(Hypertension. 1996;27:1104-1107.)
© 1996 American Heart Association, Inc.
Articles |
2-Adrenergic Agonists Increase Cellular Lactate Efflux
From Wayne State University School of Medicine (W.L., K.K., S.F.), Detroit, Mich; Veterans Administration Medical Center (W.L., S.F.), Allen Park, Mich; University of Michigan Medical School (W.L.), Ann Arbor; and Naval Health Research Center (W.L., S.F.), San Diego, Calif.
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Abstract |
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Abstract We reported previously that genetic polymorphisms of the
2-adrenergic receptor are
associated with hyperinsulinemia, diabetes
mellitus, and hypertension in blacks. The evolutionary driving force
for maintaining such deleterious mutations in the black population is
unknown. Recognizing that vascular 2-adrenergic
receptors mediate cold-induced vasoconstriction and that
temperature maintenance is a primary thrust of cellular
metabolism, we postulated that vascular
2-adrenergic receptors contribute significantly to
metabolic heat generation in homeotherms such as humans.
Using aerobic lactate production as an indicator of
thermogenesis, we measured metabolic heat
production in HT29 cells that expressed the gene encoding human
vascular 2-adrenergic receptors. Epinephrine, an
2-adrenergic receptor agonist, increased net lactate
efflux from 226±20 to 280±20 nmol/min (mean±SE) (P=.06).
Clonidine, a more specific 2-adrenergic agonist,
increased lactate efflux from 110±6 to 156±8 nmol/min
(P<.01). Similarly, in the presence of
physiological concentrations of glucose (5.5
mmol/L), insulin increased lactate production from 123±6 to
175±10 nmol/min (P<.01). Because differences in aerobic
glycolysis may also explain the heat intolerance and abnormal fuel
homeostasis found in genetically hypertensive rats, we also measured
lactate production in cultured vascular smooth muscle cells
isolated from stroke-prone spontaneously hypertensive rats (SHRSP)
and normotensive control Wistar-Kyoto rats (WKY). Vascular smooth
muscle cells from SHRSP had significantly greater lactate efflux
compared with cells from normotensive WKY (296±4 versus 172±2
nmol/min, P<.001). These differences were not due to
abnormalities in glucose uptake, as lactate efflux was greater in SHRSP
cells compared with WKY cells when dextrose was replaced with equimolar
concentrations of fructose (230±6 versus 138±2 nmol/min,
P<.001). 2-Adrenergic agonists increase
lactate efflux in HT29 cells, and abnormalities in vascular smooth
muscle lactate metabolism in genetically hypertensive rats
is independent of altered glucose uptake. These data provide support
for our hypothesis that balanced polymorphisms of the
2-adrenergic receptor could offer protection against
cold stress by increasing the thermogenic response associated with
aerobic lactate production.
Key Words: blood vessels • Na,K-ATPase • receptors, adrenergic • diabetes mellitus • temperature • clonidine • polymorphism (genetics)
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Introduction |
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Under anaerobic conditions, cells metabolize glucose to lactate at a high glycolytic rate. In the presence of oxygen, these cells decrease their utilization of glucose and production of lactate. The inhibition of glucose consumption and cessation of lactate accumulation with the onset of oxygen consumption is known as the Pasteur effect.1 For some time it has been known that despite the Pasteur effect, blood vessels demonstrate substantial aerobic glycolysis. The cellular benefit derived from this inefficient use of metabolic substrate is unknown. It has been reported that aerobic glycolysis in vascular smooth muscle could be the result of poor tissue oxygenation, decreased capacity for oxidative phosphorylation, and high concentrations of glucose in experiments designed to measure this phenomenon.1 2 However, we observed that in most biological systems, the wasteful use of fuel results in the salutary generation of heat. We hypothesized that aerobic glycolysis in blood vessels may serve to contribute to temperature conservation in humans.
The main cellular pathway for metabolic heat generation in humans is the activation of plasmalemmal Na,K-ATPase. The increased passive permeability of sodium and potassium in homeotherms is responsible for the high Na,K-ATPase activity reported in these organisms compared with "cold-blooded" poikilotherms.3 Humoral agents that increase Na,K-ATPase activity (eg, thyroxine, catecholamines, and insulin) also increase metabolic heat production.4 5
In addition to the "futile cycling" of ions via the sodium pump to maintain heat production, gluconeogenesis from lactate (the Cori cycle) also results in the significant generation of heat.6 7 8 Accordingly, aerobic glycolysis has the potential to contribute significantly to the maintenance of regional and core body temperatures in humans. It is generally accepted that blood vessels maintain core temperature by decreasing conductive heat loss through the extremities. Recognizing that blood vessels can contribute significant quantities of lactate derived from aerobic glycolysis and because of the significant thermogenic potential of lactate, it follows that blood vessels could also contribute substantially to metabolic heat production in humans.9 10 11
We have reported that polymorphisms of the genes encoding the A2AR are associated with hypertension, hyperinsulinemia, and diabetes mellitus in American blacks.12 13 14 Furthermore, genetically hypertensive rats demonstrate heat intolerance and abnormalities in glucose metabolism. Because A2AR agonists such as epinephrine are thermogenic, we hypothesized that these polymorphisms may offer some thermal protective advantage in humans. We used measurements of net lactate efflux as an indicator of aerobic glycolysis in HT29 cells, a human colonic epithelial line that continues to express the A2AR with passage. Because chromosome 2 A2AR is also expressed in blood vessels and because hyperinsulinemia and insulin resistance are found in the spontaneously hypertensive rat,15 we measured lactate produced in cultured VSMCs isolated from SHRSP and WKY.
