Fig. 1. (A) Map of Asia showing the range of greenish warblers in the breeding season. Different colors represent different subspecies as designated by Ticehurst (20) (P. t. viridanus, blue; ludlowi, green; trochiloides, yellow; obscuratus, orange; plumbeitarsus, red; and nitidus, violet). Colors grade together in areas where Ticehurst described gradual morphological change. The hatched area in central Siberia indicates the overlap zone between viri-danus and plumbeitarsus. The gap in the ring in northern China is likely due to recent habitat destruction (70). Sampling sites are indicated by symbols corresponding to major mitochondrial clades [open symbols indicate western clade, and closed symbols eastern, see fig. S1 and (70)], with the most important sites indicated by two-letter codes. (B) Geographic variation in 62 AFLP markers as summarized by principal coordinates analysis. Each symbol represents a single individual, and distance between symbols corresponds roughly to genetic distance. Colors and 2 0 25

sAm AltShocghretheonSrth<- First principal coordinate <AFLP>

ern subspecies viridanus and plumbeitarsus differ distinctly in their genetic characteristics, there is gradual genetic change through the southern chain of populations. PC1 explains 19.4% of the variance, PC2 5.6%.

flow. The simplest historical scenario for this result is that short-distance dispersal in a continuously distributed species has resulted in a pattern of isolation by distance (18).

This interpretation may at first seem inconsistent with previously published patterns of variation in mitochondrial DNA (mtDNA) (10) (fig. S1), in which there are several deep phylogeographic breaks around the ring, the deepest of which is in the western Himalayas. In fact, the mtDNA and AFLP patterns are compatible. Short-distance dispersal in a continuously distributed species is expected to cause phylogeographic structuring in mtDNA clades (19) and a pattern of isolation by distance in AFLP markers (18). The shape of the greenish warbler range is particularly suited to creating this pattern; the birds breed in a narrow string of treeline habitat through the Himalayas, where dispersal distances are likely shorter than in more broadly distributed forest habitat further north. Ticehurst (20) hypothesized that the greenish warblers were at one time confined to the Himalayas and then expanded northward along two pathways into Siberia. Theory predicts that the pattern of isolation by distance should be weaker in regions of recent range expansion compared with regions that have been inhabited over a long period of time (18). The steeper genetic change seen in AFLPs (figs. S2 and S3), mtDNA, and two microsatellite loci (10) through the Himalayas than through regions to the north is consistent with this prediction. It is also possible that these patterns were influenced by temporary breaks in gene flow due to geographic barriers in the Himalayas; however, such barriers, if they existed, did not cause reproductive isolation to evolve in that region.

Greenish warblers provide the only known example of a smooth genetic gradient between two genetically differentiated and reproductively isolated forms, providing rare insight into how speciation can occur. Patterns of variation in ecologically and

Fig. 2. Genetic distance based on AFLP markers increases with geographic distance measured around the southern ring (that is, if one assumes no direct gene flow between viridanus and plumbeitarsus or across the uninhabited area in the center of the ring). Corrected average pairwise distances between populations were calculated as the mean number of pairwise differences between two populations minus the average distance between individuals within those populations. For purposes of illustration, a least-squares regression line is fit to all points.

Fig. 2. Genetic distance based on AFLP markers increases with geographic distance measured around the southern ring (that is, if one assumes no direct gene flow between viridanus and plumbeitarsus or across the uninhabited area in the center of the ring). Corrected average pairwise distances between populations were calculated as the mean number of pairwise differences between two populations minus the average distance between individuals within those populations. For purposes of illustration, a least-squares regression line is fit to all points.

sexually selected traits of greenish warblers suggest that latitudinal gradients in environmental characteristics, such as forest density and seasonal migration distance, during the two northward expansions into Siberia have resulted in rapid evolutionary adaptation, divergence, and reproductive isolation (8, 10, 11).

Several authors (4, 21) have suggested on theoretical grounds that ring species or "sexual continua" are unstable and will fairly quickly break into two or more species that do not exchange genes. The two models that show this effect do not apply well to the greenish warbler, because one (4) does not include local adaptation throughout a continuous geographic range and the other (21) does not include different geographic locations for different populations. We suggest that ring species such as the greenish warbler, in which local adaptation occurs along a long and nearly continuous ring of populations, could be stable indefinitely. This stability could be interrupted by processes such as habitat change, which could increase the likelihood of parapatric speciation (5), or habitat destruction, which could divide the continuous range and thereby increase the likelihood of additional species boundaries forming.

