Warren K. Palmer

Mechanism of poloxamer 407-induced hypertriglyceridemia in the rat

One 300 mg i.p. injection of the nonionic surfactant poloxamer 407 (Pluronic F-127) produces a significant increase above control of both circulating cholesterol and triglyceride (TG) concentrations. The present study was conducted to determine the effect of poloxamer 407 (P-407) on the capacity to hydrolyze circulating TG by lipoprotein lipase (LPL) in an attempt to determine the mechanism of action of P-407. The concentration of TG in the rat following a single 300 mg i.p. injection of P-407 was marked, increasing from 84 +/- 10 to 3175 +/- 322 mg/dL at 24 hr. The maximal rate of TG accumulation (5.74 mg/dL/min) in the plasma of P-407-injected rats occurred between 2 and 4 hr post-injection. In vitro incubation of LPL with P-407 significantly inhibited enzyme activity with an inhibitory concentration at which 50% of the enzymatic activity was lost of approximately 24 microM. Concentrations of P-407 exceeding 350 microM in vitro completely inhibited LPL activity. The effects of P-407 on the enzymatic activity of LPL in post-heparin plasma obtained following a single 300 mg dose of P-407 to rats demonstrated greater than 95% suppression of LPL activity 3 hr post-injection compared with controls. Inhibition of LPL activity was greater than 90% as long as 24 hr following a single i.p. injection of P-407. However, while the heparin-releasable fraction of capillary-bound LPL was inhibited in the plasma, LPL activity significantly increased in cardiac and skeletal muscle in poloxamer-injected animals compared with sham-injected controls. Although there was no significant change in LPL activity in adipose tissue, testes, and lung resulting from P-407 treatment, LPL activity increased by 37% in myocardium, 69% in soleus, and 66% in gastrocnemius muscle in P-407-injected rats when compared with controls. Our studies would suggest that the predominant mechanism by which P-407 induced an increase in circulating TG was by a reduction in the rate at which TG was hydrolyzed due to inhibition of heparin-releasable LPL by the surfactant.

Poloxamer 407-induced atherogenesis in the C57BL:6 mouse

Poloxamer 407 (P-407) induces hyperlipidemia in the rat. It was the purpose of this investigation to determine if chronic P-407 administration would produce atherogenic arterial lesions in the C57BL/6 mouse, a strain reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. One injection (i.p.) of P-407 (0.5g/kg) produced hypercholesterolemia in the mouse that peaked at 24 h and returned to control levels by 96 h following treatment. Four groups of mice were maintained: (1) saline injected (C); (2) P-407-injected (0.5g/kg every 3rd day) (P); (3) P-407 injected plus cholic acid in the diet (PC); and (4) mice fed a high cholesterol (CHOL) diet containing cholic acid (HF). Mice from each group were sacrificed following 90, 145, 200, or 300 days of treatment. Plasma lipid concentrations, hepatic CHOL concentrations (145 and 300 day), and aortic atherogenic lesion areas were measured. Plasma CHOL and triglyceride remained at control levels throughout the 300 days in the C group. CHOL of the HF animals plateaued at approximately 225 mg/dl. P-407 produced CHOL concentrations of 600 mg/dl in P mice and 1000-1500 mg/dl in PC animals. There was no lesion formation in C mice. However, by 90 days lesions were present in the three other groups. Size of the lesions progressed through day 300 with the largest lesions (184.33 + 27.99 mu2 x 10(-3)) being present in the PC mice. HF and P animals had lesions of 70.50 + 11.35 and 43.33 + 7.88 mu2 x 10(-3), respectively. This study provides an animal model where atherogenesis has been produced with hyperlipidemia induced using a chemical agent.

The poloxamer 407-induced hyperlipidemic atherogenic animal model.

