Glen E. Mott

Relationship of lipoprotein cholesterol concentrations to experimental atherosclerosis in baboons.

For 26 months, we fed 60 baboons a high saturated fat, high cholesterol diet that contained very low concentrations of four common pesticides (chlordane, parathlon, dlazlnon, and carbofuran). We detected no effect of pesticides on body weight, serum llpld, or Iipoprotein cholesterol concentrations, or experimental atherosclerosis. We then examined the associations of serum llpld and Iipoprotein cholesterol concentrations {predictor variables) with arterial lesions (naponto variables). Among predictor variables, very low density Iipoprotein plus low density Iipoprotein cholesterol concentration showed a positive association with fatty streaks in the aorta and its major branches, Including the coronary arteries, while high density Iipoprotein cholesterol concentration showed a consistently negative association. The very low density Iipoprotein plus low density llpoproteln/hlgh density Iipoprotein cholesterol ratio was more highly associated with lesions than was either value alone. These results are consistent with epidemlologic evidence suggesting that high density Iipoprotein cholesterol concentration Is Inversely related to probability of developing clinically manifest atherosclerotic disease.

Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatins effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Molecular heterogeneity of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C16:0 alkyl chain in the sn-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C15- and C18-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.

Lovastatin induces apoptosis by inhibiting mitotic and post-mitotic events in cultured mesangial cells

Lovastatin, an inhibitor of protein prenylation, was reported to inhibit DNA synthesis and induce apoptosis in cultured cells. This report describes the morphological consequences of lovastatin treatment. Lovastatin (50 microM) induced mesangial cell rounding and disassembly of actin stress fibers within 24 to 48 h. After 48 to 72 h of lovastatin treatment, the cells detached from the substratum and underwent apoptotic cell death as evidenced by condensed nuclear chromatin, nuclear fragmentation, cell blebbing and decrease in cell size. Time lapse cinematography revealed that lovastatin caused cell rounding by either inhibiting cytokinesis or cell spreading following cytokinesis. Lovastatin-induced cell rounding, detachment, and apoptosis were dependent upon cell proliferation. These effects were prevented by serum deprivation to inhibit cell proliferation or by plating cells at densities which resulted in contact inhibition of cell growth. Lovastatin-induced mesangial cell rounding and apoptosis were also prevented by the inclusion of the isoprenoids all-trans-farnesol or all-trans-geranylgeraniol in the incubation medium. These results indicate that the effects of lovastatin were mediated by inhibition of protein isoprenylation because exogenous all-trans-geranylgeraniol can be used only in protein prenylation. The small GTP-binding protein RhoA, which may be important for cell spreading and cytokinesis, accumulated in the cytosol following treatment with lovastatin, suggestive of its inactivation. This effect was also prevented by the inclusion of either farnesol or geranylgeraniol in the incubation medium. Thus, lovastatin-induced apoptosis in mesangial cells occurs by interfering with prenylation dependent mitotic and post-mitotic events.

Influence of infant and juvenile diets on serum cholesterol, lipoprotein cholesterol, and apolipoprotein concentrations in juvenile baboons (Papio sp.)☆

The long-term effects of infant diet (breast milk or formula containing 2, 30, or 60 mg/dl cholesterol) and subsequent dietary cholesterol (1 mg/kcal) and fat (saturated or unsaturated) on serum lipid and apolipoprotein concentrations were estimated using 82 juvenile baboons 4-6 years of age. A significant interaction of infant diet (breast vs formula) with type of fat (saturated vs unsaturated) at 4-6 years of age was observed on HDL cholesterol and apolipoprotein A-I (apoA-I) concentrations. That is, animals breast-fed as infants had higher HDL cholesterol and apoA-I concentrations when fed unsaturated fat from weaning to 4-6 years of age than those fed saturated fat (77 vs 68 mg/dl). In contrast, animals fed formulas in infancy followed by a diet containing unsaturated fat had lower HDL cholesterol and apoA-I concentrations at 4-6 years of age than did those fed saturated fat (67 vs 78 mg/dl). However, breast feeding or feeding formulas containing various levels of cholesterol for 3 months during infancy did not result in statistically significant differences in total serum cholesterol, VLDL + LDL cholesterol and apolipoprotein B (apoB) concentrations. Dietary cholesterol after infancy significantly increased serum total cholesterol, VLDL + LDL and HDL cholesterol, apoA-I and apoB concentrations. All of these response variables also were higher in animals fed saturated fat compared to those fed unsaturated fat on the same level of cholesterol. At 4-6 years of age, regardless of diet, females had significantly higher serum VLDL + LDL cholesterol (57 vs 43 mg/dl) and apoB concentrations (39 vs 30 mg/dl) than did males.