References and Notes

1. E. Mayr, Systematics and the Origin of Species (Dover Publications, New York, 1942).

2. J. A. Coyne, H. A. Orr, Speciation (Sinauer Associates, Sunderland, MA, 2004).

3. W. R. Rice, E. E. Hostert, Evolution 47, 1637 (1993).

4. S. Gavrilets, H. Li, M. D. Vose, Proc. R. Soc. London B Biol. Sci. 265, 1483 (1998).

5. M. Doebeli, U. Dieckmann, Nature 421, 259 (2003).

6. T. B. Smith, R. K. Wayne, D. J. Girman, M. W. Bruford, Science 276, 1855 (1997).

7. C. J. Schneider, T. B. Smith, B. Larison, C. Moritz, Proc. Natl. Acad. Sci. U.S.A. 96, 13869 (1999).

8. D. E. Irwin, J. H. Irwin, T. D. Price, Genetica 112-113, 223 (2001).

9. T. Dobzhansky, in A Century of Darwin, S. A. Barnett, Ed. (Heinemann, London, 1958), pp. 19-55.

10. D. E. Irwin, S. Bensch, T. D. Price, Nature 409, 333 (2001).

12. A. D. Richman, T. Price, Nature 355, 817 (1992).

16. U. G. Mueller, L. L. Wolfenbarger, Trends Ecol. Evol. 14, 389 (1999).

17. Materials and methods are available as supporting material on Science Online.

20. C. B. Ticehurst, A Systematic Review of the Genus Phylloscopus (Johnson Reprint Corp., New York, 1938).

21. A. J. Noest, Proc. R. Soc. London B. Biol. Sci. 264, 1389 (1997).

22. Supported by an International Research Fellowship grant from the National Science Foundation (to D.E.I.) and the Swedish Research Council (to S.B.) plus grants for fieldwork by the National Geographic Society and National Science Foundation

Benowitz-Fredericks, J. Gibson, S. Gross, G. Kelberg, A. Knorre, K. Marchetti, and B. Sheldon for assistance in the field, and P. Alstrom, K. Marchetti, U. Olsson, A. Richman, J. Tiainen, and the Burke Museum for additional samples. R. Calsbeek, M. Whitlock, and several anonymous reviewers provided helpful comments.

Supporting Online Material DC1

Materials and Methods Figs. S1 to S3 Table S1 References

14 September 2004; accepted 17 November 2004 10.1126/science.1105201

Large Sulfur Bacteria and the Formation of Phosphorite

Heide N. Schulz1* and Horst D. Schulz2

Phosphorite deposits in marine sediments are a long-term sink for an essential nutrient, phosphorus. Here we show that apatite abundance in sediments on the Namibian shelf correlates with the abundance and activity of the giant sulfur bacterium Thiomargarita namibiensis, which suggests that sulfur bacteria drive phosphogenesis. Sediments populated by Thiomargarita showed sharp peaks of pore water phosphate (<300 micromolar) and massive phosphorite accumulations (>50 grams of phosphorus per kilogram). Laboratory experiments revealed that under anoxic conditions, Thiomargarita released enough phosphate to account for the precipitation of hydroxyapatite observed in the environment.

The formation of phosphorites in marine sediments is a major long-term sink for phosphorus, removing it from the biosphere. The initial step in phosphorite formation is the precipitation of phosphate-containing minerals, for example, hydroxyapatite, followed by many other processes such as sediment transport, winnowing, and re-crystallization (1, 2). A fundamental problem in explaining massive phosphorite deposits has been identifying mechanisms that can concentrate pore water phosphate enough to drive spontaneous precipitation of phosphorus minerals. Here we suggest a new mechanism, the episodic release of phosphate into the an-oxic sediment by an abundant benthic bacterium that is specially adapted to survive under both oxic and anoxic conditions. Thiomargarita periodically contacts oxic bottom water in order to take up nitrate, and it survives long intervals of anoxia with nitrate stored internally (3). The phosphate uptake from different sources occurs when Thiomargarita forms thick mats at the sediment surface or is suspended in the oxic water column.

institute for Microbiology, University of Hannover, Schneiderberg 50, D-30167 Hannover, Germany. 2Department of Geosciences, University of Bremen, Klagenfurter Strasse, D-28359 Bremen, Germany.