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Methods |
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HT29 cells were obtained from American Type Tissue Collection. VSMCs isolated from thoracic aortas of SHRSP and WKY from the University of Michigan colony were kindly provided by David Bohr, MD. All three cell lines, the HT29, the SHRSP, and the WKY, were grown in bicarbonate-buffered Dulbecco's modified Eagle's medium with high glucose supplemented with 13% fetal calf serum, antibiotics, and glutamine. Nearly confluent VSMCs were used between passages 4 and 15, and the same passage numbers were always used when data were compared among SHRSP and WKY cells. Lactate was measured with a sensitive spectrophotometric assay.16 Because basal lactate concentration varied with the passage number and confluence of the cells, similar numbers of controls were always run with each experiment performed.
All experiments were performed in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Plates of HT29 and VSMCs were rinsed free of the Dulbecco's modified Eagle's medium/fetal bovine serum medium with PSS of the following composition (mmol/L): NaCl 130, KCl 4.7, KH2PO4 1.18, MgSO4 · 7H2O 1.17, CaCl2 · H2O 1.6, NaHCO3 14.9, dextrose 5.5, and CaNa2EDTA 0.03. In experiments with potassium-free PSS, KCl was omitted and sodium phosphate was substituted for potassium phosphate.
It has been shown that the lactate production from aerobic glycolysis is associated with Na,K-ATPase activity.2 Furthermore, epinephrine and insulin can directly stimulate Na,K-ATPase17 18 and indirectly increase lactate efflux. We were also concerned that any effect of A2AR agonists on aerobic glycolysis under our experimental conditions would be obscured by the high endogenous rates of Na,K-ATPase activity found in homeotherms.3 Therefore, we measured the effect of each agonist on lactate efflux during inhibition of Na,K-ATPase. We modified Na,K-ATPase activity by altering the potassium concentration of our incubation media using a technique we have described for isolated, helical strips of vascular smooth muscle.19 Briefly, plates were incubated in potassium-free PSS for 1 hour, the supernatant was aspirated, and potassium-free or regular PSS was added back to the cells for 5 minutes. At the end of this time interval, an aliquot of the supernatant PSS was sampled and placed on ice, and the net lactate efflux into the PSS over 5 minutes was determined. Values are expressed as nanomoles lactate per minute per plate (mean±SE). Student's t test was used to compare the mean net lactate efflux between the different experimental interventions. Next, we measured the effect of epinephrine, clonidine, and insulin on net lactate efflux, with and without Na,K-ATPase inhibition, in HT29 cells. Plates were similarly incubated in potassium-free PSS for 1 hour. After this interval, the plates were rinsed, and potassium-free PSS or regular PSS containing vehicle, 10 µmol/L epinephrine, 10 µmol/L clonidine, or 10 µmol/L insulin was added to the cells. Aliquots of the supernatant were similarly sampled after 5 minutes for lactate determinations.
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Results |
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As shown in Fig 1
, after 1 hour of incubation in
potassium-free PSS, the addition of 4.7 mmol/L K+ to
HT29 cells resulted in an increase in net lactate efflux from 155±14
to 193±14 nmol/min (P=.05). Because these results
demonstrated high lactate production in the face of active
sodium-potassium transport, we examined the effect of the A2AR
agonists epinephrine and clonidine as well as insulin during
inhibition of Na,K-ATPase activity. We found that epinephrine,
clonidine, and insulin had no effect on lactate efflux when the
cellular Na,K-ATPase was able to actively maintain ion concentration
gradients (data not shown). However, when Na,K-ATPase activity was
inhibited by placement of the cells in potassium-free PSS,
epinephrine, clonidine, and insulin increased lactate efflux.
Epinephrine (10 µmol/L) increased net lactate efflux by 27%
(Fig 1). Clonidine (10 µmol/L) and insulin (10 µmol/L) similarly
increased lactate efflux in HT29 cells that had been incubated in
potassium-free PSS for 1 hour (Fig 2).
