1 Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905; 2 Division of Clinical Physiology, Karolinska Hospital, S-171 76 Stockholm, Sweden; and 3 Departments of Medicine, Biochemistry, and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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These studies were conducted to understand the relationship between measures of systemic free fatty acid (FFA) reesterification and regional FFA, glycerol, and triglyceride metabolism during fasting. Indirect calorimetry was used to measure fatty acid oxidation in six men after a 60-h fast. Systemic and regional (splanchnic, renal, and leg) FFA ([3H]palmitate) and glycerol ([3H]glycerol) kinetics, as well as splanchnic triglyceride release, were measured. The rate of systemic FFA reesterification was 366 ± 93 µmol/min, which was greater (P < 0.05) than splanchnic triglyceride fatty acid output (64 ± 6 µmol/min), a measure of VLDL triglyceride fatty acid export. The majority of glycerol uptake occurred in the splanchnic and renal beds, although some leg glycerol uptake was detected. Systemic FFA release was approximately double that usually present in overnight postabsorptive men, yet the regional FFA release rates were of the same proportions previously observed in overnight postabsorptive men. In conclusion, FFA reesterification at rest during fasting far exceeds splanchnic triglyceride fatty acid output. This indicates that nonhepatic sites of FFA reesterification are important, and that peripheral reesterification of FFA exceeds the rate of simultaneous intracellular triglyceride fatty acid oxidation.
lipolysis; free fatty acids; glycerol; triglycerides
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INTRODUCTION |
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ADIPOSE TISSUE LIPOLYSIS is an important fuel source, providing free fatty acids (FFA) and gluconeogenic carbons in the form of glycerol. The glycerol derived from adipose tissue lipolysis may become more important when humans fast for more than 24 h, because virtually all glucose must then be derived by gluconeogenesis. Recent studies have documented that splanchnic and renal glycerol uptake in 60-h-fasted humans is largely converted to glucose (22). Intrahepatic glycerol-3-phosphate (G-3-P), which is partially derived from circulating free glycerol, can also serve another physiological need, i.e., reesterification of fatty acids for export as very low density lipoprotein (VLDL) triglyceride. The quantitative need for G-3-P for this purpose has rarely been considered in the context of hepatic fuel metabolism during fasting.
In overnight postabsorptive adults, triglyceride recycling, a measure
of lipolysis in excess of oxidative needs, is reported to be ~ 1.2 µmol · kg1 · min
1
(~86 µmol/min) (21). This triglyceride recycling rate
is similar to the rate of splanchnic triglyceride release that we have
observed (14). These concordant data are consistent with
the suggestion that FFA released in excess of fatty acid oxidation
(fatty acid recycling) primarily represents hepatic reesterification of
FFA and their subsequent export as VLDL triglyceride (12),
although recent data suggest that this may not be the case
(14). During prolonged fasting, FFA reesterification is
reported to increase as much as sixfold (9). If so, and if
VLDL triglyceride export is the major means of dealing with
"excess" lipolysis, this process could consume a significant
portion of hepatic G-3-P. Therefore, it is possible that,
during fasting, increased rates of both gluconeogenesis and fatty acid
reesterification compete for a limited hepatic G-3-P pool.
Recently, we observed that estimated FFA reesterification (lipolysis minus fatty acid oxidation as determined by indirect calorimetry) was substantially in excess of splanchnic FFA uptake in men fasted for 60 h (22). In that study, FFA release rates were estimated as 3× glycerol rate of appearance (Ra), which could be misleading if the ratio of FFA to glycerol Ra is <3:1 (25). In addition, splanchnic FFA kinetic studies were not performed (22). If visceral adipose tissue lipolysis (and therefore splanchnic FFA release) is increased substantially during fasting, hepatic FFA uptake could be much greater than predicted from net splanchnic FFA uptake.
These issues caused us to question the extent of fatty acid reesterification in the liver during fasting. This study was conducted to directly measure systemic and regional FFA uptake and release in fasting humans and to assess net splanchnic triglyceride export as a minimum estimate of hepatic G-3-P requirements for fatty acid reesterification. To place the splanchnic data into context, measures of regional substrate kinetics across the leg and kidney bed were also performed.
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MATERIALS AND METHODS |
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Subjects
Six men participated in these studies, which were performed in the Division of Clinical Physiology, Karolinska Hospital, Stockholm, Sweden. All volunteers were healthy and took no medications on a regular basis. Their characteristics are provided in Table 1. They were informed of the nature, purpose, and possible risks of the study before consenting to participate. The Institutional Human Ethics and the Isotope Radiation Committees approved the experimental protocol.
