Mechanism of aminobisphosphonate action: characterization of alendronate inhibition of the isoprenoid pathway.

Alendronate (ALN), an aminobisphosphonate compound used for the treatment of osteoporosis and other disorders of bone resorption, has been suggested to act by inhibition of the formation of GGPP. In the present study we used an S(10) homogenate fraction of rat liver to show that ALN causes a dose-dependent inhibition of [(3)H]MVA incorporation into sterols and a concomitant increase in incorporation of radiolabel into IPP and DMAPP. We further show that ALN is a potent inhibitor of cytosolic trans-prenyltransferase (FPP synthase). The inhibition is competitive with respect to allylic pyrophosphate substrates, but not IPP, suggesting that ALN acts as an allylic pyrophosphate analog and binds to the free enzyme. The K(i) is in the 0.5 microM range.

Compensatory responses to inhibition of hepatic squalene synthase.

The mechanism by which depletion of hepatic cholesterol levels, achieved by inhibition of squalene synthase, alters hepatic LDL receptor, HMG-CoA reductase, and cholesterol 7alpha-hydroxylase gene expression was investigated by measuring transcription rates, mRNA stability, rates of translation, translational efficiency, and levels of sterol response element binding proteins. It was found that the transcription of both hepatic LDL receptor and HMG-CoA reductase were increased about twofold. The increase in LDL receptor transcription occurred within 2 h after giving 2 mg/kg zaragozic acid A, a potent inhibitor of squalene synthase. This preceded the increase in transcription of HMG-CoA reductase that occurred at 4 h. Increases in the stability of both of these mRNAs were also observed. These changes account for the increases in LDL receptor and HMG-CoA reductase mRNA levels previously observed. The rate of transcription of hepatic cholesterol 7alpha-hydroxylase was decreased to about 25% of control within 3 h after administration of zaragozic acid A, which correlates with the decrease in this mRNA. The rates of translation, as determined by pulse labeling, of both hepatic HMG-CoA reductase and LDL receptor were increased two- to threefold. The translational efficiency of these two mRNAs was also increased as judged by polysome profile analysis. There was an increase in mRNA associated with the heaviest polysome fraction and a decrease in that associated with monosomes. No significant change was observed in the levels of sterol response element binding protein 2, the form that mediates induced transcription, in response to zaragozic acid A treatment, indicating that this protein might not be involved in mediating the observed transcriptional changes. An increase in sterol response element binding protein -1 was observed 30 min after giving zaragozic acid A. The results suggest that compensatory responses to depletion of squalene-derived products involve alterations in the rates of transcription, mRNA stability, and translational of key proteins involved in cholesterol homeostasis.

Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency.

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.

Isoprenoid metabolism in the vertebrate retina.

Herein, studies concerning the biosynthesis, intracellular transport and utilization of isoprenoid lipids in vertebrate retinas are reviewed, with particular regard to rod photoreceptor cells and the assembly of rod outer segment (ROS) disk membranes. Initial in vitro studies with bovine retinas showed that [3H]mevalonate is metabolized primarily to squalene and 'methylated' sterols, rather than to cholesterol. Subsequently, similar results were obtained with frog retinas using [3H]acetate as a precursor, and the absolute rate of the sterol pathway was determined in vitro with 3H2O. With the aid of vesicular transport inhibitors, energy poisons, and reduced temperature, it was demonstrated that lipid and protein trafficking mechanisms in the rod cell are separate and independent from one another. In vivo, the majority of newly synthesized squalene in the frog retina is not metabolized to sterols; rather, it is transported to the ROS, where it turns over in parallel with the disk membranes. The remaining squalene is converted slowly to cholesterol, much of which becomes incorporated into the ROS. In contrast, the in vivo metabolism of [3H]acetate to cholesterol in the rat retina is relatively efficient and rapid. However, in both frog and rat, retinal cholesterol turnover is slow (> 60 days), suggesting the existence of a retention mechanism that minimizes the need for de novo biosynthesis. The use of pharmacological approaches to assess the biological roles of isoprenoid lipids and protein prenylation in the retina and the mechanism of retinal cholesterol homeostasis are discussed.

Mitochondrial encephalomyopathy with coenzyme Q10 deficiency.