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As shown in Fig 3, reduction of the potassium
concentration in the medium of aortic cells isolated from normotensive
WKY resulted in a significant decrease in net lactate efflux from
212±6 to 172±2 nmol/min. However, the inhibition of lactate
production in potassium-free PSS was not seen in aortic
cells cultured from SHRSP (Fig 3). This result may be due to the
extremely high lactate production we found in the cells from
SHRSP compared with cultures from the aortas of WKY (310±6 versus
212±6 nmol/min, P<.001).
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Impaired glucose uptake in aortic cells from SHRSP could decrease
aerobic glycolysis and lactate production,15 and
fructose stimulates thermogenesis in the absence of
insulin-mediated glucose uptake.20 Accordingly, we
performed a set of experiments in which we substituted glucose with an
equimolar concentration of fructose. As shown in Fig 3
, equimolar
substitution of glucose with fructose decreased net lactate
production in cell cultures from both WKY and SHRSP. Again,
lactate production in SHRSP cells during inhibition and active
transport of sodium and potassium did not differ.
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Discussion |
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The A2AR is found in the pancreas and on blood vessels. We have reported that genetic polymorphisms of adrenergic receptors are associated with hypertension, hyperinsulinemia, and diabetes mellitus in blacks.12 13 14 However, to offset any deleterious effect and preserve such functional mutations, the physiological effect of "balanced" polymorphisms should offer some advantage to the fitness of humans.
The advantage for the seemingly wasteful high rate of aerobic glycolysis found in vascular smooth muscle is unknown. We were intrigued by the observations that endogenous humoral agents such as epinephrine, thyroxine, and insulin, which increase Na,K-ATPase activity, are exceedingly thermogenic.4 7 8 19 It is dogmatic that peripheral blood vessels constrict on exposure to cold, and this increase in peripheral vascular tone shunts blood from the cold extremities and decreases conductive heat loss. However, blood vessels can contribute in other ways to the maintenance of core temperature in homeotherms.21 With values from the literature,1 7 8 10 22 23 it can be calculated that maximum stimulation of vascular oxygen utilization through aerobic glycolysis can increase the metabolic rate by more than 170%. Alternatively, thermogenesis can be estimated directly from vascular lactate metabolism through the Cori cycle. We report that clonidine and insulin increased lactate efflux by nearly 50%. From measurements taken during lactate infusion in humans, a doubling of the plasma lactate concentration is associated with a 16% increase in thermogenesis over the basal metabolic rate. Aerobic glycolysis and vascular lactate generation can contribute significantly to thermogenesis.
In addition to its role in systemic and peripheral thermogenesis, it is likely that vascular lactate serves a local, paracrine function. For example, it has been demonstrated that lactate mediates the activity of ATP-dependent potassium channels in the heart.24 25 Lactate is not necessarily a wasteful by-product of anaerobic metabolism.
Vascular A2ARs mediate cold-induced vasoconstriction,9 and it is possible that much in the way that skeletal muscle contractions generate heat (eg, "shivering"), vascular smooth muscle may yield mechanical heat. Alternatively, we postulated that A2AR agonists could contribute to vascular thermogenesis by stimulating aerobic glycolysis and increasing vascular lactate production. The reconversion of lactate to glucose in various tissues (eg, kidney, liver, and perhaps blood vessels) is a thermogenic process. Although we did not directly measure oxygen tensions in our experiments, other researchers have demonstrated that the changes in lactate efflux measured under similar experimental conditions are not due to tissue hypoxia.10 11
We did not observe increases in lactate production under physiological conditions in which the sodium pump is maximally active, and other researchers have reported that aerobic glycolysis correlates with Na,K-ATPase activity.2 We found that epinephrine, clonidine, and insulin could increase lactate production despite inhibition of Na,K-ATPase. Accordingly, we believe that A2AR agonists do not have a role in thermogenesis under normal physiological conditions. However, Na,K-ATPase activity may be decreased in some individuals with hyperinsulinemia or diabetes mellitus and in genetic models of hypertension.26 27 28 In these pathological states, the thermogenic potential offered by activation of A2ARs by endogenous ligands may prove salutary and help to maintain an individual's core temperature.
Although our data are by no means conclusive, our observations warrant further investigations into the thermogenic potential of blood vessels in individuals with abnormal vascular reactivity. It also remains to be determined whether the genetic polymorphisms of the A2ARs associated with hyperinsulinemia and increased peripheral vascular resistance also affect metabolic heat production.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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These studies were supported by the American Diabetes Association; American Heart Association, Michigan Affiliate; National Institutes of Health grant HL-50849; and the Naval Medical Research and Development Command. Dr Lockette is an Established Investigator of the American Heart Association.
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Footnotes |
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Reprint requests to Warren Lockette, MD, Division of Endocrinology, 4H University Health Center, Wayne State University School of Medicine, 4201 St Antoine, Detroit, MI 48201.
Received January 3, 1996; first decision January 16, 1996; accepted January 16, 1996.
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