*To whom correspondence should be addressed. E-mail: [email protected]

The giant sulfur bacterium Thiomargarita namibiensis occurs in high biomass in surface sediments off the coast of Namibia (3). Like its close relatives Beggiatoa spp. and Thioploca spp., this bacterium gains energy by oxidizing sulfide, which accumulates in anoxic marine sediments as a result of the degradation of organic matter by sulfate-reducing bacteria. The production of sulfide is directly proportional to the amount of organic carbon in the sediment, thus these large sulfide-oxidizing bacteria are abundant in highly productive upwelling areas, where the flux of organic material to the sea floor is high. Thiomargarita and Beggiatoa dominate sediments beneath the Benguela upwell-ing area off Namibia (3), whereas Thioploca

Fig. 1. Sediment profiles from the Namibian shelf (22°10'S, 14°03'E; water depth 70 m). (A) Phosphate concentrations in the pore water (mM) at different sediment depths (cm). (B) Phosphorus content of dried sediment (g kgj1) at different sediment depths. (C) Biomass of T. namibiensis (cells mj1) at different sediment depths. Three parallel measurements are shown as indicated by the different symbols. The dashed lines show the steady-state concentration of pore water phosphate and the amount of phosphorus accumu- Ptiosptiate lating as predicted by the model calculation.

dominates sediments off the South American west coast (4) and in the Arabian Sea (5). In all of these areas, modern phosphorite formation has been reported (1, 6). All of these sulfur bacteria species contain large amounts of intracellular polyphosphates, which we found by staining cells specifically for polyphosphate with toluidine blue (7, 8). Also, these bacteria show electron-dense inclusions (3, 9, 10), which is a typical appearance of polyphosphate.

During an expedition with the German research vessel Meteor off the coast of Namibia in March 2003, we found high pore water phosphate concentrations (7) of up to 300 mM in sediments that were densely populated by T. namibiensis (Fig. 1A). The sharp phosphate peaks that were observed in sediments were restricted to a narrow sediment horizon (about 3 cm thick), which corresponded to the depths where T. namibiensis was most abundant (Fig. 1C). Because of the high phosphate concentrations, active formation of phosphorite occurred in this thin zone as indicated by the large amounts of phosphorus-containing minerals in the sediment (7) (>50 g kgj1 of dry sediment or 5% P) (Fig. 1B). The predominant phosphorus mineral phase was hydroxyapatite [Ca5OH(PO4)3], which was determined by x-ray diffraction (XRD) analysis (7). Fifty grams of P per kg of sediment is equivalent to 270 g of hydroxy-apatite per kg of sediment. Therefore, more than 25% of the solid phase in this layer was hydroxyapatite, which is one of the major

mineral precursors in the formation of phosphorite deposits.

To gain a quantitative understanding of the measured pore water and solid phase-concentration profiles, we used a spreadsheet model similar to that of Schulz (11). Diffusive transport of phosphate [diffusion coefficient in sediment Dsed = 4.1 x 10-10 m2 s-1 with a temperature of 11 °C and a porosity of 0.9 for HPO42- as a major species, following thermodynamic calculations (12)] was calculated for a one-dimensional (1D) column of 100 cells using an explicit numerical solution of Fick's laws of diffusion (7). Boundary conditions were 5 |jM phosphate in the bottom water and precipitation of hydroxyapatite when concentrations exceeded 40 |jM, which reflects saturation with respect to hydroxyapatite. Thus, the measured pore water profile of Fig. 1A reflects a steady-state situation for production of dissolved phosphate by the bacteria and simultaneous precipitation of hydroxy-apatite. Fitting the model to the measured pore water concentration (Fig. 1A, dashed line) resulted in pairs of values; fast phosphate release and fast precipitation, or slow phosphate release and slow precipitation. Laboratory experiments on apatite precipitation as well as the calculation of the necessary diffusive calcium supply for this precipitation confined the range of plausible values for simultaneous release and precipitation of phosphate. As long as a near steady-state condition persisted for ~3 to 14 months, phosphate release rates between 20 (at 3 months) and 6 (at 14 months) nmol liter-1 s-1 would lead to the observed amounts of precipitated phosphorus in the sediment. (Fig. 1B). The shape of the curve of Fig. 1A, is matched by a phosphate release between 20 and 6 nmol liter-1 s-1. Under these circumstances, 3 to 14 months of constant phosphate release would lead to the observed amounts of hydroxyapatite in the sediment (Fig. 1B, dashed line). In contrast, the dissolution of hydroxyapatite after a periodic release of phosphate would be much slower because it is controlled only by