We are attempting to develop a chemically-induced murine model for the study of atherosclerosis. Injection of poloxamer-407 (P-407) into rats and mice causes significant dose-dependent hypercholesterolemia and hypertriglyglyceridemia. The elevated triglycerides (TG) seem to result primarily from the compounds inhibition of lipoprotein lipase. P-407 also indirectly stimulates the activity of the rate limiting enzyme in cholesterol (CHOL) biosynthesis, HMG CoA reductase. In addition, P-407 promotes changes in the concentration of hepatic CHOL content. These date indicate that the hyper CHOL could be the result of increased CHOL synthesis, as well as a clearing of CHOL from the liver. Chronic injection into mice of P-407 for 145 d produced atherogenic lesions in the aortas of C57BL/6 mice. The response was equivalent to that seen in animals eating a high CHOL diet for 145 d. Cholic acid potentiated the P-407-induced atherogenesis. These data suggest that P-407 could be used as an agent for the study of hyperlipidemia-induced atherogenesis.

Effect of Poloxamer 407 on the Activity of Microsomal 3-hydroxy-3-methylglutaryl Coa Reductase in Rats

A single 300-mg i.p. injection of poloxamer 407 (P-407, also called Pluronic F-127) in rats produces a marked hypercholesterolemia for a minimum of 96 h. The purpose of this investigation was to determine mechanisms by which P-407 causes hypercholesterolemia. The enzyme targeted for investigation is the rate-limiting enzyme in cholesterolgenesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Injection of P-407 in fasted rats resulted in a significant (p < 0.05) elevation in plasma cholesterol (61.2 +/- 4.2 mg/dl) as soon as 1 h after injection compared with sham-injected controls (50.1 +/- 3.7 mg/dl). Plasma cholesterol (CHO) 24 h after injection of P-407 was 449 +/- 57 mg/dl, with the fastest rate of accumulation occurring from 1 to 12 h (approximately 16.6 mg/dl/h). Over the concentration range of 0-5 mM, P-407 did not appear significantly to affect the activity of HMG-CoA reductase in vitro. However, the enzymatic activity assayed in microsomal fractions isolated from the livers of P-407-injected rats reached a maximum of 262 +/- 42.6 pmol mevalonate/min/mg approximately 15 h after injection, with a subsequent decline to control activity (94.1 +/- 8.7 pmol/min/mg) at approximately 40 h after injection. At 48 h after injection of P-407, the activity of HMG-CoA reductase significantly (p < 0.05) decreased below control values with a mean activity of 9.4 +/- 1.2 pmol/min/mg. The CHO concentrations in hepatic tissue were significantly (p < 0.01) increased at 2 h (3.26 +/- 0.19 mg/g) and 4 h (3.75 +/- 0.38 mg/g) and significantly (p < .01) reduced at 15 h (1.56 +/- 0.19 mg/g) after injection of P-407 compared with tissue CHO concentrations determined in control animals (2.65 +/- 0.18 mg/g). However, the hepatic CHO content appeared to return to control values by 24 h (mean +/- SEM, 2.61 +/- 0.08 mg/g) after injection. These data suggest that the activity of HMG-CoA reductase is regulated by some indirect mechanism(s) after injection of P-407 in rats.

Potential downregulation of HMG-CoA reductase after prolonged administration of P-407 in C57BL/6 mice

