Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy.

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a euglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

Local secretion of parathyroid hormone-related protein by an osteoblastic osteosarcoma (UMR 106-01) cell line results in growth inhibition.

Parathyroid hormone-related protein (PTHrP) has been implicated as being important in the growth of tumor cells responsive to the peptide. We utilized a rat osteoblastic osteosarcoma cell line, UMR 106-01, which has PTHrP receptors and a PTHrP-responsive adenylate cyclase/cAMP messenger system, to produce a modified cell line that overexpresses PTHrP. The human PTHrP cDNA sequence was transfected by electroporation into UMR 106-01 cells and the stable cell lines UMR-36 and UMR-34 were established. The modified cell line, UMR-36, had increased levels of PTHrP mRNA compared with control cell lines and secreted PTHrP into the culture medium at levels of 0.01-0.1 pmol/10(7) cells in 12 h. The secreted peptide was biologically active as indicated by its ability to activate adenylate cyclase. The number of UMR-36 cells following 9 days in culture was reduced by up to 80% compared with control lines, which was associated with decreased (3)H-thymidine incorporation into genomic DNA. Addition of 1000-fold excess of the PTHrP antagonist, PTHrP(7-34), to UMR-36 cells resulted in the escape of growth inhibition and increased rate of growth. In vivo, tumors derived from UMR-36 cells were smaller in size compared with tumors derived from control cells. In conclusion, increased autocrine secretion of, and responsiveness to, PTHrP results in inhibited growth kinetics of an osteoblast-like bone tumor cell line in vitro and in vivo.

A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression.

The selective alteration of the genome using Cre recombinase to target the rearrangement of genes flanked by LOX recognition sequences has required the use of two separate genetic constructs in trans, one containing cre and the other containing the gene of interest flanked by LOX sites. We have developed a strategy in which both the cre recombinase gene and LOX recombination sites may be cloned within a single vector in cis. This method uses a modified form of Cre (CREM) that contains alterations to the 5' region including the introduction of a Kozak consensus sequence and insertion of a functional intron. This system allows for the inducible, tissue-specific activation or inactivation of gene expression in a single vector and can be utilized for the 300-fold amplification of gene expression from a weak promoter. This approach can be applied to targeting strategies for generating genetically altered mice and gene therapy.

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.

The 5' flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-alpha expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

Impaired regulation of hepatic fructose-1,6-biphosphatase in the New Zealand Obese mouse: an acquired defect.

Increased hepatic glucose production, a feature of (non-insulin-dependent diabetes mellitus [NIDDM]), is present at an early age in the New Zealand Obese (NZO) mouse and is associated with impaired suppression of the gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). The aim of this study was to further characterize the abnormality in the regulation of hepatic FBPase in NZO mice versus New Zealand Chocolate (NZC) control mice. At 20 weeks of age, NZO mice have elevated FBPase activity (65.3 +/- 7.9 v 46.7 +/- 5.0 micromol/min/mg protein, P =.07) and protein levels (31.7 +/- 3.1 v 22.5 +/- 2.8 arbitrary units, P < .05), but not mRNA levels (0.18 +/- 0.03 v 0.16 +/- 0.03 arbitrary units). Elevated FBPase activity and protein levels in NZO mice were also shown at 4 to 6 weeks of age, but not in 1-day-old mice, suggesting that the increase occurs between birth and weaning. The Km of the enzyme was the same in NZO and NZC mice (3.7 +/- 0.5 v 5.0 +/- 0.9 micromol/L, NZO v NZC). The regulation of FBPase by the competitive inhibitor, fructose-2,6-bisphosphate ([Fru(2,6)Pz] 5 micromol/L) measured over a range of substrate concentrations (2.5 to 80 micromol/L) was similar between NZO and control mice (Km in the presence of Fru(2,6)Pz, 10.8 +/- v 1.9 v 13.2 +/- 3.3 micromol/L, NZO v NZC). It is concluded that increased FBPase activity in the NZO mouse is due to elevated protein levels, and that this appears to be due to a failure of the normal decrease that occurs following birth in control animals.

Inheritance of chromosome 7 is associated with a drug-resistant phenotype in somatic cell hybrids.

A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdr1 gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump. In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterized by a clonal chromosomal duplication dup(7)(q11.23q31.2). Fourteen hybrids, chosen at random, were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope. Only two hybrids were positive for both MRK16 antibody labelling and mdr1 mRNA. Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line. Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.

