![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thiazolidinediones may exert their effects via ligand activation of peroxisome proliferatoractivated receptor (PPAR
) (7), which is expressed primarily in adipose tissue (8). PPAR
activation contributes to the triggering of differentiation of preadipocytes (9) and induces expression of genes involved in the transport and sequestration of FFA (10). These cellular and molecular effects of PPAR
agonism could influence FFA exchange between adipose and other tissues, but direct evidence based on measurements of in vivo fluxes and the metabolic fate of FFA are lacking.
We report here the effects of two thiazolidinediones, rosiglitazone and darglitazone, in obese insulin-resistant /dyslipidemic Zucker fa/fa rats. The aim was to elucidate effects on FFA and triglyceride metabolism. Because of the potentially central effect of altered FFA metabolism on the beneficial effects of thiazolidinediones, we undertook a detailed evaluation of in vivo FFA mobilization and metabolic fate using 3H-palmitate tracer methods. The results demonstrate extensive effects in adipose tissue; notably, there was an increased ability to take up and store plasma FFA, an augmented capacity to mobilize FFAs under fasting conditions, and a greatly enhanced ability of postprandial levels of insulin to suppress FFA mobilization. Investigation into the kinetic mechanisms of plasma triglyceride (TG) lowering showed that abolition of hypertriglyceridemia by thiazolidinediones involves both accelerated removal of TG from VLDL particles and decreased hepatic TG production.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment groups.
Lean (Fa/?) and obese (fa/fa) Zucker rats were studied in five groups: lean untreated controls (LN), obese untreated controls (OB), and obese treated with rosiglitazone 1 µmol · kg-1 · day-1 (ROS1), rosiglitazone 10 µmol · kg-1 · day-1 (ROS10), or darglitazone 1.3 µmol · kg-1 · day-1 (DAR). Treated rats were dosed daily at 1:00 P.M. for 3 weeks by gastric gavage. Untreated control rats were gavaged with an equal volume of vehicle (0.5% carboxymethyl cellulose, 2.5 ml · kg-1).
Anesthetized rat preparation.
On the morning of the study, food was withdrawn at 7:00 A.M. Rats were anesthetized at 10:00 A.M. with Na-thiobutabarbitol (Inactin; RBI, Natick, MA), with the lean and obese rats receiving 120 and 180 mg · kg-1, respectively. Body temperature was monitored with a rectal probe and maintained between 37.5 and 38.0°C throughout the experiment. Animals were tracheotomized, and catheters were placed in the right jugular vein and left carotid artery. Arterial catheter patency was maintained throughout the experiment by continuous infusion (10 µl · min-1) of a sterile saline solution containing sodium citrate (20.6 mmol/l). Acute experimental protocols (see below) were commenced at 2:00 P.M. after a 1.5-h postsurgery stabilization period.
Conscious, chronically catheterized rat preparation.
At 1 week before the acute study, rats were fitted with jugular and carotid catheters under isoflurane anesthesia. Prophylactic antibiotics were administered subcutaneously the day before surgery and on the day of the surgery (ampicillin 150 mg · kg-1, Doktacillin; AstraZeneca). Cannulae were exteriorized via a small cutaneous incision at the nape of the neck, and patency was maintained before study by filling the lines with 45.5% (wt/wt) polyvinylpyrrolidone (molecular weight 40,000) (Fluka Chemie AG, Buchs) dissolved in a 0.9% NaCl, 20.6 mmol/l sodium citrate solution. All rats included in the acute experiments were gaining weight and had recovered their preoperative body weights during the week after implantation. On the morning of the study, food was withdrawn at 7:00 A.M. After a 90-min posthookup settling-in period, the acute experiment was commenced at 2:00 P.M. in conscious unrestrained rats.
Experimental protocols.
Study 1: Hepatic VLDL triglyceride production and plasma clearance of triglyceride.
Five groups of anesthetized 7-h fasted rats were studied: LN, OB, ROS1, ROS10, and DAR. After a 90-min postsurgery stabilization period, basal arterial blood samples were collected. Rats then received an intravenous dose of 20% (wt/wt) Triton WR1339 (200 mg · kg-1 polymeric p-isoctylpolyoxyethylenephenol) (Tyloxapol; Sigma, St. Louis, MO) in normal saline. Arterial blood samples (0.2 ml) were collected 30, 60, 90, and 120 min after Triton administration. Plasma lipoprotein profiles (see below) were determined in a basal sample and a post-Triton (120 min) sample. Plasma TG determinations were made in all samples collected.