This study investigated the potential alteration in the amount of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase messenger RNA (mRNA) and lipoprotein lipase (LPL) mRNA in the livers of C57BL/6 mice after long-term (200 days) treatment with the nonionic surfactant called poloxamer 407 (P-407). Previously, P-407 has been used to produce a dose-controlled hyperlipidemic state in C57BL/6 mice with subsequent formation of atherosclerotic lesions. Five groups of mice were studied; controls (C); mice fed a standard chow diet enriched with only cholic acid (CH); mice fed the high-cholesterol, high-fat Paigen diet (HF); mice treated with 0.5 g/kg P-407 every third day (P); and mice administered 0.5 g/kg P-407 every third day while consuming a diet identical to that of mice in group CH (PC). Neither a significant (p < 0.05) weight loss nor alteration in liver enzymes (AST and ALT) were observed for any group throughout the study when compared with the control mice. Total plasma cholesterol (CHOL) was significantly elevated compared with controls for mice in groups HF, P, and PC, whereas total plasma triglycerides (TG) were significantly increased for mice in only groups P and PC. Long-term ingestion of a high-fat diet or a diet enriched in cholic acid resulted in a significant (p < 0.05) reduction in HDL-CHOL when compared with controls. Plasma samples assayed at 200 days for mice in groups HF and P showed a shift in the lipoprotein fraction distribution primarily to VLDL-CHOL as compared with mice in group C in which, as expected, most of the CHOL was contained in the HDL fraction. The biologic activity of HMG-CoA reductase assayed in hepatic microsomal homogenates was significantly reduced for mice in groups CH (p < 0.01), HF (p < 0.01), and PC (p < 0.05), but not for mice in group P, when compared with control. A statistical analysis of the data demonstrated significant (p < 0.05) reductions in the HMG-CoA reductase mRNA levels in hepatic tissue for all treatment groups relative to mRNA levels determined for mice in group C. In contrast, no treatment group demonstrated a significant difference in hepatic LPL mRNA levels when compared with mRNA levels determined for control animals. These data demonstrate that P-407 administration to C57BL/6 mice significantly decreased the amount of HMG-CoA reductase mRNA detected in liver.

Effects of Nicotinic Acid on Poloxamer 407‐Induced Hyperlipidemia

We attempted to determine the mechanism(s) of poloxamer (P)‐407‐induced hyperlipidemia in rats by administering a lipid‐lowering drug with a known mechanism of action. Five weight‐matched animals were assigned to each of four treatment groups. Two groups received P‐407 300 mg/ml and two received saline 1 ml. One of the P‐407 and one of the saline groups were administered nicotinic acid 100 mg/kg by intraperitoneal injection at 6–96 hours after blood sampling. Blood samples were collected at 7 points from time zero to 120 hours and analyzed for triglyceride and cholesterol concentrations. The detergent produces hypertriglyceridemia (HTG) increasing from 53.4 ± 7.0 mg/dl (time zero) to 4026.9 ± 42.1 mg/dl by 24 hours. The HTG response was significantly attenuated by nicotinic acid (at t = 24 hrs). This, however, was followed by an average triglyceride concentration increase of 2.8‐fold from 72 to 120 hours. The detergent produces a dramatic hypercholesterolemia (HCHO), increasing cholesterol from 47.5 ± 1.8 mg/dl to 468.5 ± 27.9 mg/dl by 48 hours. The HCHO was significantly affected by nicotinic acid administration during the accumulation phase. Nicotinic acid reduced cholesterol concentration from 364.4 ± 16.1 mg/dl to 276.8 ± 16.4 mg/dl at 24 hours (p<0.05). It is a potent antilipolytic agent, limiting the free fatty acids available for the synthesis of triglyceride and cholesterol. These data suggest that P‐407 may act by stimulating the release of free fatty acids from the adipocyte for at least 24 hours after injection.

Epinephrine-activation of heparin-nonreleasable lipoprotein lipase in 3 skeletal muscle fiber types of the rat.

Epinephrine was used to activate the heparin non-releasable lipoprotein lipase (LPL) in the 3 skeletal muscle fiber types of the perfused rat hindlimb. Following a 9 min washout of the capillary-bound lipoprotein lipase, the hindquarter of the rat was perfused with a buffer containing 10 nM of epinephrine. Activity of the residual LPL in soleus, red vastus lateralis, and white vastus lateralis muscles increased 75%, 96%, and 102% respectively, following epinephrine perfusion. These results suggest that skeletal muscle LPL is under hormonal control possibly through protein phosphorylation by cyclic AMP dependent protein kinase.

