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AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

Magnesium (Mg) intake has been linked to bone mass and/or rate of bone loss in humans. Experimental Mg deficiency in animal models has resulted in impaired bone growth, osteopenia, and increased skeletal fragility. In order to assess changes in bone and mineral homeostasis that may be responsible, we induced dietary Mg deficiency in adult Simonsen albino rats for 16 weeks. Rats were fed either a low Mg diet (0.002 percent) or a normal control Mg diet (0.063 percent). Blood was obtained at baseline, 4 weeks, 8 weeks, 12 weeks and 16 weeks in both groups for serum Mg, calcium, PTH, and 1.25(OH)2-vitamin D determinations. Femora were harvested at 4 weeks and 16 weeks for mineral analysis and histomorphometry. Serum Mg fell in the Mg depleted group to 0.6 mg/dl (mean) by 16 weeks (controls = 2.0 mg/dl). The serum calcium (Ca) concentration was higher in the Mg depleted animals at 16 weeks, 10.8 mg/dl (controls = 8.9 mg/dl). Serum PTH concentration fell progressively in the Mg deficient rats to 30 pg/ml by week 16 (control = 96 pg/ml). Serum concentration of 1.25(OH)2-vitamin D also fell progressively in the Mg deficient animals by 16 weeks to 14 pg/ml (control = 30 pg/ml). While the percent ash weights of Ca and phosphorus in the femur were not different at any time point, the percent ash weight of Mg progressively fell to 0.54 percent vs control (0.74 percent) by 16 weeks. The percent ash weight of potassium also fell progressively in the Mg deficient group to approximately 30 percent of control by 16 weeks. Histomorphometric analyses showed a significant drop in trabecular bone volume in Mg deficient animals by 16 weeks (percent BV/TV = 13.2 percent vs 17.3 percent in controls). Evaluation of the endosteal bone surface features showed significantly greater bone resorption in the Mg depleted group as reflected in increased number of tartrate-resistant positive osteoclasts/mm bone surface (7.8 vs 4.0 in controls) and an elevated percent of bone surface occupied by osteoclasts (percent OcS/BS = 12.2 percent vs 6.7 percent in controls. This increased resorption occurred in the presence of an inappropriate lowered bone forming surface relative to controls; a decreased number of osteoblasts per mm bone surface (0.23 vs 0.94 in control) and a decrease in percent trabecular surface lined by osteoid (percent OS/BS = 0.41 vs 2.27 percent in controls) were also noted. Our findings demonstrate a Mg-deficiency induced uncoupling of bone formation and bone resorption resulting in a loss of bone mass. While the fall in PTH and/or 1.25(OH)2-D may explain a decrease in osteoblast activity, the mechanism for increased osteoclast activity is unclear. These data suggest that Mg deficiency may be a risk factor for osteoporosis.

Adenosine levels increase at seizure foci as part of a postulated endogenous negative feedback mechanism that controls seizure activity through activation of A1 adenosine receptors. Agents that amplify this site- and event-specific surge of adenosine could provide antiseizure activity similar to that of adenosine receptor agonists but with fewer dose-limiting side effects. Inhibitors of adenosine kinase (AK) were examined because AK is normally the primary route of adenosine metabolism. The AK inhibitors 5'-amino-5'-deoxyadenosine, 5-iodotubercidin, and 5'-deoxy-5-iodotubercidin inhibited maximal electroshock (MES) seizures in rats. Several structural classes of novel AK inhibitors were identified and shown to exhibit similar activity, including a prototype inhibitor, 4-(N-phenylamino)-5-phenyl-7-(5'-deoxyribofuranosyl)pyrrolo[2, 3-d]pyrimidine (GP683; MES ED50 = 1.1 mg/kg). AK inhibitors also reduced epileptiform discharges induced by removal of Mg2+ in a rat neocortical preparation. Overall, inhibitors of adenosine deaminase or of adenosine transport were less effective. The antiseizure activities of GP683 in the in vivo and in vitro preparations were reversed by the adenosine receptor antagonists theophylline and 8-(p-sulfophenyl)theophylline. GP683 showed little or no hypotension or bradycardia and minimal hypothermic effect at anticonvulsant doses. This improved side effect profile contrasts markedly with the profound hypotension, bradycardia, and hypothermia and greater inhibition of motor function observed with the adenosine receptor agonist N6-cyclopentyladenosine and opens the way to clinical evaluation of AK inhibitors as a novel, adenosine-based approach to anticonvulsant therapy.