Fig. 2. Phosphate release under anoxic conditions from 50 cells of T. namibiensis after 24 hours of anaerobic preincubation, compared to a control (open circles) not containing T. namibiensis. Solid circles show mean values of three independent measurements. The single measurements are available in (7).

diffusion (7). Thiomargarita cells picked manually and incubated in artificial media in the laboratory (7) showed an increase in concentration between 0.011 and 0.028 pmol of phosphate literj1 s-1 cell-1 with a mean value of 0.018 pmol of phosphate liter-1 s-1 cell-1 (Fig. 2). In comparison, the predicted phosphate release of 6 to 20 nmol of phosphate liter-1 s-1, produced by 250 cells ml-1 counted in the field, equals an increase in concentration of 0.024 to 0.08 pmol liter-1 s-1 cell-1. These data confirm that T. namibiensis alone could be responsible for the observed pore water phosphate peak and the resulting precipitation of hydroxyapatite.

Polyphosphate occurs in nearly all living organisms (13), but only some bacteria and yeasts are capable of accumulating large amounts. Bacterial phosphate accumulation has been most thoroughly studied in waste-water treatment plants, where bacteria are used to remove phosphate. To initiate luxury uptake of phosphate by bacteria in a waste-water treatment plant, it is necessary to introduce an anaerobic phase whereby phosphate is released and acetate is taken up and stored, for example, in the form of polyhydroxy-alkanoate (PHA). Acetate uptake and storage require energy which, in the absence of an electron acceptor, the bacteria can gain from the breakdown of polyphosphate and consequent release of phosphate. In the aerobic phase that follows, the polyphosphate-accumulating bacteria gain energy by oxidizing the stored carbon using oxygen as the electron acceptor, and they take up an excess of phosphate, which they store as polyphosphate (14, 15). This results in a sludge rich in bacterial polyphosphate, which can be removed from the system.

Based on our incubation experiments, we hypothesize that the mechanisms of phos

Fig. 2. Phosphate release under anoxic conditions from 50 cells of T. namibiensis after 24 hours of anaerobic preincubation, compared to a control (open circles) not containing T. namibiensis. Solid circles show mean values of three independent measurements. The single measurements are available in (7).

phate uptake and release in T. namibiensis are similar to that of polyphosphate-accumulating bacteria in wastewater, even though their main energy source is considered to be the oxidation of sulfide with nitrate or oxygen as the electron acceptor (3, 16). In vitro, enhanced rates of phosphate release were induced under anaerobic conditions only when acetate was added to the medium. In addition to the many large sulfur globules that were observed in the cells, smaller inclusions were visible in differing amounts (Fig. 3, A and B). Specific staining (7) demonstrated that most of the smaller inclusions were polyphosphate (Fig. 3C). The remaining small inclusions did not stain with Nile red, a specific stain for PHA, but were stained dark brown with iodine (17) (Fig. 3D) suggesting that they consisted of glycogen or another polyglucose.

T. namibiensis appears to have a life mode that is unusual for marine bacteria. Under anoxic conditions, it takes up sulfide and, presumably, acetate, which appears to be stored as glycogen. Because there is an insufficient supply of a suitable external electron acceptor, internally stored nitrate and polyphosphate are sacrificed and sulfide is oxidized to elemental sulfur to gain energy. Under oxic conditions, the bacterium can gain energy from the oxidation of both sulfur and, presumably, glycogen. At the same time, it invests energy in the accumulation of polyphosphate and nitrate, the latter of which is stored in a central vacuole at concentrations of up to 0.8 M (3). Thus, T. namibiensis is able to take up each of these chemical compounds under conditions where the chemicals are readily available and use them under different redox conditions, when they are a valuable energy source that would otherwise be impossible to obtain at that time. The observation

