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Endocrinology Vol. 142, No. 6 2702-2706
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulin Inhibits Peroxisomal Fatty Acid Oxidation in Isolated Rat Hepatocytes1

Frederick G. Hamel2, Robert G. Bennett2, Jennifer L. Upward and William C. Duckworth

Research Service (F.G.H., R.G.B., J.L.U.), Department of Veterans Affairs Medical Center, Omaha, Nebraska 68105; Department of Internal Medicine (F.G.H., R.G.B.), Department of Pharmacology (F.G.H.), University of Nebraska Medical Center, Omaha, Nebraska 68198; and Carl T. Hayden Medical Center (W.C.D.), Department of Veterans Affairs, Phoenix, Arizona 85012

Address all correspondence and requests for reprints to: Frederick G. Hamel, Ph.D., Department of Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, Nebraska 68105.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibition by insulin of long chain fatty acid oxidation in mitochondria is mediated in part by elevating malonyl-CoA levels, which inhibit carnitine palmitoyl-transferase I. Whether insulin alters peroxisomal oxidation has not been studied. We present data which show that insulin inhibits the oxidation of palmitic acid by peroxisomes (IC50 = 8.5 x 10-11 M) at hormone concentrations 100-fold less than those needed for mitochondrial inhibition (IC50 = 1.3 x 10-8 M). We used a purified peroxisome preparation to study the mechanism of insulin action. Insulin had a direct effect in the peroxisome preparations to decrease oxygen consumption, fatty acyl-CoA oxidizing system activity and acyl-CoA oxidase by approximately 40%, 30% and 15%, respectively. Since insulin degrading enzyme (IDE) is an insulin-binding protein known to be in peroxisomes, we studied the effect of an inhibitory anti-IDE antibody on the ability of insulin to inhibit the fatty acyl-CoA oxidizing system. The antibody eliminated the inhibitory effect of insulin. We conclude that insulin inhibits peroxisomal fatty acid oxidation by a mechanism requiring IDE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES mellitus is no longer seen as simply a disease of carbohydrate metabolism (1, 2). Lipid metabolism, especially that of FFA, plays an important role (1, 3, 4). The interaction of FFA and glucose metabolism was first noted by Randle’s group in the 1960s (5), in what is called the glucose-fatty acid cycle. Essentially, increased availability of one substrate would suppress oxidation of the other. Increased FFA oxidation results in increased acetyl CoA and citrate, which inhibit pyruvate dehydrogenase and phosphofructokinase 1, respectively. More recent studies have confirmed that increased FFAs cause more lipid oxidation and less carbohydrate utilization (6, 7, 8, 9). Thus, insulin control of FFA metabolism may be a critical factor in the etiology of type 2 diabetes.

Insulin decreases fatty acid oxidation, in part, by decreasing triglyceride breakdown, and thereby reducing substrate availability. Insulin also has direct effects on fatty acid oxidation. Fatty acids are metabolized in mitochondria and peroxisomes. Long chain fatty acids require coupling to carnitine for transport into the organelle’s matrix before oxidation (10, 11, 12). This coupling is catalyzed by carnitine palmitoyl-transferase I (CPT I). Malonyl-CoA, a by-product of glucose metabolism, is a potent inhibitor of CPT I (13). Insulin’s ability to decrease mitochondrial fatty acid oxidation is thought to be mediated by this mechanism. However, much less is known about the mechanism whereby insulin reduces peroxisomal oxidation.

Peroxisomes oxidize a variety of lipids through ß-oxidation, including medium, long-chain, and very long-chain fatty acids, branched fatty acids, dicarboxylic acids, prostaglandins, and some bile acids (14). Normally peroxisomal ß-oxidation of fatty acids is preferential for very long-chain fatty acids (>C20), although oxidation of shorter chains occurs. Peroxisomal ß-oxidation has many similarities to the analogous mitochondrial process (14), but peroxisomal ß-oxidation is not coupled to the electron transport chain. Instead, the by-products are heat, FADH2, and H2O2. We have examined insulin’s effect on peroxisomal FA oxidation in isolated hepatocytes. Further, we have done studies on isolated peroxisomes to explore the mechanism of the insulin effect. Our results indicate that peroxisomal FA oxidation is controlled by insulin in a manner different from mitochondria FA oxidation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Porcine crystalline insulin and insulin mono- 125-iodinated on residue A14 were gifts of Ronald Chance and Bruce Frank, both of Eli Lilly & Co. (Indianapolis, IN). Palmitic acid [14C-1] was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Octanoic acid [14C-1] and arachidonic acid [14C-1] were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Percoll was from Amersham Pharmacia Biotech. Leuco-DCF (2,7-dichlorodihydrofluorescein diacetate) was from Molecular Probes, Inc. (Eugene, OR). Palmitoyl Co-A was from Sigma (St. Louis, MO). All other chemicals were reagent grade or better. BSA (Fraction V) was defatted by treatment with activated charcoal under acidic conditions as previously described (15).

