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Background: Most patients with primary hyperparathyroidism are postmenopausal women. The presence of osteopenia in persons with mild primary hyperparathyroidism is considered an indication for parathyroidectomy. No prospective, controlled trials have assessed medical therapies for osteopenia in primary hyperparathyroidism.Objective: To examine the effects of estrogen-progestin therapy (hormone replacement therapy) on bone mineral density and biochemical indices in postmenopausal women with mild primary hyperparathyroidism.Design: Double-blind, randomized, placebo-controlled trial.Setting: University teaching hospital.Patients: 42 postmenopausal women with mild primary hyperparathyroidism.Intervention: Patients were randomly assigned to receive either conjugated estrogens, 0.625 mg/d, and medroxyprogesterone, 5 mg/d, or placebo.Measurements: Bone mineral densities of the total body, lumbar spine, proximal femur (femoral neck, Ward triangle, trochanter), and proximal forearm were measured every 6 months using dual-energy x-ray absorptiometry. Biochemical indices of bone turnover and calcium metabolism were measured at baseline, 6 months, and 2 years.Results: In the placebo group, bone mineral densities of the total body and the proximal forearm decreased significantly from baseline (mean +/- SE, -2.3% +/- 0.7% [p = 0.005] and -3.5% +/- 1.2% [p = 0.01], respectively). At the other sites, bone mineral density also tended to decline. In the hormone replacement therapy group, bone mineral density increased from baseline in the total body (1.3% +/- 0.4%; P = 0.004), lumbar spine (5.2% +/- 1.4%; p = 0.002), and femoral neck (3.4% +/- 1.5%; p = 0.05). The between-group differences in bone mineral density at the end of the study ranged from 3.6% to 6.6% and were significant at all sites (P > 0.001 and P < 0.05) except for the Ward triangle (p = 0.06). In the hormone replacement therapy group, serum alkaline phosphatase levels decreased by 22% (p = 0.0004 compared with baseline), urinary hydroxyproline excretion decreased by 42% (p = 0.0004), urinary N-telopeptide excretion decreased by 54% (p = 0.001), and urinary calcium excretion decreased by 45% (p = 0.007). Hormone replacement therapy did not change levels of serum ionized calcium or intact parathyroid hormone.Conclusions: Although hormone replacement therapy has little effect on serum calcium levels, it suppresses bone turnover, reduces urinary calcium excretion, and increase bone mineral density throughout the skeleton in postmenopausal women with mild primary hyperparathyroidism. This therapy is thus an important management option for these patients. [ABSTRACT FROM AUTHOR]

Objective: We describe how a single intraperitoneal injection of an indoline-derived drug (SN 28127) reduced mouse body weight (25-45% loss) and adipose tissue mass (∼75%)., Methods and Results: The reductions in body weight peaked at ∼21-28 days post drug injection and were maintained throughout the study (160 days). The mice ate as much as vehicle-treated control mice. A more potent SN 28127 analog (SN 29220) reversed high-fat diet-induced obesity and type 2 diabetes in C57BL/6J mice on a high-fat diet. Insulin induced a sustained reduction in blood glucose in fasted SN 29220-treated mice compared with the vehicle-treated mice. All drug-treated mice exhibited a transient increase in water intake from ∼10 days post drug injection that lasted for ∼70 days. Following a single injection of (3)H-labeled SN 29220, radioactivity accumulated within 4 h in the liver, bile duct and ileum with little detected in the brain; within 1-2 days, most of the radioactivity was found in the pancreas, spleen, liver, bile duct, stomach, kidneys and white adipose tissue. High levels of glucose were detected in urine collected from SN 29220 but not vehicle-treated C57BL/6J mice at ∼60 days post injection, while fecal triacylglycerols and cholesterol were not different between SN 29220 and vehicle-treated mice. These data lead us to hypothesize that the hepatic system is the primary drug target. Genes involved in fatty acid synthesis (FASn, SCD1 and PPARγ) and appetite stimulation (AGRP) were upregulated at 160 days post drug treatment, indicative of adaptation to reduced body weight., Conclusion: We hypothesize that indoline-derived drug-induced chronic toxicity to the hepatic system leads to a reduction in white adipose tissue mass. The mice adapt to this drug-induced toxicity with reduced steady-state body weight. Understanding molecular mechanisms underlying these responses has potential to identify novel targets for prevention and treatment of obesity.