Fig. 3. T. namibiensis. (A) A single cell of T. namibiensis with many smaller inclusions apart from the large sulfur globules. Inset: higher magnification image of the inclusions. (B) A single cell of T. namibiensis with few smaller inclusions. Inset: higher magnification image of the inclusions. (C) Small inclusions stained dark red for polyphosphate with tolu-idine blue. Many unstained inclusions can be seen. (D) Small inclusions stained with iodine, showing a dark brown color typical for glycogen.

that phosphate release could be induced only when acetate was added to the medium shows that the breakdown of polyphosphate is an auxiliary metabolism, which explains why it occurs only episodically and why phosphorus does not continuously accumulate with increased depth.

A connection between polyphosphate-accumulating bacteria and phosphorite formation was proposed two decades ago (2, 18-20). The main arguments in favor of a bacterial involvement were microfossils resembling sulfur bacteria enclosed in phosphorite deposits, for example, in the Miocene Monterey Formation (18), and the finding of low C:P ratios in recent Beggiatoa mats (20). Early diage-netic precipitation of phosphorite minerals has also been reported from the Santa Barbara Basin, where elevated pore water nitrate concentrations after sediment centrifugation suggest an involvement of large sulfur bacteria (21). There are also reports of phosphatized bacteria from the Namibian shelf (22), which seem to resemble Tftiomargarita. Because recent phosphorite formation and high biomass of large sulfur bacteria largely occur in the same areas, phosphorite formation through the activity of large sulfur bacteria could be a widespread phenomenon and is likely to also have been important in the past.

References and Notes

2. K. P. Krajewski et al., Eclogae Geol. Helv. 87, 701 (1994).

5. R. Schmaljohann etal., Mar. Ecol. Prog. Ser. 220, 295 (2001).

6. S. J. Schenau, C. P. Slomp, G. J. DeLange, Mar. Geol. 169, 1 (2000).

7. Materials and methods are available as supporting material on Science Online.

9. S. Maier, H. Volker, M. Beese, V. A. Gallardo, Can. J. Microbiol. 36, 438 (1990).

10. J. M. Larkin, M. C. Henk, Microsc. Res. Tech. 33, 23 (1996).

11. H. D. Schulz, in Marine Geochemistry, H. D. Schulz, M. Zabel, Eds. (Springer-Verlag, Heidelberg, New York, 2000), pp. 417-442.

12. D. L. Parkhurst, C. A. J. Appelo, "User's guide to PHREEQC (Version 2)—A computer program for speciation, batch-reaction, one-dimensional transport, and inverse geochemical calculations'' (U.S. Geological Survey, 1999); available at projects/GWC_coupled/phreeqc/html/final.html.

15. M. C. M. van Loosdrecht, G. J. Smolders, T. Kuba, J. J. Heijnen, Antonie Leeuwenhoek 71, 109 (1997).

16. H. N. Schulz, D. de Beer, Appl. Environ. Microbiol. 68, 5746 (2002).

18. L. A. Williams, C. Reimers, Geology 11, 267 (1983).

19. Y. Nathan, J. M. Bremner, R. E. Loewenthal, P. Monteiro, Geomicrobiol. J. 11, 69 (1993).

20. C. E. Reimers, M. Kastner, R. E. Garrison, in Phosphate Deposits of the World, W. C. Burnett, S. R. Riggs, Eds. (Cambridge Univ. Press, Cambridge, 1990), vol. 3, p. 300.

21. C. E. Reimers, K. C. Ruttenberg, D. E. Canfield, M. B. Christiansen, J. B. Martin, Geochim. Cosmochim. Acta 60, 4037 (1996).

22. G. N. Baturin, in Coastal Upwelling, J. Thiede, E. Suess, Eds. (Plenum, New York, 1983), vol. B, p. 11.

23. We thank the crew of the research vessel Meteor and the participants of the expedition, especially V. Brüchert. We also thank K. Enneking S. Hessler, K. Wien, J. Birkenstock, C. D. Fraley, and M. Wendschuh for technical and scientific assistance, and B. B. J0rgensen, S. B. Joye, J. Peckmann, M. Zabel, and an anonymous reviewer for comments. The study was supported by the Deutsche Forschungsgemeinschaft. This is publication no. 0240 of the Research Center Ocean Margins of the University of Bremen (Germany). The data are electronically available through the database PANGAEA.