Preparation of isolated rat hepatocytes
Hepatocytes were isolated from fasted male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) (150–250 g) by collagenase perfusion using a modification of the method of Peavy et al. (16). Before assay, the cells were resuspended at 2 x 106 cells/ml in Krebs Improved Ringer II buffer with 2% BSA and incubated for 30 min at 37 C with constant flow of O2/CO2 (95/5).

Cellular fatty acid oxidation
Fatty acid oxidation was measured by a modification of the method of Mannaerts et al. (17). Isolated hepatocytes (0.25 ml) were incubated for 1 h in scintillation vials in Krebs Improved Ringer II buffer with 2% defatted BSA (except where noted), with 1 mM fatty acid (palmitic, arachidonic or oleic; specific activity 1 µCi/µmol). Insulin was included at various concentrations as shown in the figures. The peroxisomal component was determined by adding 3 mM KCN to corresponding vials to inhibit mitochondrial oxidation. Radiolabeled CO2 was collected in center wells with Whatman #1 filter paper and 300 µl of 1 M methylbenzethonium hydroxide in methanol. At the end of the incubation, 300 µl of 5 M H2SO4 was added to volatilize the remaining CO2, and the solution incubated another 30 min. The center wells were then placed in other scintillation vials and 8 ml of aqueous scintillant added and counted on a ß counter.

Preparation of rat liver peroxisomes
Male Sprague Dawley rats (150–250 g) were fasted overnight, killed by decapitation, livers removed, and peroxisomes were prepared as described (18). Briefly, the livers were homogenized, and subcellular fractionation was performed to produce nuclear/heavy mitochondrial (N/M), light mitochondrial (L), microsomal (P), and cytosolic (S) fractions. The L fraction, containing light mitochondria, lysosomes, and peroxisomes, was further purified by Percoll density gradient centrifugation (18). Fractions containing peroxisomal marker activity were pooled, and Percoll was removed by centrifugation. The Percoll-purified peroxisome (PPP) fractions were used for further studies of peroxisomal ß-oxidation and the effect of insulin.

Enzyme assays
Marker enzymes catalase (19), fatty acyl-CoA oxidizing system (FAOS), (20), and acyl-CoA oxidase (AOX) (21) (peroxisomes), acid phosphatase (22) (lysosomes), lactate dehydrogenase (19) (cytosol), cytochrome-c oxidase (19) (mitochondria), 5'-nucleotidase (23) (plasma membrane), glucose-6-phosphatase (23) (endoplasmic reticulum), and {alpha}-mannosidase II (19) (Golgi) were measured as described. Oxygen consumption by isolated peroxisomes was performed as described (24) with a Gilson oxygraph equipped with a Clark electrode, using palmitoyl CoA as the substrate. Insulin degradation was measured as described (25). Protein was measured by the method of Bradford (26). Some of the studies used our monoclonal antibody C20–3.1A, which we have previously shown inhibits IDE (27, 28).

Statistical analyses
One-way ANOVA with Bonferroni’s post test on selected pairs or t test, (as appropriate) were performed using GraphPad Software, Inc. Prism version 3.02 for Windows, (San Diego, CA, www.graphpad.com). Curve fitting of means to a one site competition model was also done with Prism. All values shown are means ± SEM. The numbers of independent experiments are indicated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin inhibited total palmitic acid oxidation to CO2 by 50% at 10-7 M, the maximum concentration tested, with an apparent IC50 of 1.3 x 10-8 M (Fig. 1Go, upper panel). However, insulin inhibition of peroxisomal oxidation was more sensitive with a IC50 of 8.5 x 10-11 M. Peroxisomal oxidation was inhibited by 36%, which represents a 22% decrease of the total fatty acid oxidation. Thus, both peroxisomal and mitochondrial oxidation were affected by insulin. Insulin had no effect of on the oxidation of octanoic acid (Fig. 1Go, middle panel), which does not require carnitine for metabolism by mitochondria and is not metabolized in peroxisomes.



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  		Figure 1. The effect of insulin on [14C-1] fatty acid oxidation in hepatocytes. The graphs show the amount of fatty acid oxidized to CO2 (relative to the maximal oxidation for each preparation) at various insulin concentrations. The activities at various insulin concentrations were compared with that at 10-13 M insulin and those statistically significantly different are indicated (*, P < 0.05; ***, P < 0.001). Upper panel, The points represent the means ± SEM of at least six independent experiments with palmitic acid (C16:0). The means were curve fit to a one site competition and are shown for total (solid line, r2=0.996) and peroxisomal (dashed line, r2=0.992) oxidation. Middle panel, The points represent the means ± SEM of at least four independent experiments with octanoic acid (C8:0). Lower panel, The points represent the means ± SEM of at least four independent experiments with arachidonic acid (C20:4). The means were curve fit to a one site competition and are shown for total (solid line, r2=0.995) and peroxisomal (dashed line, r2=0.997) oxidation.