Coenzyme Q10 (CoQ10) transfers electrons from complexes I and II of the mitochondrial respiratory chain to complex III. There is one published report of human CoQ10 deficiency describing two sisters with encephalopathy, proximal weakness, myoglobinuria, and lactic acidosis. We report a patient who had delayed motor milestones, proximal weakness, premature exertional fatigue, and episodes of exercise-induced pigmenturia. She also developed partial-complex seizures. Serum creatine kinase was approximately four times the upper limit of normal and venous lactate was mildly elevated. Skeletal muscle biopsy revealed many ragged-red fibers, cytochrome c oxidase-deficient fibers, and excess lipid. In isolated muscle mitochondria, impaired oxygen consumption was corrected by the addition of decylubiquinone. During standardized exercise, ventilatory and circulatory responses were compatible with a defect of oxidation-phosphorylation, which was confirmed by near-infrared spectroscopy analysis. Biochemical analysis of muscle extracts revealed decreased activities of complexes I+II and I+III, while CoQ10 concentration was less than 25% of normal. With a brief course of CoQ10 (150 mg daily), the patient reported subjective improvement. The triad of CNS involvement, recurrent myoglobinuria, and ragged-red fibers should alert clinicians to the possibility of CoQ10 deficiency.

Regulatory adaptation of isoprenoid biosynthesis and the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency.

In a search for the pathophysiologic mechanisms, we estimated isoprenoid synthesis and concentration, cellular growth, and the activity of the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency (MKD), a severe multisystemic disorder of cholesterol and non-sterol isoprenoid biosynthesis. In response to different concentrations of LDL and non-lipoprotein-bound cholesterol, MKD cells partially counteracted their enzyme defect by increased activities of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (results from earlier studies) and the LDL receptor pathway, responses similar to the pharmacologic effects seen upon administration of HMG-CoA reductase inhibitors. Rates of N-linked protein glycosylation, estimated as the amount of [14C]galactose-labeled macromolecules secreted into cell culture medium, were significantly decreased in MKD fibroblasts in comparison with control cells which may indicate alterations in the dolichol or dolichol phosphate pool. In response to exogenous cholesterol, the major feedback inhibitor of isoprenoid biosynthesis, growth velocities of MKD fibroblasts declined in comparison with control cells, further suggesting an impairment of non-sterol isoprenoid biosynthesis in MKD. Our results suggest an imbalance in the multilevel regulation of the biosynthesis of cholesterol and non-sterol isoprenoids in MKD, representing an additional causative factor responsible for the pre- and postnatal pathology of MKD.

Squalene synthase inhibition alters metabolism of nonsterols in rat liver.

We have used the potent squalene synthase inhibitor squalestatin I to investigate the regulation of isoprenoid metabolism in rat liver Fresh-frozen liver pieces from normal rats and rats infused with squalestatin I at 16 micrograms h-1 for 16 h were assayed for farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) by HPLC after dephosphorylation. Levels of FPP and GGPP were 5.4 +/- 1.6 nmol g-1 and 1.6 +/- 0.7 nmol g-1 (n = 13) wet wt., respectively, in control livers and 110 + 41 nmol g-1 and 3.0 +/- 2.2 nmol g-1 (n = 13) in livers from squalestatin I infused rats. In order to determine the relative level of isopentenyl pyrophosphate, liver slices from normal and squalestatin I infused rats were labeled to steady-state with [3H]acetate. Analysis of isoprenoid pyrophosphate intermediates by radio-HPLC after dephosphorylation indicated that squalestatin I brought about a 20-fold increase in the relative level of FPP (confirming direct analysis) and a 5-fold increase in the relative level of IPP. No change in either of these compounds was observed in livers from cholesterol-fed rats. To determine if squalestatin I altered the synthesis of nonsterol products, rats were subjected to long term subcutaneous infusion. After 14 days of infusion of 15 micrograms h-1, the median chain length of hepatic dolichol and dolichyl phosphate increased from C95 to C115 and the levels of these lipids increased approximately 3-fold. In addition, dolichyl phosphate mannose synthase activity in microsomes from squalestatin I treated rats was increased relative to controls when assayed in the absence of dolichyl phosphate. Squalestatin I affected ubiquinone metabolism to a lesser extent: chain lengths shifted from a Q10/Q9 ratio of 0.118 +/- 0.021 in the normal rat to 0.185 +/- 0.016 in the squalestatin I treated animals, and levels rose by approximately 90%. These results suggest that the isoprenoid pyrophosphate intermediates are shared by the cholesterol, dolichol and ubiquinone pathways and further show that the dolichol and ubiquinone pathways are not saturated. Apparently, under normal conditions, the levels of these intermediates are maintained relatively constant by coordinate enzyme regulation, thereby ensuring a constant rate of synthesis of nonsterols.