Quaternary salt analogues based on the DNA minor groove binder and adenine N3 alkylating agent 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) show remarkable effects on the body weight of mice (a long-term failure to gain weight relative to matched controls with no loss of appetite or perceptible deterioration in health) following administration of a single (non-toxic) dose between about 0.5-5 μmol/kg. The nature of the quaternizing group was not important, but a related hydroxyCBI analogue was much less effective. Compounds where the chloro group was replaced by a hydrogen or hydroxy group (thus abrogating DNA alkylating capability) showed no weight control activity. It is speculated, based on other studies, that the marked long-term weight control effect is due to inhibition of bile flow into the intestine and reduced absorption of triglycerides, together with accelerated cell death in spleen and white adipose tissues due to drug accumulation there. This class of compound may serve as interesting tools for further study of these phenomena., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The study of spontaneous mutations in mice over the last century has been fundamental to our understanding of normal physiology and mechanisms of disease. Here we studied the phenotype and genotype of a novel mouse model we have called the New Zealand Ginger (NZG/Kgm) mouse. NZG/Kgm mice are very large, rapidly growing, ginger-colored mice with pink eyes. Breeding NZG/Kgm mice with CAST/Ei or C57BL/6J mice showed that the ginger coat colour is a recessive trait, while the excessive body weight and large body size exhibit a semidominant pattern of inheritance. Backcrossing F1 (NZG/Kgm x CAST/Ei) to NZG/Kgm mice to produce the N2 generation determined that the NZG/Kgm mouse has two recessive pigmentation variant genes (oca2(p) and tyrp-1(b)) and that the tyrp-1(b) gene locus associates with large body size. Three coat colors appeared in the N2 generation; ginger, brown, and dark. Strikingly, N2 male coat colour associated with body weight; the brown-colored mice weighed the most followed by ginger and then dark. The male brown coat-colored offspring reached adult body weights indistinguishable from NZG/Kgm males. The large NZG/Kgm mouse body size is a result of excessive lean body mass since these mice are not obese or diabetic. NZG/Kgm mice exhibit an unusual pattern of fat distribution; compared with other mouse strains they have disproportionately higher amounts of subcutaneous and gonadal fat. These mice are susceptible to high-fat diet-induced obesity but are resistant to high-fat diet-induced diabetes. We propose NZG/Kgm mice as a novel model to delineate gene(s) that regulate 1) growth and metabolism, 2) resistance to Type 2 diabetes, and 3) preferential fat deposition in the subcutaneous and gonadal areas.

Desacetyl alpha-MSH predominates over alpha-MSH during development, but whether it is biologically active and has a physiological role is unclear. We compared the effects of 0.3 microg.g(-1).day(-1) desacetyl alpha-MSH with that of 0.3 microg.g(-1).day(-1) alpha-MSH on postnatal body growth by administering the peptides subcutaneously daily for postnatal days 0-14 and also used a two-dimensional gel electrophoresis gel-based proteomic approach to analyze protein changes in hypothalami, the relay center for body weight and growth regulation, after 14 days of treatment. We found that the growth rate between days 1 and 10 was significantly decreased by desacetyl alpha-MSH but not by alpha-MSH, but by day 14, a time reported for development of a mature pattern of hypothalamic innervation, both peptides had significantly increased neonatal growth compared with PBS-treated control rats. Desacetyl alpha-MSH significantly increased spleen weight, but alpha-MSH had no effect. alpha-MSH significantly decreased kidney weight, but desacetyl alpha-MSH had no effect. Both desacetyl alpha-MSH and alpha-MSH significantly decreased brain weight. By 14 days, both peptides significantly changed expression of a number of hypothalamic proteins, specifically metabolic enzymes, cytoskeleton, signaling, and stress response proteins. We show that peripherally administered desacetyl alpha-MSH is biologically active and induces responses that can differ from those for alpha-MSH. In conclusion, desacetyl alpha-MSH appears to be an important regulator of neonatal rat growth.

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

Measurement of insulin-like growth factor II (IGF-II) in human serum is complicated by the presence of IGF binding proteins and usually involves cumbersome extraction procedures followed by radioimmunoassay. We have utilized an extraction process developed for measuring insulin-like growth factor II in ovine serum using Sephacryl HR100, and have applied this to the extraction of human samples followed by radioimmunoassay for human IGF-II. The assay yielded 98% recovery of unlabelled IGF-II, parallelism between dilutions of eluate and the standard curve, complete removal of binding proteins and near-complete removal of IGF-I, and intra- and interassay coefficients of variation of 5% and 9%, respectively. The normal range for serum IGF-II in women was 490-1056 micrograms/L, and IGF-II levels were positively correlated with serum concentrations of insulin-like growth factor binding protein-3 (IGFBP-3) but not with IGF-I levels. Mean serum concentrations of IGF-II were reduced below normal in a number of hypopituitary patients and children with short stature and IGF-II concentrations in these subjects correlated positively with IGF-I and IGFBP-3. In acromegalic patients IGF-II levels were usually normal and were negatively correlated with IGF-I concentrations. From our experience with the above results the present assay appears particularly suitable for clinical measurements and research projects where high sample throughput is required.