Supporting Online Material DC1

Materials and Methods Figs. S1 to S3 Table S1 References Models S1 to S4

22 July 2004; accepted 24 November 2004 10.1126/science.1103096

Cardiovascular Risk Factors Emerge After Artificial Selection for Low Aerobic Capacity

Ulrik Wisloff,1,2*. Sonia M. Najjar,3* 0yvind Ellingsen,1,2 Per Magnus Haram,1 Steven Swoap,4 Qusai Al-Share,3 Mats Fernstrom,3 Khadijeh Rezaei,3 Sang Jun Lee,3 Lauren Gerard Koch,5 Steven L. Britton5

In humans, the strong statistical association between fitness and survival suggests a link between impaired oxygen metabolism and disease. We hypothesized that artificial selection of rats based on low and high intrinsic exercise capacity would yield models that also contrast for disease risk. After 11 generations, rats with low aerobic capacity scored high on cardiovascular risk factors that constitute the metabolic syndrome. The decrease in aerobic capacity was associated with decreases in the amounts of transcription factors required for mitochondrial biogenesis and in the amounts of oxidative enzymes in skeletal muscle. Impairment of mitochondrial function may link reduced fitness to cardiovascular and metabolic disease.

Several investigations link aerobic metabolism to the pathogenesis of cardiovascular disease. Large-scale epidemiological studies of subjects with and without cardiovascular disease demonstrate that low aerobic exercise capacity is a stronger predictor of mortality than other established risk factors (1-4). In patients with type 2 diabetes, low aerobic capacity is associated with reduced expression of genes involved in oxidative phosphorylation (5). In insulin-resistant elders, there is a 40% reduction in mitochon-drial oxidative and phosphorylation activity, largely attributable to impaired skeletal muscle glucose metabolism (6). These observations are consistent with impaired regulation of mitochondrial function as an important mechanism for low aerobic capac-

1Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Olav Kyrres gt. 3, 7489 Trondheim, Norway. 2Department of Cardiology, St. Olavs Hospital, 7006 Trondheim, Norway. 3Department of Pharmacology, Cardiovascular Biology, and Metabolic Diseases, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 436145804, USA. 4Department of Biology, Williams College, Williamstown, MA 01267, USA. 5Department of Physical Medicine and Rehabilitation, University of Michigan, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0718, USA.

*These authors contributed equally to this work. .To whom correspondence should be addressed. E-mail: [email protected]

ity and cardiovascular risk factors linked to the metabolic syndrome. These risk factors include weight gain, high blood pressure, reduced endothelial function, hyperinsulin-emia, and increased triglyceride concentration in blood. The working hypothesis of the present study was that rats selected on the basis of low versus high intrinsic exercise performance would also differ in maximal oxygen uptake, mitochondrial oxidative pathways, and cardiovascular risk factors linked to the metabolic syndrome.

In previous work, we began large-scale artificial selection for low and high aerobic treadmill-running capacity with the genetically heterogeneous N:NIH stock of rats as the founder population (7). Eleven generations of selection produced low-capacity runners (LCRs) and high-capacity runners (HCRs) that differed in running capacity by 347% (Fig. 1A). The founder population had a capacity to run for 355 ± 144 m (23.1 min) until exhausted. On average, the treadmill-running capacity decreased 16 m per generation in LCRs and increased 41 m per generation in HCRs in response to selection. At generation 11, the LCRs averaged 191 ± 70 m (14.3 min), and the HCRs ran for 853 ± 315 m (41.6 min). For this study, we used young adult rats (ages 16 to 24 weeks) derived from generations 10 and 11 to test our hypothesis that risk factors for common dis eases segregate with variation in intrinsic aerobic capacity (8).

High blood pressure is associated with increased risk for stroke and ischemic heart disease (9). We found that, relative to the HCRs, the LCR rats had higher mean blood pressures during the day (105 ± 13 mm Hg compared with 89 ± 8 mm Hg), at night (98 ± 3 mm Hg compared with 91 ± 7 mm Hg), and for the combined 24-hour period (102 ± 6 mm Hg compared with 90 ± 7 mm Hg) (Fig. 1B). Extrapolating from human data (9), this 13% higher 24-hour blood pressure suggests that the LCRs are twice as likely to develop cardiovascular disease as the HCRs.