 
We also examined the effect of insulin on oxidation of a polyunsaturated fatty acid. Total oxidation of arachidonic acid to CO2 was inhibited by insulin in a dose-dependent manner by about 60% at the maximum concentration tested, 10-7 M, with an apparent IC50 of 1.3 x 10-9 M (Fig. 1Go, lower panel), 10-fold less than that for palmitate. Arachidonic acid oxidation in peroxisomes was somewhat affected, and at a similar concentration (IC50 of 2.5 x 10-9 M). The reasons for the quantitative differences compared with palmitate are not clear but may be related to the relative concentrations of endogenous fatty acids or alternative metabolic pathways (e.g. cyclooxygenase).

To examine possible mechanisms of insulin inhibition of peroxisomal fatty acid oxidation, we prepared purified peroxisomes for additional studies (Table 1Go). Peroxisomal markers were enriched about 10-fold, which is similar to previous studies. Insulin degrading enzyme has a type-1 peroxisomal targeting sequence and has been reported to be present in this organelle. A Western blot with 9B12 anti-IDE antibody (Fig. 2Go) confirms its presence in peroxisomes.


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  		Table 1. Enrichment of organelle marker enzymes in purified liver peroxisomes

 


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  		Figure 2. Anti-IDE Western blot of Percoll purified peroxisomes. Peroxisomal proteins were run on SDS-PAGE and a Western blot analysis performed with 9B12 antibody. A band is present at 110 kDa, the known molecular mass of insulin degrading enzyme.

 
Peroxisomal function was assessed by measuring O2 consumption, which reflects overall lipid ß-oxidation. The addition of insulin to purified peroxisomes reduced O2 consumption, indicating inhibition of lipid oxidation (Fig. 3Go). Insulin also inhibited peroxisomal AOX and FAOS activities but not catalase (Fig. 4Go). This selectivity of effect on enzymatic activities indicates that insulin does not have a general, nonspecific action on peroxisomal enzymes.



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  		Figure 3. Insulin inhibits O2 consumption by purified liver peroxisomes. Basal O2 consumption levels by Percoll-purified liver peroxisomes were established, then palmitoyl CoA was added where indicated. After a steady rate of O2 consumption was established, insulin (1 µM) was added where indicated. Data are expressed as nmol oxygen present in the chamber vs. time (minutes). Rates were calculated from the slopes observed.

 


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  		Figure 4. The effect of insulin on peroxisomal fatty acid oxidizing activities. Fatty acid oxidation by Percoll-purified liver peroxisomes was measured by AOX (n = 7), FAOS (n = 8), and O2 consumption (n = 5) in the presence and absence of 1 µM insulin. As a control, the insulin effect on catalase activity was also determined (n = 5). Data are expressed as % of enzyme activity with addition of vehicle only. *, P < 0.05; ** P < 0.005 compared by t test with the insulin effect on catalase.

 
To examine a potential role for IDE in mediating the insulin effect on peroxisomes, an anti-IDE monoclonal antibody that inhibits insulin degradation was added to these organelles and the effect of insulin assessed (Fig. 5Go). The antibody increased basal FAOS activity slightly and blocked the inhibitory effect of insulin on FAOS activity. A similar experiment was done for catalase and showed no effect of the antibody on enzyme activity (data not shown).



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  		Figure 5. Anti-IDE antibody blocks the insulin inhibition of peroxisomal FAOS. The FAOS activity in purified liver peroxisomes was measured in the presence and absence of anti-IDE antibody C20–3.1A with (filled bars) and without (open bars) 1 µM insulin. Data are expressed as % FAOS activity compared by t test with addition of vehicle only. *, P < 0.05; n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin exerts control over a wide variety of cellular functions, including glucose, protein, and lipid metabolism. Although attention has focused on glucose turnover, the primary metabolic derangement associated with type 2 diabetes may pertain as much to alterations in lipid metabolism as to glucose control (1, 2, 29, 30). One effect of insulin is to decrease fatty acid oxidation. It does this in part by decreasing triglyceride breakdown, thereby decreasing substrate availability and increasing glucose metabolism. The increased glucose metabolism results in an increase in malonyl CoA, an inhibitor of CPT I, which is required for long chain fatty acid oxidation by mitochondria (10, 11, 12, 13). However, long and especially very long chain fatty acids are also metabolized by peroxisomes (14). Our results show that peroxisomal fatty acid oxidation is inhibited at a much lower insulin concentration than is mitochondrial oxidation. This suggests a different mechanism for control of peroxisomal fatty acid oxidation.