Highly porous matrices of poly-L-lactide (PL) and polyglycolide (PG), 24, 50, or 95 mg/cc in the form of 10 x 10 x 3 mm wafers, were implanted subcutaneously (two per rat) in the flanks of 8-12-week-old female Lewis rats (n = 120). Matrices were harvested, two rats per week, for 15 weeks and examined histologically. At weeks 1 and 2, a thin fibrous capsule was present and matrices showed capillary beds and host-cell infiltration along the implant margins. By week 4, the PL specimens had some arterioles while the PG specimens still had only capillary beds. At week 7, PL had well developed arterioles, venules, and capillaries while PG began to show modest vascular beds of capillaries only. In terms of cellular ingrowth, PL remained unchanged from 7 to 15 weeks. Giant cell formation was observed wherever polymer was present. There was a loss of thickness and cell mass for both matrices over time (PGPL) despite initial host-cell ingrowth. As both polymers degraded and were absorbed, the ingrown cells mass regressed. There was little remaining PG at 15 weeks, leaving no trace of cells that previously had ingrown and no evidence of scar tissue.

The skeleton, the major site for Pb accumulation, is responsible for the largest fraction of the total body burden, but long-term effects of low-level exposure in adults remain unclear. In this study rats were exposed to low (0.01%; 100 ppm, LoPb) or high (0.5%, 5,000 ppm, HiPb) Pb, low calcium, feeding regimes for 1-12 months. Both LoPb and HiPb animals showed significant 12-month blood Pb levels [LoPb 21 +/- 3 micrograms/dl; HiPb 59 +/- 18; controls 3 +/- 1 (mean +/- SEM), p = 0.001]. Dual energy X-ray densitometry of the femur detected a significant decrease in bone density in HiPb animals by 3 months which remained significantly lowered through 12 months [3 months: HiPb: 0.498 +/- 0.011 (6) vs. control: 0.546 +/- 0.012 (6), p0.003]. By 12 months' density was also significantly lowered in LoPb animals (p = 0.001). Mineral analyses of ashed femurs showed a significant lead content after 1, 3, 9 and 12 months' exposure [1 month: LoPb, 0.020 +/- 0.002 (4) (% ash weight) vs control 0.008 +/- 0.0004 (4); HiPb 0.016 +/- 0.001 (8); control 0.007 +/- 0.0004 (6) (por = 0.002)]. Ca levels (% ash weight) were significantly lowered at 9 months in HiPb and 12 months in both groups (por = 0.04). Quantitative histomorphometry documented significantly elevated osteoid and resorptive trabecular surface features in both Pb groups. The LoPb design produced no overt renal functional abnormalities and resulted in blood Pb values comparable to those in man with modest environmental Pb exposure. The HiPb design resulted in development of lead nephropathy (more severe from months 6-12) and produced blood lead levels comparable to those seen in past industrial exposure. Findings show that Pb is incorporated into bone mineral after only 1 month's exposure to LoPb with significant osteopenia after 12 months' exposure; HiPb caused osteopenia by 3 months. No normal compensatory mechanism was elicited to maintain bone mass. Results stress renewed concern about the effects of cumulative, low-level lead exposure in our elderly population.