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Experimental Design
Protocol.
Each volunteer fasted for 60 h as an outpatient before reporting
to the laboratory, as in our previous studies (22). Oxygen consumption (O2) and CO2
production (
CO2) (indirect calorimetry) were measured with the subject resting quietly in bed. A series of
indwelling catheters was then placed for infusions and blood sampling.
A forearm intravenous catheter was used for the infusion of isotopic
tracers of palmitate and glycerol. The same catheter was used to infuse
para-aminohippurate (PAH) to measure renal and indocyanine green (ICG)
to measure splanchnic blood flow. Catheters were placed using standard
techniques in the brachial artery and femoral, hepatic, and renal veins
for blood sampling while the subjects rested quietly. Infusions of
[9,10-3H]palmitate (0.3 µCi/min) and
[2-3H]glycerol (0.3 µCi/min) were begun, and after 60 min, for isotopic equilibration, a series of blood samples was obtained
from each catheter at 10-min intervals over 30 min. The catheters were
then removed, and the subjects were allowed to eat.
Materials and assays. [9,10-3H]palmitate was purchased from NEN Research Products (Wilmington, DE) and bound to human albumin for infusion. [2-3H]glycerol was purchased from Amersham (Arlington Heights, IL). ICG (Pulsion Medical System, Munich, Germany) and PAH (Merck, West Point, PA) were used in these studies.
Plasma palmitate and FFA concentrations and specific activity (SA) were determined by a modification (19) of a published HPLC technique (24). Femoral, renal, and hepatic venous and arterial glycerol and plasma triglyceride concentrations were measured twice in triplicate using a microfluorometric method (13). Plasma free glycerol SA was measured using HPLC (20). Plasma ICG (6) and PAH (5) concentrations were measured on the day of the study. Blood glucose concentrations were measured with a Beckman Glucose Analyzer (Beckman Instruments, Fullerton, CA).Calculations
Systemic FFA and glycerol fluxes were measured using the mean steady-state plasma FFA and glycerol SA (dpm/µmol) and the tracer infusion rates (dpm/min) (15); because steady-state conditions were present for all studies, Ra and rates of disappearance (Rd) are equal (20). Net fatty acid oxidation rates were calculated using each individual'sSplanchnic plasma flow (SPF) (4) was measured using the
arterial and hepatic venous plasma ICG concentrations. Renal plasma flow was measured using the arterial and renal venous concentrations of
PAH (8). Splanchnic and renal blood flow were calculated as plasma flow divided by (1 hematocrit). We estimated leg
plasma flow and leg blood flow in this study by use of the mean values for men from previous studies (17), because we could not
justify placing an additional catheter into the femoral artery for dye infusion purposes.
Net balance of FFA and glycerol across the splanchnic, renal, and leg tissue beds was calculated using the venous-arterial concentration gradients together with plasma or blood flow values. Plasma FFA and glycerol concentrations and SA were constant over the sampling interval, as were splanchnic and renal blood flow; therefore, steady-state plasma FFA SA and concentration were used together with measures of renal and hepatic plasma flow to measure regional (leg and splanchnic) FFA uptake and release (15, 23). Regional glycerol kinetics were similarly measured, except that because glycerol is distributed in whole blood (3) (glycerol equilibrates freely between plasma and erythrocytes), regional glycerol kinetics were performed using blood flow values. The formulas used to determine regional glycerol kinetics have been published (15).
Arterial plasma triglyceride concentrations were subtracted from hepatic venous concentrations, and the difference was multiplied by splanchnic plasma flow to determine net splanchnic triglyceride release.
Statistics
All results are expressed as means ± SE. Comparisons between substrate concentrations in arterial, femoral venous, hepatic venous, and renal venous blood were made using a Student's paired t-test. ![]() |
RESULTS |
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Subject Characteristics
Data regarding the characteristics, indirect calorimetry, and substrate concentrations are provided in Table 1. The expected adaptations to fasting were observed: moderate hypoglycemia, elevated plasmaSystemic Lipid Kinetics
The arterial plasma FFA and glycerol concentrations for the subjects are provided in Table 2. Concentrations and SA of palmitate and glycerol were stable over the sampling interval (Fig. 1). Free fatty acid flux was 739 ± 85 µmol/min, and systemic glycerol flux was 301 ± 38 µmol/min. Fatty acid oxidation rates measured using indirect calorimetry were 373 ± 22 µmol/min, resulting in a calculated fatty acid reesterification rates of 366 ± 93 µmol/min.