Study 2: Mobilization and fate of plasma FFAs
Tracer preparation.
Tracer infusates were freshly prepared each day. For every rat, 75 µl ethanol containing 2 x 108 dpm [9,10-3H]palmitic acid (3H-P; Amersham, Solna, Sweden) and 152 nmol Na-palmitate (Sigma) was added dropwise to 300 µl of continuously stirred 4% (w/v) essentially fatty acidfree bovine serum albumin (Sigma) in normal saline. The infusate was increased to a final volume of
3 ml by the addition of normal saline.
General tracer methodology.
The albuminpalmitate3H-P complex was infused at a constant rate (1 · 106 dpm · min-1, 17 µl · min-1) into the jugular vein. To obtain plasma FFA and 3H-P concentrations, 150 µl arterial blood samples were collected 10, 20, 40, 60, 80, 100, and 120 min after the start of tracer infusion. At 120 min, tracer infusion was stopped and additional blood samples were collected at 124, 128, 132, and 140 min. Plasma was rapidly separated in a refrigerated centrifuge, and a 25-µl aliquot was placed directly into 2 ml lipid extraction mixture (described below). After collection of the 140-min blood sample, rats were given a lethal dose of Na-thiobutabarbitol. Samples (
100 mg) of the following tissues were collected: red quadriceps muscle (RQ), white quadriceps (WQ), inguinal adipose tissue (WAT), interscapular brown adipose tissue (BAT), and liver. Tissue samples were weighed and placed in small cardboard cones for determination of 3H activity after combustion (see below).
Whole-body FFA metabolism in anesthetized rats.
Five groups of anesthetized 7-h fasted Zucker rats were studied: LN, OB, ROS1, ROS10, and DAR. Experiments were performed to assess FFA metabolism only at the whole-body level and were thus performed according to the experimental procedure detailed in the section above (General tracer methodology) until collection of the 120-min blood sample, at which time the experiment was terminated.
Whole-body and tissue-specific FFA metabolism in conscious rats.
Two groups of conscious obese Zucker rats were studied: OB and DAR. Rats that were fasted for 7 h were studied in either the basal state or under conditions of the euglycemic-hyperinsulinemic glucose clamp. The tracer methodology described above was commenced in the basal studies after collection of the basal blood sample and in the clamp studies after establishing a glucose steady state (60 min after starting the insulin infusion).
Clamp studies were performed at postprandial levels of hyperinsulinemia. Human insulin (Actrapid; Novo Nordisk A/S, Copenhagen) was infused via the jugular catheter using a syringe pump (Model 22 I/W; Harvard Apparatus, South Natic, MA). Arterial blood glucose was measured every 5 min using a glucose analyzer (YSI 2700; YSI, Yellow Springs, OH) and was clamped within 10% of the target level by manually adjusting a variable rate infusion of 30% (w/v) glucose in normal saline solution. Blood loss was minimized by a special direct sampling procedure from the arterial line (15 µl/sample). Glucose was infused with a syringe pump (CMA 1100; Carnegie Medicin, Solna, Sweden).
Analysis of plasma and tissue samples.
Plasma lipids, glucose, and insulin.
Colorimetric kit methods were used for the measurement of plasma FFA (NEFA C; Wako, Richmond, VA), triglycerides (Triglycerides/GB; Boehringer Mannheim, Indianapolis, IN), and glucose (Glucose HK; Roche, Stockholm). These methods were performed on a centrifugal analyzer (Cobas Bio; F. Hoffmann-La Roche & Co., Basle, Switzerland). Insulin concentrations were measured using radioimmunoassay (rat insulin RIA kit; Linco Research, St. Charles, MO).
Resolution of plasma 3H-P and 3H2O.
To discriminate 3H-P from total plasma 3H activity, a lipid extraction and separation procedure based on the method of Hagenfeldt (11) was performed on plasma samples. This involved an initial acid lipid extraction using a mixture of isopropanol-hexane-0.5 mol/l H2SO4 (40:10:1) followed by a polarity separation step under alkaline conditions. This step partitioned neutral lipids (including esterified fatty acids) into a hexane phase, and it partitioned polar lipids (including 3H-P) into an alcohol phase. 3H2O was estimated as the 3H activity lost during evaporation of the lower (isopropanol-water) phase of the lipid extraction procedure.
Measurement of tissue 3H activity.