EFFECT OF GLUCOSE AND INSULIN ON TRIACYLGLYCEROL METABOLISM IN ISOLATED NORMAL AND DIABETIC SKELETAL MUSCLE

The effects of insulin and glucose on triacylglycerol (TG) metabolism in normal and diabetic isolated skeletal muscle were investigated in this study. Intracellular TG was continuously synthesized and hydrolyzed in both normal and diabetic skeletal muscle. In the absence of insulin and glucose, normal and diabetic skeletal muscle TG content and synthesis were decreased. In contrast, in the presence of insulin and glucose, the normal and diabetic TG contents were unchanged and triacylglycerol synthesis was increased as compared with the respective control values. However, insulin and glucose increased intramuscular TG content to a greater extent than could be accounted for by their stimulation of TG synthesis, indicating that insulin and glucose appear to inhibit TG hydrolysis in diabetic muscle, as well as in normal muscle. In addition, these data suggest that diabetes causes a defect in the ability of insulin and glucose to stimulate TG synthesis, as the increase in diabetic muscle TG synthesis in the presence of insulin and glucose was less than in normal muscle.

The Effect of Pravastatin on Hepatic 3-Hydroxy-3-methylglutaryl CoA Reductase Obtained from Poloxamer 407-Induced Hyperlipidemic Rats

A single 300‐mg intraperitoneal injection of poloxamer 407 (P‐407) to rats produces a marked hypercholesterolemia for a minimum of 96 hours and increases the activity of hepatic 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase compared with the enzyme activity found in microsomal homogenates of control animals. We attempted to determine whether inhibition of microsomal HMG‐CoA reductase by pravastatin sodium would yield similar values for the maximum reaction in velocity (Vmax) and the HMG‐CoA reductase‐pravastatin dissociation constant (Ki) when the enzyme was in the activated state compared with the control state. Knowledge of the respective values for Vmax and Ki would allow us to determine whether P‐407‐induced hypercholesterolemia in the rat was refractory to pravastatin treatment. Over a pravastatin concentration range of 0.5–50 nM, enzyme activity in vitro decreased as the drugs concentration increased. A standard Dixon plot of mean values of reciprocal reaction velocity versus pravastatin concentration yielded Ki of 3.7 and 4.1 nM for the control and activated states, respectively. The Vmax for conversion of HMG‐CoA to mevalonate in vitro in the presence of pravastatin was 3.5‐fold greater when assayed in microsomal homogenates obtained from P‐407‐injected rats compared with control animals. Dixon plot analysis of the data resulted in Vmax of 58.1 and 202 pmol•min−1•mg−1 for the control and activated states, respectively. These data suggest that whereas the Vmax is affected, injection of P‐407 to rats does not alter the binding affinity of pravastatin for receptor(s) contained in HMG‐CoA reductase as reflected by similar Ki values. This experimental animal model may be an additional screen with which to rank order the relative potency of HMG‐CoA reductase inhibitors by determining the drugs effectiveness when HMG‐CoA reductase is in an activated state.

Effects of pravastatin on plasma lipid concentrations in poloxamer 407-induced hyperlipidemic rats

A new animal model of hyperlipidemia is being developed using the nonionic surfactant poloxamer 407 (P‐407). We investigated the impact of pravastatin on P‐407‐induced hyperlipidemia. Twenty rats received P‐407 300 mg intraperitoneally to induce hyperlipidemia, and 20 control rats received saline injection. Pravastatin was administered orally to an equal number of rats in both groups using three different regimens. A fourth group did not receive pravastatin. At 24 hours after injection, total cholesterol levels in two of the pravastatin groups were 28% and 34% lower than those in animals that did not receive pravastatin (p≤0.01). At 48 hours, triglyceride levels were significantly lower in all pravastatin groups (21–44%) versus animals not receiving pravastatin. Pravastatin diminished the effects of P‐407 on lipoproteins. This new animal model may be useful in screening for investigational antihyperlipidemic agents.