Purpose: To assess the effect of the antiestrogenic agent tamoxifen on bone mineral density in normal late postmenopausal women., Methods: A randomized, double-blind, placebo-controlled trial was performed with 57 healthy, late postmenopausal women (mean 11 +/- 7 years since menopause). Subjects were assigned to take either tamoxifen 20 mg/d or placebo for 2 years. Total body, lumbar spine, and proximal femoral (femoral neck, Ward's triangle, trochanter) bone mineral densities were measured every 6 months using dual-energy x-ray absorptiometry. Serum and urine indices of bone turnover were measured at baseline, 6 months, and 2 years., Results: In the women given tamoxifen, the mean bone mineral density of the lumbar spine increased by 1.4%, while that in the women given placebo declined by 0.7% (P < 0.01 for difference between groups). Total body bone mineral density declined in both groups, but less so in the tamoxifen-treated women (P < 0.05). At both sites, the effect of tamoxifen was maximal after 1 year, with no further separation of the groups thereafter. There was no significant effect of tamoxifen on bone mineral density in the proximal femur. Tamoxifen produced significant falls in serum alkaline phosphatase (P < 0.0001), ionized calcium (P < 0.0001), and phosphate (P < 0.01), and in urinary excretion of hydroxyproline, n-telopeptides, and calcium (P < 0.05 for each)., Conclusions: In normal late postmenopausal women, tamoxifen at a dose of 20 mg/d exerts a small protective effect on bone mineral density, comparable in magnitude to that of calcium supplementation and less than that of either estrogen or the bisphosphonates. Tamoxifen is unlikely to supersede any of these therapies in the management of postmenopausal osteoporosis.

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.

The biological properties of three covalently-linked insulin dimers were studied in greyhounds. Constant infusions showed that the plasma distribution kinetics were slower for the dimers than for insulin. The metabolic clearance rates of the three dimers (10.3 +/- 0.4, 8.8 +/- 0.5, 8.2 +/- 0.5 ml X min-1 X kg-1; mean +/- SEM) were significantly lower than that of insulin (19 +/- 0.8 ml X min-1 X kg-1), and their hypoglycaemic effects (11.2%, 3% and 0.3%) were markedly reduced compared with their lipogenic potencies in vitro (80%, 30% and 13%, respectively). A low dose infusion of insulin or an equipotent dose of one of the dimers significantly prolonged the effects of an insulin bolus on plasma glucose but not on non-esterified fatty acids. The apparent distribution space (106.4 +/- 11.9 ml/kg) and clearance rate (14.7 +/- 0.5 ml X min-1 X kg-1) of an insulin bolus were significantly reduced by one dimer (44.5 +/- 8.4 ml/kg and 10.7 +/- 2.8 ml X min-1 X kg-1) but not by the equipotent insulin infusion (102.7 +/- 8.2 ml/kg and 16.4 +/- 0.07 ml X min-1 X kg-1). The apparent partial competitive antagonism of insulin by the dimers that has been reported in vitro can be observed in vivo, in that antagonism of insulin metabolism was directly demonstrated with one of the dimers.

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.

The effects of NaCl concentration and pH on binding to purified rat liver plasma membranes were compared for labelled insulin and two covalently-linked insulin dimers. Specific binding of both dimers and insulin increased as NaCl concentration increased from 0 to 1M in 0.05M Tris/HCl buffer. The initial rise in binding was much greater for dimers than for insulin. Specific binding of dimers was sensitive to pH changes in 0.029M barbital sodium acetate buffer, but this effect was more marked with insulin, which reached a sharp binding peak at pH 7.7 to 8.0. Both total and non-specific dimer binding were approximately 2-fold greater at pH 5.0 than at pH 8.0. Specific-binding fraction determinations of both dimers relative to insulin have been shown to be critically dependent on the experimental conditions of Na+/Cl- ion concentration and pH. These observations allow a better understanding of the nature of ligand-receptor interaction.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

The C-terminus of the insulin B chain is essential for dimerisation and expression of negative cooperativity. In order to evaluate the possible physiological role of these phenomena, we have studied the properties in vivo and in vitro of despentapeptide insulin (B26-30 deleted), derived from beef insulin, and deshexapeptide insulin (B25-30 deleted), derived from pork insulin. These materials do not dimerise and have 15% and 0% retention of negative cooperativity respective. Lipogenesis potencies in rat adipocytes were: despentapeptide insulin 19.9 +/- 0.3%; deshexapeptide insulin 19.9 +/- 1.5%. Binding potencies in adipocytes were: despentapeptide insulin 22.6 +/- 7.8%; deshexapeptide insulin 13.2 +/- 3.3%. Metabolic clearance rates were reduced compared to insulin (insulin = 19.1 +/- 0.9; despentapeptide insulin = 9.7 +/- 0.8; deshexapeptide insulin = 6.4 +/- 0.6 ml . min-1 . kg-1 at plasma concentration 0.5 nmol/l). Hypoglycaemic potencies were reduced for both analogues (40% and 30%) when calculated on the basis of plasma concentration although both analogues and insulin were equally effective at lowering plasma glucose concentration in equimolar doses. Plasma half-disappearance time was prolonged (despentapeptide insulin = 7.3 +/- 0.5; deshexapeptide insulin = 9.1 +/- 0.2 min). Both analogues were full agonists and conformed to the general relationship between in vitro and in vivo properties seen with a wide range of modified insulins. They resemble other analogues with modifications which reduce receptor affinity without impairing dimerisation or negative cooperativity. The results do not support a physiological role for dimerisation or negative cooperativity.