Freshly collected tissue samples were desiccated in a freeze dryer to remove 3H2O. Non-3H2O-associated 3H activity was then determined using a Packard System 387 automated sample preparation unit (Packard Instrument, Meriden, CT), which completely oxidized the sample and collected the formed 3H2O into scintillant (Monophase S; Packard Bioscience B.V., Groningen, The Netherlands) for counting. 3H activity was measured using liquid scintillation spectrometry (Wallac 1409 counter; Wallac OY, Turku, Finland).
Lipoprotein profiles.
Cholesterol distribution profiles were measured in 10-µl plasma samples by a size-exclusion high-performance liquid chromatography system using a Superose 6 PC 3.2/30 column (Amersham Pharmacia Biotec, Uppsala, Sweden), as previously described (12). For simplicity, the various peaks in the profiles are designated as VLDL, LDL, and HDL, analogous with the nomenclature used for the human profile.
Liver triglyceride content.
Liver samples (4050 mg) were extracted in 1 ml isopropanol. After centrifugation, 4-µl aliquots of supernatant were added to 300 µl reagent (Unimate 5 TRIG; F. Hoffman-La Roche AG, Basel) for enzymatic colorimetric determination of triglyceride concentration.
Calculations.
Study 1: Estimation of hepatic triglyceride output.
Triton WR1339 effectively blocks the clearance of plasma TG. When applied in the postabsorptive state, the rate of hepatic TG output (HTGO) can be calculated from the linear rate of accumulation of TG in plasma (13), assuming a lipoprotein distribution space of 4% of lean body mass. The plasma TG clearance rate (KTG), an index of the combined ability of the tissues to remove TG from the circulation, was calculated as the ratio of HTGO-to-CTG, where CTG refers to the basal plasma TG immediately before Triton administration.
Study 2: Estimation of whole-body and tissue-specific rates of FFA metabolism.
Rates of plasma FFA clearance and appearance.
Plasma FFA mobilization was assessed using a constant infusion of 3H-palmitate (3H-P). After attainment of isotopic steady states (<40 min after the start of tracer infusion), the plasma clearance rate of 3H-P (KP) was calculated as
![]() |
where iP is the tracer infusion rate (dpm · min-1) and cP is the steady state arterial concentration of 3H-P (dpm · ml-1). The rate of appearance of plasma FFAs (Ra) was calculated as
![]() |
where CP is the arterial plasma FFA (µmol · ml-1).
Rate of plasma FFA oxidation.
An estimate of the whole-body rate of FFA oxidation (Rox) was calculated from the plasma accumulation of 3H2O as
![]() |
where cw is the plasma concentration of 3H2O (at 140 min), Vw is the total water space of the rat (assumed to be 70% of lean body mass), and Tin is the tracer infusion time (120 min).
Tissue-specific FFA metabolism.
Indexes of the tissue-specific clearance rates of plasma FFA incorporation into storage products (Kfs) were calculated as
|
where mP is the tissue content of 3H activity associated with non3H2O-associated products of 3H-P (at t = T), cP is the arterial plasma concentration of 3H-P, and T is the time of tissue collection (140 min). Collection of tissues 20 min after infusion of 3H-P had stopped (see protocol above) ensured low plasma 3H-P at T and therefore minimal contribution of free unmetabolised 3H-P to tissue 3H content. The integral representing the area under the 3H-P curve was calculated using a trapezoidal approximation. An index of the tissue-specific rate of plasma FFA incorporation (Rfs) was estimated as
![]() |
Statistics.
Analyses of group data were based on four contrasts designed to specifically test the principle a priori questions of the study: LN versus OB (obesity effect), ROS1 versus OB, ROS10 versus OB, and DAR versus OB (treatment effects). Statistical significance of the contrasts were evaluated on the basis of F tests using the program SPSS (SPSS, Chicago, IL). Results are reported as means ± SE. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Study 2: Mobilization and fate of plasma FFA.
Whole-body FFA metabolism in anesthetized rats.