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Regional Substrate Kinetics
Splanchnic blood flow was 1.22 ± 0.04 l/min, and plasma flow was 0.67 ± 0.02 l/min. Renal blood flow was 1.64 ± 0.07 l/min, and plasma flow was 0.88 ± 0.03 l/min, respectively. Leg blood and plasma flows were estimated to be 0.42 and 0.23 l/min.Concentrations of FFA, glycerol, and triglycerides in the hepatic,
renal, and femoral veins are provided in Table 2. The net balances as
well as regional uptake and release rates of FFA and glycerol are
provided in Table 3. The fractional
uptake of systemic FFA by the splanchnic bed was 38 ± 1%. Leg
FFA uptake and release were calculated using estimated leg plasma flow,
and glycerol uptake and release were calculated using estimated blood flow. Splanchnic glycerol uptake accounted for 54 ± 5%, and
renal glycerol uptake accounted for 40 ± 8% of systemic glycerol
removal.
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Splanchnic FFA release was 15 ± 3% of systemic Ra, whereas renal FFA release was not significantly different from zero. Estimated leg FFA release (both legs) accounted for 22 ± 3% of FFA release. Free glycerol was released from both the splanchnic and the renal beds.
Net splanchnic glucose release was 0.40 ± 0.01 mmol/min, and renal glucose release was 0.22 ± 0.04 mmol/min. The net splanchnic triglyceride release was 21 ± 2 µmol/min (64 ± 6 µmol/min of fatty acids), and the arteriovenous triglyceride concentration difference across the renal bed was not significantly different from zero.
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DISCUSSION |
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These studies were designed to address unresolved issues regarding the physiology of lipid substrate metabolism in fasting, namely the relationship between splanchnic uptake of lipid derived substrates and estimates of fatty acid reesterification. We combined indirect calorimetry with measures of regional and systemic substrate kinetics to assess the contribution of leg, splanchnic, and renal tissues to FFA and glycerol utilization in relationship to systemic fatty acid oxidation and reesterification. As expected, the FFA reesterification rates with fasting were quite high. There was no commensurately elevated splanchnic triglyceride release; in fact, splanchnic triglyceride release was less than one-half of what we previously found in overnight, postabsorptive adults (14). Thus the estimates of systemic FFA reesterification obtained by combining indirect calorimetry and tracer techniques do not appear to reflect hepatic export of fatty acids in triglyceride.
A previous study (22) of systemic and regional glycerol kinetics during fasting suggested that FFA reesterification should be occurring at the rate of ~820 µmol/min. If all FFA are reesterified in the liver, that would require ~270 µmol/min of intrahepatic G-3-P, substantially more than the splanchnic glycerol uptake (120 µmol/min) observed (22). We reasoned that if the liver were the only site of FFA reesterification during fasting, hepatic triglyceride export would result in net utilization of gluconeogenic carbons in amounts greater than those derived/delivered from lipolysis. This was inconsistent with the finding that ~90% of glycerol uptake was converted to glucose (22). In the present study, splanchnic glycerol uptake alone was about sixfold the amount of G-3-P needed for the splanchnic triglyceride released.
We also observed high rates (366 ± 93 µmol/min) of FFA
reesterification, as assessed by subtracting fatty acid oxidation
(indirect calorimetry) from FFA Rd. Net splanchnic
triglyceride export (an index of hepatic VLDL triglyceride secretion in
fasting humans) was 64 ± 6 µmol/min, accounting for ~17% of
FFA reesterification. The relationships among FFA flux, fatty acid
oxidation and reesterification, and splanchnic triglyceride fatty acid
output are depicted in Fig. 2. The
presence of extrahepatic sites of FFA reesterification is the most
likely explanation for the difference between systemic FFA
reesterification rates and splanchnic triglyceride fatty acid output.
We have recently reported direct evidence that muscle is capable of
reesterifying significant amounts of FFA in humans (11),
suggesting that muscle could be an important site of FFA reesterification after 60 h of fasting. It should be noted,
however, that the current findings apply to resting conditions. During ambulation, muscle lipid oxidation likely increases immediately and
substantially, whereas FFA availability increases only gradually (11). Thus modest net utilization of intramyocellular
triglyceride fatty acids during physical activity likely offsets the
net accumulation that occurs at rest.