Bone healing was investigated histologically in a rat fibular osteotomy model subjected to microgravity (shuttle flight STS-29) and the tail suspension microgravity simulation model. Exposure to microgravity or tail suspension occurred during the last 5 days of a 10-day healing period. Periosteal osteogenesis and the development of vascular channels in both experimental groups were similar to that observed in a weightbearing control group. Chondrogenesis was more advanced in weightbearing rats than in either flight or tail-suspended rats. Angiogenesis in the osteotomy gap was more advanced in weightbearing and tail-suspended rats than in the flight group. These findings suggest that bone healing may be impaired during space travel. Interpretation of the findings is complicated by observations that flight and suspended rats lost weight during the flight period and that suspended rats consumed less water than control rats. Tail suspension did not produce the same pattern of healing as spaceflight; therefore, long-term studies of bone healing, conducted entirely in the microgravity environment, are needed to distinguish metabolic from mechanical influences and to determine whether effective fracture consolidation will occur in the absence of gravitational forces.

Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)2D3 levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D3 levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin, bone ALP, periosteal MAR, and trabecular bone volume. However, rats receiving lower doses of phenytoin (i.e., 1 mg/kg/day) did not show significant increases in the serum bone formation parameters. In contrast, metaphyseal osteoblast surface, osteoblast number, osteoid thickness, surface, and volume were all significantly increased in rats treated in 1 mg/kg/day but not with 5 mg/kg/day phenytoin, suggesting that the tibial diaphysis and metaphysis bone formation parameters might have different dose-dependent responses to phenytoin treatment. Administration of the test doses of phenytoin did not significantly affect the histomorphometric bone resorption parameters. In conclusion, these findings represent the first in vivo evidence that phenytoin at low doses (i.e., between 1 and 5 mg/kg/day) is an osteogenic agent in the rat.

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.

Cartilage and bone in the lumbar vertebrae of magnesium-deficient (LoMg) rats were studied after 4 and 8 weeks of Mg restriction. LoMg animals receiving 0.03% Mg intake had significantly reduced serum Mg levels compared to controls (receiving 0.2% Mg) at both 4 weeks [0.66 +/- 0.06 mg% mean +/- SE (n = 10)] and 8 weeks [0.74 +/- 0.02 mg% (n = 3)] of study. Mean width of the vertebral growth plate was significantly decreased in LoMg animals at 4 weeks [54.7 +/- 3.5 microns (n = 9) vs. control 68.0 +/- 3.0 microns (n = 6)] and 8 weeks [39.5 +/- 2.8 microns (n = 3) vs. control 57.5 +/- 3.6 microns (n = 3)]. The mean number of cells/cartilage column in the growth plate was also less at 4 weeks [5.8 +/- 0.18 (n = 5) vs. control 7.2 +/- 0.19 (n = 5)] and 8 weeks [4.9 +/- 0.19 (n = 3) vs. control 6.2 +/- 0.08 (n = 3)]. A significant proportion of LoMg animals possessed decreased pericolumnar diastase-PAS reactivity at 4 and 8 weeks of study; this indicates a decrease in cartilage glycoprotein content during Mg deficiency. Quantitative histomorphometric analysis determined that vertebral trabecular bone possessed significantly decreased percentage of osteoid surface (2.49 +/- 0.54 vs. control 6.98 +/- 2.8) and significantly decreased percentage of osteoid area (0.18 +/- 0.05 vs. control 0.82 +/- 0.38) after 4 weeks of Mg deficiency. These findings document significant alterations in both bone and cartilage histologic features following relatively long-term Mg deficiency.

Fluoride has often been used as a treatment for osteoporosis, a metabolic bone disease of considerable importance in the elderly population. The techniques currently used to monitor a patient's response to fluoride are outlined. New findings concerning (1) a mechanism for interaction of fluoride with osteoblasts (via mitogenic signals or growth factors); (2) toxicity and carcinogenesis; (3) recent clinical trial data; and (4) the importance of dosage, administration regimens, and side effects in an effective fluoride treatment protocol are reviewed. Some recent clinical data challenge the efficacy of fluoride in the treatment of postmenopausal osteoporosis. Because of the implications of these recent studies with respect to fracture incidence during fluoride therapy, fluoride cannot be recommended at this time for general use in the treatment of osteoporosis.

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.