Whole-body FFA mobilization (Ra) and KP (an index of the combined ability of the bodys tissues to take up FFA) were estimated based on steady-state tracer dilution using a constant infusion of H3H-palmitate (Table 3). Obese untreated Zucker rats showed substantially elevated systemic FFA availability, with higher plasma FFA levels and Ra than the lean controls. Unexpectedly, thiazolidinedione treatment of the obese animals actually increased systemic FFA availability under fasting conditions with low insulin levels. Thus, in both the DAR and ROS10 groups, Ra was much higher (fourfold and 2.5-fold, respectively) than in the untreated controls. These large increases in Ra translated into relatively modest increments in plasma FFA due to large treatment-induced increases in KP (DAR 2.4-fold and ROS10 twofold relative to untreated obese controls). Individual parameters in the ROS1 group did not achieve statistical significance; however, the means for Ra, plasma FFA, and KP for this group lay between the corresponding means for the control and ROS10 groups, compatible with genuine effects at the lower dose.
|
In the basal 7-h fasted state in conscious animals, darglitazone treatment approximately doubled both Ra (Fig. 3) and KP (Table 4), qualitatively confirming the findings made in the anesthetized animals. In line with the results from the anesthetized animals, mean FFA levels tended to be higher with darglitazone treatment (Fig. 3), although this difference did not achieve statistical significance. Darglitazone treatment increased basal Rox by 50% (Fig. 3), although the treatment-induced increase in Ra was mostly directed into nonoxidative FFA metabolism (Ra Rox for DAR versus obese controls was 45.5 ± 4.6 vs. 19.4 ± 1.9 µmol · min1, P < 0.001). At the tissue level, darglitazone treatment induced a substantial elevation in the Rfs in adipose tissue in both BAT and WAT depots (Fig. 3). Darglitazone also induced a small increase in the Rfs of the glycolytic skeletal muscle WQ and tended to increase Rfs in RQ and liver. To exclude the influence of differences in FFA levels and isolate local tissue level effects, Kfs was calculated (Table 4). Of the various tissues examined, treatment only induced significant Kfs changes in adipose tissues.
|
|
|
Hepatic triglyceride depots.
Liver weights and triglyceride contents are summarized in Table 6. Untreated obese Zucker rats had elevations in both liver weight and hepatic TG content (as compared with lean controls) that were partly reversed by thiazolidinedione treatment.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that thiazolidinediones can eliminate the extreme hypertriglyceridemia of the obese Zucker rat. One component of this antihypertriglyceridemic action was increased plasma TG clearance. An accelerated conversion of VLDL to triglyceride-poor remnants lowered VLDL and raised remnant concentrations in the plasma. This mode of thiazolidinedione action has previously been indicated in experiments in normal animals (15). The present study extends those observations by examination of a dyslipidemic animal model using an informative experimental paradigm (the combined use of Triton WR 1339 and lipoprotein analysis) that enabled functional identification of the VLDL and remnant lipoprotein fractions.
In addition to the treatment-induced enhancement of plasma TG clearance, the thiazolidinediones also lowered plasma TG levels by reducing the rate of HTGO. A higher dose requirement for the HTGO-lowering action than for the plasma TG clearanceenhancing mechanism was indicated in a comparison of the effects of the two rosiglitazone doses (Table 2) and may explain apparent discrepancies regarding kinetic mechanisms of TG lowering by different members of this group of agents. Thus, studies demonstrating effects only on clearance (1314151617) may have examined responses to effectively lower doses than those used in the current study and in another study (2). The HTGO-lowering action was associated with (and may have been at least partly mediated by) treatment-induced reductions in intrahepatic hepatic TG stores (Table 6), an important source of lipids for VLDL assembly (18).
To elucidate the effects of thiazolidinediones on the kinetic processes that determine plasma FFA levels over a physiological range of insulin levels, studies were conducted in both the basal 7-h fasted state and during euglycemic clamps (performed at insulin levels corresponding to those observed postprandially). Increased plasma FFA clearance was a robust thiazolidinedione action observed in both the basal and clamp situations. In contrast, the effects of thiazolidinediones on Ra were totally opposite in basal and clamp states. In the basal state, there was a substantial increase in Ra. Although initially surprising, this effect was considered consistent with the actions of the thiazolidinediones to induce adipocyte proliferation (19) and upregulate adipose tissue genes involved in FFA exchange with plasma (10,20). At the low fasting insulin levels in the treated animals, these actions may have provided the molecular machinery for this enhanced Ra.
Whatever the mechanism, the increase in Ra was sufficient to cancel out the influence of enhanced FFA clearance. In fact, fasting FFA levels tended to be higher in the treated animals. Under clamp conditions, treatment resulted in substantially lower plasma FFA levels because of the combined effects of much greater suppression of Ra (a manifestation of insulin sensitization in adipose tissue) and the increase in FFA clearance. Altogether, the results of the clamp and basal studies showed that thiazolidinediones induced a substantially larger change in plasma FFA levels across a physiological range of insulin levels. This implies more rapid and larger changes in FFA levels in the transition from the fed state to fasting. There is a practical implication of this situation on the assessment of thiazolidinedione effects on FFA levels. In fed animals, FFA levels are expected to be consistently lower, but as the length of fasting increases after food withdrawal, this treatment effect may become nonapparent or even reversed.