Impaired glucose tolerance and increased weight gain in transgenic rats overexpressing a non-insulin-responsive phosphoenolpyruvate carboxykinase gene.

The effects of an overexpressed, non-insulin-responsive gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32), on glucose homeostasis were investigated. Transgenic rats harboring a metallothionein-driven PEPCK gene (lacking the entire PEPCK upstream-regulatory region) expressed transgene PEPCK mRNA in the key gluconeogenic tissues, liver and kidney. Female transgenic rats, studied at 10 weeks of age, showed mild fasting hyperglycemia (6.9 +/- 0.2 vs. 5.9 +/- 0.1 mM P = 0.002 n = 6), hyperinsulinemia (92.2 +/- 4.0 vs. 54.0 +/- 6.6 pM, P = 0.001, n = 6), impaired glucose tolerance and increased weight gain (178.3 +/- 3.2 vs. 153.4 +/- 2.5 g, P = 0.001, n = 16 and n = 13 transgenic and control rats, respectively). Despite hyperinsulinemia at this age, kidneys of transgenic rats maintained a significant 20% elevation of total PEPCK enzyme activity, while total liver PEPCK activity was not reduced. This study suggests that an insulin-resistant step in the gluconeogenic pathway can lead to glucose intolerance and an increase in weight. These rats offer the unique opportunity to study the metabolic consequences of chronic, mild excess glucose supply, as seen in non-insulin-dependent diabetes.

Impaired regulation of hepatic fructose-1,6-bisphosphatase in the New Zealand obese mouse model of NIDDM.

The New Zealand obese mouse, a model of NIDDM, is characterized by hyperglycemia, hyperinsulinemia, and hepatic and peripheral insulin resistance. The aim of this study was to investigate the biochemical basis of hepatic insulin resistance in NZO mice. Glycolytic and gluconeogenic enzyme activities were measured in fed and overnight fasted 19- to 20-wk-old NZO and control New Zealand chocolate mice. The NZO mice were twice as heavy as the NZC mice. The activity of the glycolytic enzymes glucokinase and pyruvate kinase was higher, whereas that of the gluconeogenic enzymes PEPCK and glucose-6-phosphatase was lower in fed and fasted NZO mice. These enzyme changes are consistent with a normal response to the hyperinsulinemia in NZO mice. In contrast, the activity of the third regulated gluconeogenic enzyme, fructose-1,6-bisphosphatase, was similar in fed and fasted NZO and NZC mice despite the higher insulin and glucose levels in the NZO mouse. This enzyme is primarily regulated by the powerful inhibitor fructose-2,6-bisphosphate. The levels of this metabolite were measured and found to be increased in both the fed and fasted states in the NZO mouse, suggesting that the activity of the bifunctional enzyme that regulates the level of inhibitor (6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase) is normally regulated in the NZO mouse. We conclude that most insulin-responsive gluconeogenic and glycolytic enzymes are normally regulated in the NZO mouse, but an abnormality in the regulation of fructose-1,6-bisphosphatase may contribute to the increase hepatic glucose production in these mice.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene.

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Surgical damage stimulates proliferation of dopamine uptake sites in normal mouse brain.

To test the hypothesis that proliferation of host dopaminergic tissue in response to injury plays an important role in the response to intrastriatal grafting, we transplanted autologous adrenal medullary to striatum in normal C57-black mice and compared this procedure with transplantation of non-dopaminergic tissue (frontal cortex) or a non-cellular matrix (Gelfoam). [3H]Mazindol autoradiography revealed that all three protocols resulted in a marked proliferation of dopamine uptake sites 10 months after transplantation.

Calcitonin increases transcription of parathyroid hormone-related protein via cAMP.

Transcriptional regulation of the human parathyroid hormone-related protein (PTHrP) gene by calcitonin was examined in a lung cancer line (BEN cells). Northern analysis demonstrated that calcitonin caused a rapid 4.5-fold elevation in PTHrP mRNA. Transient transfection of a construct containing 1119 base pairs of the human PTHrP gene 5' to the ATG start site of translation, fused to the CAT reporter sequence, was used to demonstrate a five-fold increase in transcription by calcitonin. Similar increases were also observed when transfected cells were exposed to a number of cAMP agonists including forskolin, as well as isobutyl-methylxanthine. A putative cAMP responsive element (5'-TGACTTCA-3') present within exon 4 was placed upstream of the heterologous SV40 promoter. Expression of this construct was elevated 4.5-fold in response to calcitonin and 7-fold in response to forskolin. Similar responses to calcitonin occurred with a smaller construct (pZMR30) containing 530 bp of sequence upstream of the ATG start site. Thus we postulate that calcitonin acts at least partially via cAMP through this element in exon 4 of the human PTHrP gene.