Fast atom bombardment-mass spectrometric identification of molecular species of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes.

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.

Effects of two forms of hypertension on atherosclerosis in the hyperlipidemic baboon.

We examined the relationship of hypertension and plasma renin activity to atherogenesis in 48 moderately hyperlipidemic (total serum cholesterol was about 200 mg/dl) baboons (Papio sp.). We used renal artery stenosis (two-kidney, one clip model) to produce hypertension associated with elevated plasma renin activity, and used cellophane wrapping of both kidneys (bilateral perinephritis model) to produce hypertension with normal renin activity. Renal artery stenosis and bilateral perinephritis increased both systolic and diastolic blood pressure by about 30 mm Hg. Renal artery stenosis approximately doubled, but bilateral perinephritis did not change plasma renin activity.

Pleiotropy and genotype by diet interaction in a baboon model for atherosclerosis: A multivariate quantitative genetic analysis of HDL subfractions in two dietary environments

We investigated dietary effects on pleiotropic relationships among 3 HDL cholesterol (C) subfractions (HDL1-C, HDL2-C, and HDL3-C; levels quantified by gradient gel electrophoresis) for 942 pedigreed baboons (Papio hamadryas) who were fed a basal (Chow) diet and a high cholesterol, saturated fat (HCSF) challenge diet. Using multivariate maximum likelihood methods we estimated heritabilities for all 6 traits, genetic and environmental correlations (rhoG and rhoE) between them, and the additive genetic variance of each subfractions response to the diets. On the Chow diet, genetic correlations between the 3 subfractions were significant, and we observed complete pleiotropy between HDL1-C and HDL3-C (rhoG=-0.81). On the HCSF diet, only the genetic correlation between HDL1-C and HDL3-C (rhoG=-0.61) was significant. Genetic correlations between individual subfractions on the Chow and HCSF diets did not differ significantly from 1.0, indicating that the same additive genes influenced each subfractions levels regardless of diet. However, the additive genetic variance of response to the diets was highly significant for HDL1-C and HDL2-C, but not for HDL3-C. Similar sets of genes influence variation in the 3 HDL subfractions on the Chow diet, and the same set influences variation in each subfraction on the HCSF diet. However, the expression of genes influencing HDL1-C and HDL2-C is altered by the HCSF diet, disrupting the pleiotropy observed between the 3 subfractions on the Chow diet.

Effect of cyclin E Overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells

The HMG‐CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin‐induced growth arrest and cell rounding. Lovastatin treatment (10 μM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin‐dependent kinase inhibitor p27kip1, inhibition of both cyclin E‐ and cyclin A‐dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E‐ or cyclin A‐dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27kip1. However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane‐bound Rho proteins (i.e., GTP‐bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E‐dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane‐associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E‐dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA. J. Cell. Biochem. 74:532–543, 1999.

Cholesterol metabolism in juvenile baboons. Influence of infant and juvenile diets.

The deferred effects of infant diets and the effects of juvenile diets on cholesterol metabolism were estimated in 83 baboons (Papio sp.) at 3.5 years of age. As infants, the animals were breast-fed or fed one of three formulas containing approximately 2, 30, or 60 mg/dl cholesterol. After weaning at 14 weeks of age, the animals were fed one of four juvenile diets high or low in cholesterol with saturated (P/S = 0.37) or unsaturated (P/S = 2.1) fats. Cholesterol absorption and cholesterol turnover were measured by fecal isotopic methods, and variables of cholesterol metabolism were estimated from a two-pool model. Among juvenile animals breast-fed during infancy, the percentage of cholesterol absorption was higher, while the fluxes of cholesterol from Pool A (QAA, QA, and QAB) and the cholesterol mass of Pool B were lower, compared to those fed formulas. The level of cholesterol in formulas fed during infancy did not influence cholesterol metabolism during the juvenile period. During the juvenile period, saturated fat significantly decreased the cholesterol production rate (QA) and increased the rate constants for cholesterol flux between Pool A and Pool B (KAB and KBA) compared to unsaturated fat. High cholesterol intake increased bile acid and neutral steroid excretion, cholesterol turnover rate, the mass of Pool A, and the rate constant KA and fluxes QA and QAA for removal of cholesterol from Pool A. However, KAB, t1/2B (half-time of Pool B), and the percentage of cholesterol absorbed were decreased. Dietary cholesterol and saturated fat had similar effects on serum cholesterol and lipoprotein concentrations in these animals, but they had opposite effects on several aspects of cholesterol metabolism.