulla) (12.6 +/- 1.0 pmol/g wet wt, N = 7, mean +/- SEM). High concentrations of immunoreactive adrenomedullin were also present in the tumor tissues of pheochromocytoma (4.5 +/- 1.5 pmol/g wet wt, N = 11). Immunoreactive adrenomedullin was detected in some adrenocortical tumors, but these concentrations were much lower than those in the normal adrenal glands and pheochromocytomas. Reverse phase high-performance liquid chromatography of the normal adrenal gland and pheochromocytoma showed a peak eluting in the position of synthetic adrenomedullin 1-52. The present study has shown the presence of high concentrations of immunoreactive adrenomedullin in the normal adrenal glands and pheochromocytomas.

Specific adrenomedullin binding sites in the human brain.

Binding sites for adrenomedullin in human brain were investigated and characterized by radioligand binding. Specific binding sites for adrenomedullin were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons and medulla oblongata) obtained at autopsy. Despite the homology with calcitonin gene-related peptide (CGRP), CGRP was a poor inhibitor of [125I]adrenomedullin binding (IC50 > 1 microM) compared with adrenomedullin(1-52) (IC50 = 1.2 +/- 0.5 nM, mean +/- SEM, n = 3). Three adrenomedullin fragments, adrenomedullin(1-12), adrenomedullin(22-52), and adrenomedullin(13-52), were also poor inhibitors of the binding (IC50 = 0.3 microM), suggesting that the whole molecule of adrenomedullin(1-52) is required for binding to the receptor. Scatchard plots of [125I]adrenomedullin binding in human brain (cerebral cortex) gave a dissociation constant of 0.17 +/- 0.03 nM and maximal binding of 99.3 +/- 1.9 fmol/mg protein (n = 5). These findings suggest that specific adrenomedullin binding sites that differ from the CGRP receptors exist in human brain. This indicates a possible novel neurotransmitter/neuromodulator role for adrenomedullin in human brain.

Immunoreactive brain natriuretic peptide in human adrenal glands and adrenal tumors.

The presence of brain natriuretic peptide (BNP) in tissues of human adrenal glands and adrenal tumors was investigated by radioimmunoassay. Immunoreactive BNP concentrations were 0.203 +/- 0.061 pmol/g wet tissue (mean +/- SEM) in normal parts of adrenal glands (cortex and medulla, N = 8), 0.205 +/- 0.037 pmol/g wet tissue in pheochromocytomas (N = 8), 0.230 +/- 0.062 pmol/g wet tissue in aldosteronomas (N = 11) and 0.180 +/- 0.054 pmol/g wet tissue in adrenocortical adenomas with Cushing's syndrome (N = 4). Sephadex G-50 superfine column chromatography and reverse-phase high-performance liquid chromatography showed that most (> 70%) of the immunoreactive BNP in the normal part of adrenal glands was eluted in the position of human BNP-32. Sephadex G-50 superfine column chromatography of immunoreactive BNP in the pheochromocytoma and aldosteronoma showed four peaks: one in the position of gamma-BNP, one in the position of BNP-32, one between gamma-BNP and BNP-32 and one in the smaller molecular weight region. The present study has shown that immunoreactive BNP is present both in normal human adrenal glands and in adrenal tumors. Multiple molecular forms of BNP were found to be present in the tumor tissues of pheochromocytoma and aldosteronoma.

Urinary immunoreactive brain natriuretic peptide in patients with renal disease.