Angiotensin II receptor binding associated with nigrostriatal dopaminergic neurons in human basal ganglia.

In the human brain, receptor binding sites for angiotensin are found in the striatum and in the substantia nigra pars compacta overlying dopamine-containing cell bodies. In contrast, angiotensin-converting enzyme occurs in the substantia nigra pars reticulata and is enriched in the striosomes of the striatum. In this study, using quantitative in vitro autoradiography, we demonstrate decreased angiotensin receptor binding in the substantia nigra and striatum of postmortem brains from patients with Parkinson's disease. In the same brains the density of binding to angiotensin-converting enzyme shows no consistent change. We propose, from these results, that angiotensin receptors in the striatum are located presynaptically on dopaminergic terminals projecting from the substantia nigra. In contrast, the results support previous studies in rats demonstrating that angiotensin-converting enzyme is associated with striatal neurons projecting to the substantia nigra pars reticulata. These findings raise the possibility that newly emerging drugs that interact with the angiotensin system, particularly converting enzyme inhibitors and new nonpeptide angiotensin receptor blockers, may modulate the brain dopamine system.

Distribution of catecholamine uptake sites in human brain as determined by quantitative [3H] mazindol autoradiography.

Because of the importance of the catecholamine system in Parkinson's disease and its relevance to a variety of clinical movement disorders, catecholamine uptake sites were mapped in the human brain using [3H] mazindol autoradiography. Displacement studies with known dopamine (DA) and noradrenaline (NA) uptake blockers showed that binding in the striatum was to dopamine uptake sites; binding in the locus coeruleus was to noradrenergic uptake sites. By using the selective noradrenergic uptake blocker desmethylimipramine (DMI), a comprehensive map of both DA and NA uptake sites was generated. In general, catecholamine uptake sites were better seen in terminals than in cells of origin or axonal projections. In some areas, such as the locus coeruleus, punctate binding could be seen over individual pigmented cells. A variegated pattern of binding was seen in caudate nucleus and putamen and some correspondence of patches of low binding with striosomes was observed in the caudate. The highest levels of binding to DA uptake sites was observed in the striatum, where regional differences in binding occurred. The most dense binding was seen in the ventral striatum, and a rostral-to-caudal decrement in binding levels in caudate nucleus and putamen was evident. Binding was more intense in the putamen compared to the caudate and within the caudate lower values were seen laterally. The highest levels of binding to noradrenergic uptake sites were in the locus coeruleus and dorsal raphé, although these sites may be on terminals from other projections. Whereas uptake sites were more often evident in known catecholamine pathways, [3H] mazindol binding was seen in some areas where catecholamine neurons or terminals had not been identified previously. These maps of the catecholamine uptake system add further information concerning the nature of the distribution of catecholamines in human brain and provide an important baseline for the study of disease and ageing processes.

Evidence for plasticity of the dopaminergic system in parkinsonism.

A series of compensatory mechanisms within the dopaminergic system have been shown to maintain clinical function in the presence of dopamine loss. Experimental evidence for increased presynaptic dopamine turnover owing to increased dopamine synthesis, release, and reduced reuptake exists. Direct evidence that these mechanisms maintain extracellular dopamine levels is provided by intracerebral microdialysis techniques. Postsynaptic denervation supersensitivity clearly occurs with D2 dopamine receptors, although this is less evident with D1 receptors. Similarly, mechanisms of plasticity have been shown to be relevant in human postmortem and Positron Emission Tomographic studies of patients with Parkinson's disease. However, although presynaptic increases in dopamine turnover are well documented, postsynaptic D1 and D2 receptor changes have been more difficult to establish, mainly because of methodological difficulties. D2, but not D1, receptor increases have been documented in drug naive Parkinsonian patients with PET techniques. In transplantation of adrenal gland to striatum in animal models and patients with Parkinsonism where clinical improvement occurs, plasticity of host response may be as important as plasticity of the graft. Although some elements of the compensatory mechanism of dopamine plasticity may be deleterious, such as dyskinesias owing to dopamine receptor supersensitivity, the overall effect of delay and minimization of the clinical expression of disease is advantageous. An even greater understanding of the mechanisms involved may assist in developing future therapeutic strategies.