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Our measures of the splanchnic uptake of FFA and glycerol should be considered minimum estimates of hepatic FFA and glycerol uptake. Splanchnic FFA release was ~100 µmol/min, which is the net effect of omental and mesenteric (visceral) adipose tissue lipolysis and hepatic uptake of FFA from the portal circulation. Given that splanchnic FFA uptake averaged 38% of systemically delivered FFA and that the majority of splanchnic FFA uptake occurs in liver (2), this suggests that visceral adipose tissue FFA release was ~165 µmol/min. Assuming that FFA released from visceral adipose tissue lipolysis are taken up by the liver in comparable proportions to the uptake of systemic FFA, hepatic FFA uptake could have been as much as 360 µmol/min. The export of 64 µmol/min as VLDL triglyceride would allow ~300 µmol/min available for hepatic energy needs and ketone body production. Considering that ketone body production rates in fasting humans can be quite high (1), this is likely an important fate for FFA taken up by the liver.
In these men fasted for 60 h, FFA concentrations and FFA flux were approximately double that usually found in overnight-postabsorptive men (16). The splanchnic FFA release into the systemic circulation accounted for 15 ± 3% of systemic FFA appearance, whereas leg adipose tissue FFA release was estimated to account for 22 ± 3% of systemic FFA. These proportions are remarkably similar to those we observed in overnight-postabsorptive men (16) and suggest that mobilization of FFA from adipose tissue in prolonged fasting is not regionally different from that which occurs after an overnight fast.
We found that splanchnic and renal glycerol uptake accounted for 86 ± 15% of systemic glycerol uptake, whereas in a previous study of 60-h-fasted men (22) these two tissue beds accounted for only ~44% of glycerol uptake. The splanchnic and renal glycerol uptake rates were reasonably comparable in the two studies. However, systemic glycerol flux was ~40% higher in the previous study (22). A possible explanation for the difference between the studies is that, at higher glycerol release rates, tissues other than kidney and liver play an increasing important role in systemic glycerol removal.
As with many studies, these experiments have limitations. We did not
measure urinary ketone excretion rates in this experiment. On the basis
of upon published data (1) regarding the relationship between plasma ketone body concentrations and urinary ketone losses, we
could expect ~20-30 µmol/min of ketonuria in our volunteers. This would result in the consumption of oxygen without concomitant CO2 production, resulting in a lower respiratory quotient
(10). In our subjects, the ketonuria could be expected to
result in 0.6-1.2 ml/min of oxygen consumed without production of
CO2. Given the O2 rates of
these volunteers (Table 1) this error would be only an ~0.5%
underestimate of the RQ, which would not materially affect our
estimates of fatty acid oxidation. Another limitation is the
calculation of uptake and release in the face of small arteriovenous
differences in substrate concentration or SA. Despite good assay
performance [coefficient of variation (CV) of concentration and SA of
1-2% (24)], the need to use arterial and venous
concentration and SA data compounds the potential error. These
uncertainties are then multiplied by blood or plasma flow values. Thus,
despite the collection of four samples over 30 min, it is likely that uptake and release values for each individual is accurate only to
within ~10%.
In summary, FFA reesterification in 60-h-fasted men are about sixfold in excess of net splanchnic triglyceride fatty acid release, which should be a reasonable estimate of hepatic VLDL triglyceride export. FFA reesterification was also greater than splanchnic FFA uptake. These data suggest that FFA are reesterified in tissues other than liver during fasting, and adds to the expressed concern (7) that VLDL production rates cannot be readily estimated using FFA reesterification rates (12). Our measures of splanchnic/hepatic FFA uptake during fasting are consistent with the needs of the liver for oxidative fuel and ketone body production, with the remainder relegated to VLDL triglyceride release. The kidney takes up a significant fraction of systemic glycerol removal in fasting adults; however, leg glycerol uptake also appears to occur.
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ACKNOWLEDGEMENTS |
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We acknowledge the invaluable contributions of Dr. John Wahren, the technical assistance of Rita Nelson, and the editorial assistance of Susan Leachman.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40484 and DK-14507 from the US Public Health Service and by the Mayo Foundation.
Address for reprint requests and other correspondence: M. D. Jensen, Endocrine Research Unit, 5-194 Joseph, Mayo Clinic, Rochester, MN 55905.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 2000; accepted in final form 1 June 2001.
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