The oxidation of octanoic acid does not require the action of CPT I for its transport into the matrix. In our studies octanoic acid metabolism was unaffected by insulin. Thus, mitochondrial function per se was not altered by insulin. Palmitate oxidation, however, was affected in both mitochondria and peroxisomes. Peroxisomal oxidation was much more sensitive to insulin, by a factor of 150, indicating that it was not working by the same mechanism in the two different organelles. The mitochondrial inhibition was probably primarily mediated by malonyl CoA inhibition of CPT I. Arachidonic acid metabolism was affected by insulin, but the effect was primarily on mitochondrial oxidation with a smaller effect on peroxisomal oxidation, and with different IC50s than for palmitate.

Insulin degrading enzyme (IDE) has a type-1 peroxisomal targeting sequence (31, 32, 33), and some data suggest that it may be concentrated in these organelles (34). A Western blot (Fig. 2Go) demonstrates its presence in our preparations. Furthermore, our data show IDE has a role in modulating peroxisomal enzyme activity. Insulin incubated with purified peroxisomes inhibited fatty oxidation and lowered O2 consumption. An inhibitory anti-IDE antibody abolished the insulin effect. Thus, enzymatically active IDE is required for the effect. Whether the enzyme is involved directly or whether insulin degradation products are required is still to be determined. However, this function of IDE is similar to one we have described for its interaction with the proteasome (27, 28, 35, 36, 37). In that system, insulin, binding to IDE, inhibited two peptidolytic activities of the proteasome. This results in a typical insulin action, a decrease in cellular protein degradation. A similar situation is found with IDE and interactions with the androgen and glucocorticoid receptors (38, 39). In the current system, another typical insulin action, decreased fatty acid oxidation, is also achieved by insulin interacting with IDE in association with a subcellular organelle. We propose that IDE mediates these effects of insulin, acting as an intracellular receptor for the hormone. This idea is especially interesting in light of the recent identification of IDE as a candidate susceptibility gene in GK rats, a model of type 2 diabetes (40). The exact mechanism remains to be elucidated, but since insulin and the anti-IDE antibodies have effects in permeabilized peroxisomes, IDE apparently does not work by altering fatty acid transport into peroxisomes, but rather has a more direct action.

Fatty acids or their acyl-CoA thioesters are known to be ligands for peroxisome proliferator-activated receptors (PPARs) and other transcription factors and to alter gene expression (41, 42, 43, 44, 45, 46). Fatty acids or their acyl-CoA thioesters are also known to alter a number of enzyme activities, such as acetyl-CoA carboxylase, tricarboxylate carrier, and mitochondrial uncoupling proteins (UCP) (47, 48). Thus, hormonal control of peroxisomal activity and the subsequent changes in the levels of fatty acids could result in significant alteration in cellular metabolism both short-term (enzyme activity) and long term (gene transcription). Insulin’s action in peroxisomes at relatively lower concentrations may be directed at altering the relative composition of available intracellular FFA, rather than altering total cellular fatty acid oxidation. These changes in fatty acid composition would work as another signal transduction mechanism of insulin, and one where dietary status could directly affect insulin action. While highly speculative, excessive dietary fatty acids would increase intracellular fatty acid levels and alter their relative composition, overwhelming this subtle control by insulin. These changes could contribute to insulin resistance by altering normal fatty acid-mediated control of the PPARs. The thiazolidinediones may act to reverse these changes.

In summary, insulin decreases fatty acid oxidation in peroxisomes. This action requires insulin degrading enzyme and thus uses a mechanism distinct from the one that decreases fatty acid oxidation in mitochondria in response to insulin; malonyl CoA inhibition of CPT I. While the effect of insulin in peroxisomes on total fatty acid oxidation by the cell is relatively minor, the resulting change in the fatty acid composition available for metabolism and regulatory control would provide an additional mechanism for insulin-directed regulation of cellular activities.


   Acknowledgments
 
The authors thank Jennifer L. Larsen for critical reading of the manuscript.


   Footnotes
 
1 This work supported by the Medical Research Service of the Department of Veterans Affairs and the Bly Memorial Research Fund, University of Nebraska Medical Center. Back

2 These authors contributed equally to this work. Back

Received September 22, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Cabrero, M. Merlos, J. C. Laguna, and M. V. Carrera
Down-regulation of acyl-CoA oxidase gene expression and increased NF-{kappa}B activity in etomoxir-induced cardiac hypertrophy
J. Lipid Res., February 1, 2003; 44(2): 388 - 398.
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