Farnesol is not the nonsterol regulator mediating degradation of HMG-CoA reductase in rat liver.

A recent report, in which cultured tumor cells were used, identified farnesol as the nonsterol mevalonate-derived metabolite required for the accelerated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (C. C. Correll, L. Ng, and P. A. Edwards, 1994, J. Biol. Chem. 269, 17390-17393). We examined this proposed linkage in animals by measuring hepatic farnesol levels and rates of HMG-CoA reductase degradation under conditions previously shown to alter the stability of the reductase. In normal rats, the hepatic farnesol level, quantified by high-pressure liquid chromatography, was 0.10 +/- 0.08 microgram/g and the half-life of HMG-CoA reductase was 2.5 h. Administration of mevalonolactone at 1 g/kg body wt to provide all nonsterol metabolites in addition to cholesterol increased farnesol levels 6-fold without significantly affecting the half-life of the reductase. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, raised hepatic farnesol levels 10-fold and decreased the half-life of HMG-CoA reductase to 0.25 h. However, feeding lovastatin to rats did not lower hepatic farnesol levels despite a marked stabilization of HMB-CoA reductase protein. Moreover, intubation of rats with 500 mg/kg body wt of farnesol failed to decrease the half-life of HMG-CoA reductase protein, alter the levels of enzyme activity, or change of the levels of immunoreactive protein despite an increase of 1000-fold in hepatic farnesol levels. These observations indicate that farnesol per se does not induce accelerated degradation of HMG-CoA reductase in rat liver.

Metabolism of [3H]farnesol to cholesterol and cholesterogenic intermediates in the living rat eye.

Adult rats were injected intravitreally with all-trans [1-3H]farnesol, with or without co-injection of the squalene epoxidase inhibitor NB-598. Retinas were isolated 16 h later and their lipids were extracted, saponified, and analyzed by radio-HPLC. Most (> or = 90%) of the nonsaponifiable radioactivity was recovered as unmetabolized [3H]farnesol; however, about 6-8% of the radioactivity in control retinas exhibited the chromatographic behavior of sterols, including cholesterol. Unlike the controls, the NB-598-treated retinas exhibited substantial accumulation of both [3H]squalene and squalene mass. Calculations indicate that most of the squalene mass was derived from metabolism of endogenous precursors, with an in vivo biosynthetic rate of 46 +/- 17.5 pmol/retina/h. Retinas from eyes injected with all-trans [1-3H]geranylgeraniol yielded only the unmetabolized precursor in the nonsaponifiable extracts. These results suggest that farnesol can be "activated" in vivo (presumably to the corresponding allylic pyrophosphate) in the retina and subsequently metabolized to sterols and sterol precursors.

Isoprenoid lipid metabolism in the retina: dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes.

Frogs were injected intravitreally with [3H]acetate, and the formation of [3H]-labeled squalene and cholesterol in the retina and their incorporation into rod outer segment (ROS) membranes were evaluated biochemically over a 60-day time course. ROS [3H]squalene specific activity was maximal by 1-3 days, then declined with a half-time of approximately 20-30 days. In contrast, the specific activity of ROS [3H]cholesterol initially increased to a level substantially less than that of [3H]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS cholesterol. When [3H]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% of those recovered from the ipsilateral retina; hence, systemic contributions to de novo synthesis were obviated. Long-term (> or = 8 hr) in vitro incubations of isolated retinas with [3H]acetate resulted in incorporation of [3H]-labeled sterols and squalene into ROS, at levels comparable to those observed in ROS from companion incubated eyecup preparations and from retinas 8 hr after intravitreal injection of [3H]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with respect to isoprenoid lipid metabolism, and suggest that de novo synthesis within the neural retina is responsible for generating most, if not all, of the [3H]squalene and [3H]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy poisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited marked suppression of incorporation of newly synthesized protein into the ROS, while [3H]squalene incorporation was only minimally reduced, compared with frogs maintained at 22 degrees C. These results are consistent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane proteins.

Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A.

In vivo biosynthesis of cholesterol in the rat retina.

Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of [3H]acetate. HPLC analysis of retinal non-saponifiable lipid extracts revealed co-elution of radioactivity with endogenous cholesterol mass within 4.5 h post-injection. Incorporation of [3H]acetate into cholesterol was markedly reduced by co-injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB-598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo.

Regulation of isoprenoid metabolism in rat liver: near constant chain lengths of dolichyl phosphate and ubiquinone are maintained during greatly altered rates of cholesterogenesis.

When rat liver slices were incubated with varying concentrations of [3H]mevalonolactone, the chain lengths of radiolabeled dolichyl phosphate and ubiquinone varied according to the initial mevalonolactone concentration, indicating that product chain length is dependent on the level of isoprenoid diphosphate intermediates. However, when livers were analyzed from rats which had been maintained on diets of either colestipol (which induces cholesterogenesis 3-fold), or normal chow, or cholesterol (which suppresses cholesterogenesis to 5% of normal) there were only minor changes in the isoprene distribution of either dolichyl phosphate or ubiquinone. In contrast, when rats were maintained on 2% cholesterol plus mevalonolactone (conditions prone to increase the levels of intermediates), the isoprene distributions of both of these compounds were greatly shifted to the higher homologs. However, under none of these conditions were the hepatic levels of these compounds changed significantly. It is concluded that under conditions of greatly altered cholesterogenesis, regulatory mechanisms exist which stabilize the levels of isoprenoid diphosphate intermediates, and that even when levels are increased (e.g., by dietary manipulation), the effect is only to alter isoprene distribution and not the rate of synthesis of dolichyl phosphate and ubiquinone.

Rapid synthesis of isoprenoid diphosphates and their isolation in one step using either thin layer or flash chromatography.

A rapid procedure for the preparation of short-chain (C s-C.) isoprenoid diphosphates is described . It is based on the method of Cornforth and Popjak [Methods Enzymol., 15 (1969) 359-390] which utilizes bis-triethylammonium phosphate in trichioroacetonitriie as the phosphorylating reagent . The reaction takes place in 15 min, and product isolation, previously requiring several steps, is done in a single step using either preparative thin-layer chromatography or flash chromatography on silica . From a single TLC plate, up to 50 µ mol of pure farnesyl diphosphate (i .e., ca. 20 mg) can be isolated, while up to 1200 µ mol can be isolated using a standard flash chromatography column.

GTPases.

GTPases represent a large family of proteins involved in a wide variety of cellular processes. They function as switches in which they are "on" in the GTP bound state and "off" in the GDP bound state. The "on" and "off" states are regulated by proteins which interact with the GTPases and enhance their rate of either GTP hydrolysis or GDP-GTP exchange. Thus far, GTPases have been shown to be involved in protein synthesis, signal transduction, control of cell growth, and intracellular transport.

Feedback regulation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol is not due to altered mRNA levels.

Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for "cellular nucleic acid-binding protein" which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7 alpha-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feed-back regulation of hepatic HMG-CoA reductase gene expression does not occur at the level of transcription.

Incorporation of squalene into rod outer segments.

We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [3H]acetate in short term incubations of frog retinas (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254). In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254), these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms.

Lipid crystals in malignant melanoma of the choroid.

The crystallization of extracellular lipid (other than cholesterol) in histologic preparation is a rarely observed phenomenon. The following case report describes lipid crystals within a necrotic uveal melanoma. Ultrastructural findings suggest that lipids released from necrotic lipidized melanoma cells were the source of the extracellular deposits. No unique lipids were identified from extracts of the tumor using thin layer chromatography. Energy dispersion microanalysis revealed elemental concentrations within the crystals comparable to intracellular organelles.