Catecholamine uptake sites in mouse brain: distribution determined by quantitative [3H]mazindol autoradiography.

Because of the importance of the mouse brain catecholamine system in the study of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and because little information is available concerning the chemical neuroanatomy of the mouse, catecholamine uptake sites were mapped in C57 black mouse brain using [3H]mazindol autoradiography. Displacement studies with known dopamine (DA) and noradrenaline (NA) uptake blockers showed that binding in the striatum was entirely to DA uptake sites, while binding in the locus coeruleus was to NA uptake sites only. By using the selective noradrenergic uptake blocker desmethylimipramine (DMI), a complete map of both DA and NA uptake sites was generated. The mesostriatal DA system was the most clearly labelled and uptake sites were seen better in striatal terminals than the substantia nigra. Within the noradrenergic system, highest binding levels were seen over the locus coeruleus, although it was unclear whether these uptake sites were on cell bodies or terminals from the lateral tegmental noradrenergic system. These maps of the catecholamine uptake system in mouse brain provide a baseline for study of newly discovered neurotoxins and ageing processes.

Regional and temporal effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on dopamine uptake sites in mouse brain.

When the regional effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on brain dopamine uptake sites in C57 Black mice were studied using [3H]mazindol autoradiography, marked regional differences in effect were seen: the mesolimbic system was less affected than the nigrostriatal tract and within each system the effect was more severe in the terminal fields of the striatum than in the cells of origin. Within the striatum itself there was inhomogeneity of effect, with relative sparing of the dorsomedial aspect compared to the remainder. Complete recovery of [3H]mazindol binding to striatal membranes occurred over 12 months, while dopamine levels recovered more slowly. This supports the concept that MPTP has a highly selective effect within dopaminergic systems and that the initial effect is more pronounced on distal terminals compared to cell bodies. The possibility that recovery of mazindol binding with time may be associated with terminal regrowth needs to be investigated further.

Motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse.

Motor function was assessed by use of a swim test in C57 Black mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Thirty minutes after the last MPTP injection significant motor impairment was observed while striatal dopamine was reduced to 13.9% of control levels. At 24 h and 7 days post MPTP injection dopamine levels were still reduced to 17.3% and 26.4% of control values but swimming abilities of the mice were unimpaired. Histofluorescence of catecholaminergic neurons confirmed the presence of catecholamine depletion but showed little evidence of neuronal destruction. The use of MPTP as a non-invasive means of nigrostriatal dopamine depletion in rodents and higher animals allows a re-evaluation of the role of the dopaminergic system in the modulation of movement.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on presynaptic dopamine uptake sites in the mouse striatum.

The effects of the specific dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were studied on the kinetics of [3H]mazindol binding to striatal membranes of C57 black mice. This radioligand was used to label dopamine uptake sites and when administered in vivo, MPTP caused an irreversible, non-competitive inhibition of mazindol binding, consistent with damage to dopaminergic terminals. This effect was abolished by pretreatment with pargyline, a MAOB inhibitor, suggesting that oxidation of MPTP to the pyridinium moiety, MPP+, is a necessary step for toxicity when mazindol binding is used as an end point. In keeping with these findings, pretreatment of mice with mazindol protected against the dopamine-depleting effects of MPTP in vivo. This data suggests that MPTP exerts its toxic effects via MPP+ which is concentrated intraneuronally via the dopamine uptake system. During this process the neurotoxin irreversibly inactivates the dopamine uptake sites.

The neurochemical and clinical effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in small animals.

The recent discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a syndrome of Parkinsonism in man and monkey has stimulated the search for a small animal model of Parkinsonism. In this study, MPTP was administered to a series of small animals and observations made on clinical and neurochemical changes. The clinical effects of MPTP in rabbits, guinea pigs, and rats were short lived, and no chronic Parkinsonian syndrome developed. The C57 black mouse, however, although also not showing clinical changes, proved to be an ideal neurochemical model in which to study the effects of MPTP since striatal dopamine levels were reliably reduced to 13% of control values after 4 intraperitoneal injections of 30 mg/kg MPTP. Pathological study of the striatum and substantia nigra in the mouse model failed to show any alteration in neuronal morphology or numbers. Although the effect of MPTP on striatal dopamine lasted for up to 2 weeks after the last MPTP injection, the possibility exists that no neurotoxic effects occur and the observed dopamine depletion is pharmacological only.