We propose that the actions of thiazolidinediones on adipose tissue FFA release provide a basis for more widespread tissue effects that lead to an improvement in whole-body metabolic flexibility, as evidenced by increased fatty acid oxidation under fasting conditions and improved glucoregulation under hyperinsulinemic conditions. Therefore, the augmented FFA mobilization may drive the enhanced whole-body FFA oxidation in the fasting state. We did not assess peripheral glucose utilization, but based on the reciprocal relationship between glucose and fatty acid catabolism (21), the elevation of FFA oxidation under fasting conditions may well be accompanied by a reduction in glucose oxidation. Under conditions of insulin stimulation, the much more effective suppression of FFA mobilization possibly plays an important indirect role in the observed improvements in whole-body glucoregulation. Decreased fatty acid availability and oxidation could theoretically increase both glucose utilization in skeletal muscle and insulin suppression of hepatic glucose production (HGP) (22). Tissue-specific glucose metabolism was not assessed in the current study. However, previous studies of thiazolidinediones have clearly established that major components of the whole-body insulin sensitization are increased insulin-stimulated muscle glucose utilization (23242526) and an enhanced ability of insulin to suppress HGP (24,27).
Thiazolidinedione treatment enhanced the ability of adipose tissue to take up and store FFA (Table 4). This enhanced ability allied with the large adipose tissue mass of the obese Zucker rats increased plasma FFA clearance. Figure 4 represents a breakdown of the tissue distribution and fate of plasma FFA calculated from the whole-body and tissue-specific flux determinations (Fig. 3) as well as estimates of tissue weights. Darglitazone treatment in both the basal fasting and clamp situations thus resulted in much greater trafficking of FFA into adipose tissues and away from oxidative metabolism as well as greater nonoxidative disposal in the liver. Adding together the estimated contributions of whole-body oxidative disposal (Rox) and nonoxidative disposal in skeletal muscles, liver, and white adipose tissue, we were able to account for >79% of the FFA leaving the plasma in all groups. Nonoxidative disposal into other tissues (including those not examined at all and BAT, whose tissue mass is unknown) make up the remaining fraction of whole-body FFA disposal. The current demonstration that thiazolidinediones enhance FFA uptake by adipose tissue fits well with reports that PPAR agonists upregulate adipose tissue gene expression of molecules involved in FFA transport and metabolism, including a putative fatty acid transporter protein, fatty acid translocase, and acyl-CoA synthetase (10,20). To our knowledge, the present data provide the first in vivo evidence that these changes in gene expression are of functional significance for FFA metabolism.
|
In conclusion, thiazolidinediones induced remarkable changes in the handling and fate of lipids in the obese Zucker rat. The results point to a critical role of adipose tissue FFA metabolism in both the general metabolic dysregulation of the obese animals and the subsequent thiazolidinedione-induced amelioration of this condition. The present data indicate that treatment with thiazolidinediones allows much greater metabolic flexibility to utilize lipids and carbohydrates in the fasting and postprandial states, respectively. In addition, we have shown that thiazolidinedione-induced abolition of hypertriglyceridemia in this model was attributable to the combination of enhanced VLDL-TG catabolism and reduced hepatic TG secretion.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
Address correspondence and reprint requests to N.D. Oakes, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. E-mail: Nick.Oakes{at}astrazeneca.com.
Received for publication 19 May 2000 and accepted in revised form 9 February 2001.
BAT, brown adipose tissue; DAR, rats treated with darglitazone; FFA, free fatty acid; GIR, glucose infusion rate; HGP, hepatic glucose production; K, rate of FFA clearance; KP, clearance rate of 3H-P; Kfs, rate of FFA incorporation into storage products; KTG, TG clearance rate; LN, lean controls; OB, obese controls; PPAR, peroxisome proliferatoractivated receptor
; Ra, rate of FFA appearance; Rfs, rate of nonoxidative FFA disposal; Rox, rate of FFA oxidation; ROS, rats treated with rosiglitazone; HTGO, hepatic TG output; RQ, red quadriceps muscle; TG, triglyceride; WAT, inguinal adipose tissue; WQ, white quadriceps.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|