Endothelial dysfunction is an independent predictor of long-term cardiovascular disease progression and cardiovascular event rates (10). To assess endothelial function in the two strains of rats, we assayed nitric oxidemediated (acetylcholine) vascular relaxation in isolated ring segments of carotid arteries. In this assay, higher vessel relaxation is interpreted as better endothelial function. For maximal absolute relaxation, the HCR rats demonstrated a 48% increase compared with the LCR rats. Furthermore, the concentration of acetylcholine that provoked a half-maximal response [median effective concentration (EC50)] was 7.8-fold greater in LCR than HCR rats (Fig. 1C and fig. S1).

LCR rats were insulin-resistant compared with the HCR rats, as demonstrated by higher fasting insulin levels and impaired glucose tolerance (Table 1 and fig. S2). Insulin C-peptide levels were normal in LCR rats, indicating that insulin secretion was preserved. However, insulin clearance was reduced in the LCR rats, as indicated by lower steady-state C-peptide/insulin molar ratios. These data indicate that hyperinsulinemia results mainly from reduced insulin clearance. Consistent with the clinical scenario of the metabolic syndrome, the LCR rats also had more visceral adiposity, higher plasma triglycerides, and elevated plasma free fatty acids compared with the HCR rats (Table 1).

Because individuals with cardiovascular disease often show diminished capacity for adaptation to exercise training (11), we measured 12 variables to assess the general exercise capacity and left ventricular function both in sedentary control (C) and in exercise-trained (T) LCR and HCR rats (Table 2). Each rat was trained for 6 weeks on a treadmill at an intensity relative to its own individual maximal oxygen consumption (VO2max) (12). Consistent with a low tolerance for exercise, the C-LCR rats had a 58% lower VO2max, a 17% lower economy of running (i.e., higher oxygen cost of running), 23% less left ventricular weight, and a trend (P = 0.07) toward shorter left ventricular cell length compared with the C-HCR rats. Isolated left ventricular cells from C-HCR rats had better systolic and diastolic function relative to the C-LCR rats (Table 2). In response to training, both T-LCR and T-

HCR rats showed significant improvement in all 12 of the measures of capacity (Table 2), with a uniformly greater training response in the T-HCR relative to the T-LCR rats for each measure except cell width.

Mitochondrial dysfunction is associated with a wide range of human diseases (5). In view of the lower aerobic capacity and reduced cardiovascular function of LCR rats, we hypothesized that they have compromised mitochondrial oxidative function relative to the HCR rats. To test this hypothesis, we measured the cellular content of proteins required for mitochondrial biogenesis and function (5,13) in soleus muscle, which is composed largely of highly oxidative fibers. The amounts of peroxisome proliferative activated receptor g (PPAR-g), PPAR-g co-activator 1 a (PGC-1a), ubiquinol-cytochrome c oxidoreductase core 2 subunit (UQCRC2), cytochrome c oxidase subunit I (COXI), uncoupling protein 2 (UCP2), and ATP synthase H+-transporting mitochondrial F1 complex (F1-ATP synthase) were markedly reduced in the LCR rats in comparison with the HCRs. The uniform decline in these proteins is consistent with the hypothesis that reduced aerobic metabolism

Table 1. LCR and HCR rats differed significantly for carbohydrate and lipid metabolic measures. Measurements were taken from male LCR (n = 8) and HCR (n = 8) rats. Blood was drawn at 0900 hours with food and water ad libitum to measure random blood sugar. Other metabolic measures were made on blood drawn after 12 hours of food and water deprivation.

LCR HCR % Difference LCR vs. HCR P value

Table 1. LCR and HCR rats differed significantly for carbohydrate and lipid metabolic measures. Measurements were taken from male LCR (n = 8) and HCR (n = 8) rats. Blood was drawn at 0900 hours with food and water ad libitum to measure random blood sugar. Other metabolic measures were made on blood drawn after 12 hours of food and water deprivation.

LCR HCR % Difference LCR vs. HCR P value

Random glucose (mg/dl)

86 :

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