Urinary immunoreactive brain natriuretic peptide (BNP) was studied by radioimmunoassay in patients with renal disease. Urinary immunoreactive human BNP excretion measured in 11 normal subjects was 3.82 +/- 0.62 pmol/day (mean +/- SEM). Significantly increased 24-h urinary secretion of immunoreactive human BNP was noted in patients with chronic renal failure (11.07 +/- 1.73 pmol/day, n = 9, P < 0.05 to normal subjects). A significant correlation was noted between 24-h urinary excretion of immunoreactive human BNP and creatinine clearance in patients with various renal diseases (r = -0.43, P < 0.01, n = 45). Gel chromatography of the urine extracts obtained from normal subjects and patients with chronic renal failure showed multiple immunoreactive peaks; two eluting earlier, one in the position of human BNP-32 and others eluting later. Reverse-phase high-performance liquid chromatography of the urine extracts showed a peak in the position of human BNP-32 and a peak eluting earlier. These findings indicate that: (1) immunoreactive human BNP is present in human urine; (2) urinary immunoreactive human BNP consists of multiple components, i.e., human BNP-32 itself or a substance very similar to it, smaller molecular forms which are probably metabolic products of human BNP-32, and larger molecular forms; and (3) 24-h urinary excretion of immunoreactive human BNP is increased in patients with renal dysfunction.

Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland.

Adrenomedullin is a potent vasodilator peptide that was isolated from pheochromocytoma. Localization of adrenomedullin-like immunoreactivity was studied by immunocytochemistry in the human hypothalamus and adrenal gland. Adrenomedullin-immunoreactive cell bodies were found in the paraventricular, supraoptic and infundibular nuclei of the hypothalamus. Both magnocellular and parvocellular cells of the paraventricular nucleus were positively immunostained. Adrenomedullin-like immunoreactivity was localized in the adrenal medulla. No positive immunostaining was observed in the vascular endothelium, vascular smooth muscle cell or adrenal cortex. The preabsorption of the antiserum with synthetic human adrenomedullin (1-52) abolished the immunostaining. These findings indicate that adrenomedullin-like immunoreactivity is localized in the paraventricular, supraoptic and infundibular nuclei as well as in the adrenal medulla, and suggest that adrenomedullin acts as a neurotransmitter, a neuromodulator or a neurohormone in the human hypothalamus.

Three cases of clinical or preclinical Cushing's syndrome due to adrenocorticotropic hormone-independent bilateral adrenocortical macronodular hyperplasia: pituitary-adrenocortical function and immunohistochemistry.

We performed pituitary-adrenocortical function test and analysis of plasma steroids in three cases of adrenocorticotropic hormone-independent bilateral adrenocortical macronodular hyperplasia, and examined the plasma cortisol response to meal and oral glucose in two cases. On pathological examination, nodules were mainly composed of clear cells and some nests of compact cells were observed. Immunohistochemistry of steroidogenic enzymes showed positive P-450C17 only in the compact cells and positive 3 beta-hydroxysteroid dehydrogenase only in the clear cells. These cases were similar to clinical or preclinical Cushing's syndrome due to adenoma in both pituitary-adrenocortical function and plasma steroid patterns, but different from adenoma cases in the distribution of steroidogenic enzymes.

[Cerebellin in the cerebellum in spinocerebellar degeneration].

To clarify the possible pathophysiological role of cerebellin in spinocerebellar degeneration, immunoreactive cerebellin was measured in the postmortem brain of 4 patients with spinocerebellar degeneration and 4 controls. Three other representative neuropeptides, corticotropin releasing hormone (CRH), neuropeptide Y and somatostatin, were also measured. Significant decreases in the concentration of immunoreactive cerebellin and immunoreactive CRH were found in the cerebellar hemisphere in spinocerebellar degeneration, suggesting an important pathophysiological role of cerebellin and CRH in this group of diseases. No such decreases were found in neuropeptide Y or somatostatin.

Decrease in cerebellin and corticotropin-releasing hormone in the cerebellum of olivopontocerebellar atrophy and Shy-Drager syndrome.

Four neuropeptides; cerebellin, corticotropin-releasing hormone (CRH), neuropeptide Y and somatostatin were studied by radioimmunoassay in the postmortem human brains obtained from three patients with olivopontocerebellar atrophy (OPCA) and one with Shy-Drager syndrome. Significant decreases in cerebellin and CRH concentrations were found in the cerebellar hemisphere of these diseases compared with controls. These findings suggest important pathophysiological roles of cerebellin and CRH in these cerebellar diseases. Such significant decreases were not found in neuropeptide Y and somatostatin.

Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma.

Adrenomedullin is a potent vasodilator peptide that was isolated from human pheochromocytoma. But the presence of adrenomedullin in the brain has not been clarified. We studied the presence of adrenomedullin in the human brain obtained at autopsy from 6 subjects by radioimmunoassay, as well as in the human adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. Immunoreactive adrenomedullin was detected in every region of human brain examined (0.26-1.4 pmol/g wet weight) with the highest concentrations found in thalamus (1.40 +/- 0.39 pmol/g wet weight, mean +/- SEM) and hypothalamus (1.28 +/- 0.48 pmol/g wet weight). Reverse phase high performance liquid chromatography showed that the immunoreactive adrenomedullin in the human brain was eluted in the position of synthetic human adrenomedullin 1-52. High concentrations of immunoreactive adrenomedullin were found in human adrenal glands (12.6 +/- 1.0 pmol/g wet weight, n = 7), pheochromocytoma (4.5 +/- 1.5 pmol/g wet weight, n = 11), ganglioneuroblastoma (2.0 +/- 1.3 pmol/g wet weight, n = 4) and neuroblastoma (0.55 +/- 0.21 pmol/g wet weight, n = 3). The present study has shown that adrenomedullin is present in the human brain in high concentrations, suggesting that adrenomedullin acts as a neurotransmitter, neuromodulator or neurohormone in man.

Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion, loss of beta-cell GLUT 2 and glucose incompetence.

Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.

To elucidate the mechanism of the basal hyperinsulinemia of obesity, we perfused pancreata from obese Zucker and lean Wistar rats with substimulatory concentrations of glucose. Insulin secretion at 4.2 and 5.6 mM glucose was approximately 10 times that of controls, whereas beta-cell volume fraction was increased only 4-fold and DNA per islet 3.5-fold. We therefore compared glucose usage at 1.4, 2.8, and 5.6 mM. Usage was 8-11.4 times greater in Zucker islets at 1.4 and 2.8 mM and 4 times greater at 5.6 mM; glucose oxidation at 2.8 and 5.6 mM glucose was > 12 times lean controls. To determine if the high free fatty acid (FFA) levels of obesity induce these abnormalities, normal Wistar islets were cultured with 0, 1, or 2 mM long chain FFA for 7 days. Compared to islets cultured without FFA insulin secretion by FFA-cultured islets (2 mM) perifused with 1.4, 3, or 5.6 mM glucose was increased more than 2-fold, bromodeoxyuridine incorporation was increased 3-fold, and glucose usage at 2.8 and 5.6 mM glucose was increased approximately 2-fold (1 mM FFA) and 3-fold (2 mM FFA). We conclude that hypersecretion of insulin by islets of obese Zucker fatty rats is associated with, and probably caused by, enhanced low Km glucose metabolism and beta-cell hyperplasia, abnormalities that can be induced in normal islets by increased FFA.

Immunoreactive endothelin in the human kidney: comparison with natriuretic peptides.

The presence of immunoreactive endothelin (IR-ET) was studied by radioimmunoassay in human kidney with and without clinical renal failure, and the levels were compared with those of three natriuretic peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Kidney tissues were obtained at autopsy from three subjects with renal disorders (diabetic nephropathy, renal tubular necrosis, and end-stage kidney disease). Normal renal tissue was obtained at surgery from two patients with renal cell carcinoma. IR-ET was detected in three diseased kidneys obtained at autopsy (0.101 +/- 0.043 pmol/g wet weight, mean +/- SD) but not in tissues of two normal kidneys obtained at surgery (< 0.015 pmol/g wet weight). Reverse-phase HPLC showed that most of the IR-ET in the kidney (> 90%) was eluted in the position of ET-1. IR-ANP (0.23 +/- 0.06 pmol/g wet weight), IR-BNP (1.15 +/- 0.94 pmol/g wet weight), and IR-CNP (0.44 +/- 0.16 pmol/g wet weight) were detected in all samples examined. Higher concentrations of IR-BNP were found in three diseased kidneys obtained at autopsy (1.70 +/- 0.83 pmol/g wet weight vs. mean in two normal kidney tissues, 0.32 pmol/g wet weight). Such increases were not observed in IR-ANP or IR-CNP. These findings indicate that IR-ET is present in the human diseased kidney even in end-stage disease, with high concentrations comparable to those of natriuretic peptides. This raises the possibility that the production of ET-1 and BNP is increased in kidneys of patients with renal disorders.

Post-GLUT-2 defects in beta-cells of non-insulin-dependent diabetic obese rats.

Zucker diabetic fatty (ZDF) rats develop non-insulin-dependent diabetes mellitus concomitantly with loss of glucose responsiveness and GLUT-2, the high-Michaelis constant glucose transporter of beta-cells. To determine the integrity of beta-cell glucose metabolism distal to the level of glucose transport and phosphorylation, we examined the insulin responses of isolated pancreata to 5, 10, and 20 mM D-glyceraldehyde and monomethylsuccinate, as well as to glucose. The insulin response of diabetic pancreata to glucose was 90% below the response prior to the onset of diabetes, whereas the responses to glyceraldehyde and succinate had declined to 65 and 44%, respectively, below the prediabetic responses. D-[14C]glyceraldehyde oxidation by diabetic islets was 74% below that of islets from lean nondiabetic controls. We conclude that 1) the insulin responses to glyceraldehyde and monomethylsuccinate, as well as to glucose, are impaired in the diabetes of ZDF rats and 2) the impairment of the glucose response was greater than that of the glyceraldehyde response, which was, in turn, greater than that of the monomethylsuccinate response; this decrescendo pattern of impairment is consistent with defects at multiple sites in glucose metabolism; if the defect were entirely due to a postmetabolic signaling defect, the impairment to glucose and its metabolites should be comparable.

Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships.

Hyperinsulinemia, loss of glucose-stimulated insulin secretion (GSIS), and peripheral insulin resistance coexist in non-insulin-dependent diabetes mellitus (NIDDM). Because free fatty acids (FFA) can induce these same abnormalities, we studied their role in the pathogenesis of the NIDDM of obese Zucker diabetic fatty (ZDF-drt) rats from 5 weeks of age (before the onset of hyperglycemia) until 14 weeks. Two weeks prior to hyperglycemia, plasma FFA began to rise progressively, averaging 1.9 +/- 0.06 mM at the onset of hyperglycemia (P < 0.001 vs. controls). At this time GSIS was absent and beta-cell GLUT-2 glucose transporter was decreased. The triacylglycerol content of prediabetic islets rose to 10 times that of controls and was correlated with plasma FFA (r = 0.825; P < 0.001), which, in turn, was correlated with the plasma glucose concentration (r = 0.873; P < 0.001). Reduction of hyperlipacidemia to 1.3 +/- 0.07 mM by pair feeding with lean littermates reduced all beta-cell abnormalities and prevented hyperglycemia. Normal rat islets that had been cultured for 7 days in medium containing 2 mM FFA exhibited increased basal insulin secretion at 3 mM glucose, and first-phase GSIS was reduced by 68%; in prediabetic islets, first-phase GSIS was reduced by 69% by FFA. The results suggest a role for hyperlipacidemia in the pathogenesis of NIDDM; resistance to insulin-mediated antilipolysis is invoked to explain the high FFA despite hyperinsulinemia, and sensitivity of beta cells to hyperlipacedemia is invoked to explain the FFA-induced loss of GSIS.

More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling.

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic beta-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802-5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose-->malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS)