Reduced adiposity and liver steatosis by stearoyl-CoA desaturase deficiency are independent of peroxisome proliferator-activated receptor-alpha.

Stearoyl-CoA desaturase catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids, which are required for normal rates of synthesis of triglycerides, cholesterol esters, and phospholipids. Mice with a targeted disruption of the stearoyl-CoA desaturase 1 (SCD1) isoform are protected against diet and leptin deficiency-induced adiposity, have increased energy expenditure, and have up-regulated expression of hepatic genes encoding enzymes of fatty acid beta-oxidation. Because peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key transcription factor that induces the transcription of fatty acid beta-oxidation and thermogenic genes, we hypothesized that the increased fatty acid oxidation observed in SCD1 deficiency is dependent on activation of the PPARalpha pathway. Here we show that mice nullizygous for SCD1 and PPARalpha are still protected against adiposity, have increased energy expenditure, and maintain high expression of PPARalpha target genes in the liver and brown adipose tissue. The SCD1 deficiency rescued hepatic steatosis of the PPARalpha(-/-) mice. The SCD1 mutation increased the phosphorylation of both AMP-activated protein kinase and acetyl-CoA carboxylase, thereby increasing CPT activity and stimulating the oxidation of liver palmitoyl-CoA in the PPARalpha null mice. The findings indicate that the reduced adiposity, reduced liver steatosis, increased energy expenditure, and increased expression of PPARalpha target genes associated with SCD1 deficiency are independent of activation of the PPARalpha pathway.

Stearoyl-CoA desaturase (SCD) 1 catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acid from saturated fatty acids. SCD, in conjunction with the cofactors NADH, cytochrome b 5 reductase, and cytochrome b 5 , introduces a single double bond into its substrates, palmitic acid (16:0) and stearic acid (18:0), to generate the products, palmitoleic (16:1) and oleic acid (18:1). These products are the most abundant fatty acids found in triglycerides, cholesterol esters, and phospholipids. Four mouse isoforms of SCD (SCD1, SCD2, SCD3, and SCD4) (1)(2)(3)(4) and two human isoforms have been characterized (5,6). The mouse SCD1 isoform is the best studied and is highly regulated by several dietary and hormonal factors including insulin, leptin, glucose, fructose, cholesterol, and polyunsaturated fatty acids (7,8). We recently found that mice with a disruption in the SCD1 gene have increased energy expenditure, reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced obesity (9,10). The SCD1 Ϫ/Ϫ mice also have reduced hepatic triglycerides and cholesterol esters and are resistant to the generation of liver steatosis (11)(12)(13)(14). Associated with the reduced body fat in SCD1deficient mice, the expression of several genes encoding enzymes of lipid ␤-oxidation is up-regulated, whereas genes encoding enzymes of lipid synthesis including fatty-acid synthase and glycerol 3-phosphate acyltransferase are downregulated (9).
Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear receptors (15) that consist of three distinct isoforms, ␣, ␤/␦, and ␥, with different ligand specificities and tissue distribution. PPAR␣ is primarily expressed in brown adipose tissue and the liver and, to a lesser extent, in kidneys, skeletal muscle, and heart (16), whereas PPAR␥ is abundant in adipose tissue functioning as the key transcription factor of adipocyte differentiation (17,18). PPAR␣ null (19), PPAR␤/␦ null (20,21), and conditional PPAR␥ null mice (22)(23)(24)(25)(26)(27) have been generated, and studies with these mice have demonstrated that PPAR␣ controls the expression of genes involved in mitochondrial and peroxisomal ␤-oxidation, fatty acid uptake, or binding and lipoprotein assembly and transport while PPAR␤/␦ controls genes of fatty acid oxidation in muscle and brown adipose tissue (28,29). Using adipose tissue-specific PPAR␦-transgenic mice, it was demonstrated that PPAR␦ activation in adipose tissue leads to enhanced fatty acid oxidation, improved lipid profiles, reduced adiposity, and resistance to both diet-induced obesity and genetically predisposed obesity (28,29). Additionally, the disruption of PPAR␥ in the liver has been shown to improve the fatty liver phenotype of leptin-null mice, suggesting that PPAR␥ can also regulate lipid metabolism in the liver in addition to its known role in adipogenesis (26). One of the physiological stimuli of PPAR␣ is the fasting state, and it is hypothesized that the free fatty acids that are delivered from adipose tissue to the liver act as ligands and activate the receptor (30). The fatty acids are also oxidized in conjunction with increased synthesis of ketone bodies. Several in vivo studies have also shown that synthetic PPAR␣ ligands such as fibrates markedly induce the transcription of hepatic ␤-oxidation genes and BAT-thermogenic genes including UCP1, UCP2, and UCP3, (31)(32)(33)(34)(35)(36). The increased expression of thermogenic genes is negatively correlated with obesity in rodents (37)(38)(39).
Despite the great expansion of our understanding of the function of PPAR␣, it remains unclear when and how the PPAR signaling pathways are triggered in an intact organism and how they specifically affect lipid and carbohydrate metabolism. Recently, we presented evidence that SCD1 deficiency protects mice against diet and leptin deficiency-induced adiposity, reduces hepatic triglyceride concentration, and induces the expression of PPAR␣ target genes in the liver. We hypothesized that the elimination of the SCD1 gene in mice generates a signal (e.g. a fatty acid or fatty acid derivative) that activates the PPAR␣ pathway, because fatty acids are known to activate PPARs (40 -42). To test this possibility, we generated mice that are deficient in both SCD1 and PPAR␣ (SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ ). These mice would be expected to show reduced expression of PPAR␣ target genes and reduced energy expenditure as well as accumulation of hepatic triglyceride. We report here that the expression of PPAR␣ target genes in the liver and brown adipose tissue remain elevated in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. The reduced adiposity and increased energy expenditure due to SCD1 deficiency were not ameliorated. The SCD deficiency rescued hepatic steatosis of the PPAR␣ Ϫ/Ϫ mice. The rate of fatty acid oxidation and activity of CPT-1 as well as the phosphorylation of AMPK and ACC were higher in the SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ mice relative to PPAR␣ null controls. The levels of malonyl-CoA were lower in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. Taken together, these data demonstrate that the increased fatty acid ␤-oxidation and reduced adiposity due to SCD1 deficiency are independent of the PPAR␣ pathway.

EXPERIMENTAL PROCEDURES
The generation of the SCD1 Ϫ/Ϫ mice in 129S6/SvEv background and PPAR␣ Ϫ/Ϫ mice in 129/Sv Jae was described previously (19,43,44). To obtain PPAR␣ Ϫ/Ϫ on a background of SCD1 deficiency, SCD1 Ϫ/Ϫ mice were crossed with PPAR␣ Ϫ/Ϫ mice and the F1 progeny of these matings (SCD1 ϩ/Ϫ /PPAR␣ ϩ/Ϫ ) were then crossed to obtain mice that had both mutations of PPAR␣ and SCD alleles (SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ ). Animals were housed individually in a pathogen-free facility operating at room temperature in 12-h light/dark cycles. All of the animals were allowed free access to water and were fed ad libitum except during food restriction procedures. Animals were fed a high fat diet (21 w/w % fat, Harlan Takelad Madison, WI) at 8 weeks of age. Food consumption was monitored daily, and body weight was recorded every week. Animals were sacrificed when 16-weeks-old. All of the studies were approved by the animal care research committee of the University of Wisconsin-Madison.

Preparation of Mitochondria and Measurement of Mitochondrial
Fatty Acid Oxidation-Mitochondria were isolated essentially as described by Vance (46). The mixture used to measure mitochondrial fatty acid oxidation contained in a final volume of 2.5 ml: 2 ml of modified Krebs-Henseleit buffer, pH 7.4, 0.2 mM [ 14 C]palmitoyl-CoA bound to 7.2 mg/ml bovine serum albumin, 4 mM ATP, 0.5 mM L-carnitine, 0.05 mM coenzyme A, 2 mM dithiothreitol, and 0.5 ml of suspended mitochondria (5 mg of protein/ml). Palmitoyl-CoA oxidation was determined in the presence and absence of 2 mM KCN, and the cyanide-sensitive part of the oxidation was taken as mitochondrial oxidation (47). Labeled CO 2 was trapped in 10 M KOH. An aliquot of the neutralized extracts was analyzed for the [ 14 C] content of the ketone bodies (48), and another aliquot was adjusted to pH 4 with 3 M acetate buffer and extracted twice with petroleum ether to remove traces of [ 14 C]palmitoyl-CoA and the aqueous phase was then counted (acid-soluble labeled oxidation products) (48).
Blood and Tissue Sampling-Mice were fasted for 16 h and sacrificed by CO 2 asphyxiation and/or cervical dislocation. Blood was collected aseptically by direct cardiac puncture and centrifuged (13,000 ϫ g, 5 min, 4°C) to collect plasma. Plasma cholesterol and triglyceride levels were measured by using commercial kits (Roche Applied Science). Plasma free fatty acids and liver triglycerides were measured by gasliquid chromatography as previously described (51) Plasma glucose concentrations were measured by using commercial kits (Sigma). Liver malonyl-CoA levels were measured as described previously (52). Retroperitoneal, reproductive, mesenteric, and subcutaneous fat pads were used to determine total fat pad content. Histology was performed on livers, and white adipose tissue was fixed in 10% formalin and stained with hematoxylin and eosin.

SCD1 Mutation Reduced Hepatic and Plasma Triglyceride in
SCD1 Mutation Rescues Liver Steatosis of PPAR␣ Ϫ/Ϫ Mice-Histological liver sections of male PPAR␣ Ϫ/Ϫ mice fed a high fat diet showed large lipid-filled vacuoles, whereas those of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice showed little or no vacuoles and were similar to those of SCD1 Ϫ/Ϫ mice (Fig. 4A). The liver steatosis of the female PPAR␣ Ϫ/Ϫ mice was reduced in the SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ (Fig. 4B). Fig. 4C shows that the liver triglyceride content was 50% higher in the male PPAR␣ Ϫ/Ϫ mice relative to the wild-type mice upon feeding a high fat diet but reduced by greater than 80% in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. The triglyceride content was reduced by 65% in the female SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ relative to the PPAR␣ Ϫ/Ϫ mice. BAT sections of male PPAR␣ Ϫ/Ϫ mice fed the high fat diet showed large lipidfilled vacuoles, whereas those of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice showed small vacuoles and were similar to those of SCD1 Ϫ/Ϫ mice (Fig. 4D). Fig. 4E shows that BAT triglyceride content was reduced by greater than 60% in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ relative to the PPAR␣ Ϫ/Ϫ mice. These observations suggest that the protection against liver steatosis and reduction in triglyceride content in BAT caused by SCD1 deficiency is independent of PPAR␣ activation.

SCD1 Mutation Increases Expression of PPAR␣ Target Genes in Liver and BAT of PPAR␣
Ϫ/Ϫ Mice-To investigate whether the SCD1 mutation increased the expression of PPAR␣ target genes in PPAR␣ Ϫ/Ϫ mice, we measured the mRNA levels of AOX, VLCAD, and liver CPT in the livers and BAT and also BAT-thermogenic genes (UCP-1, UCP2, and UCP3) of wildtype, SCD1 Ϫ/Ϫ , PPAR␣ Ϫ/Ϫ , and SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. Fig.  5A shows that the mRNA levels for AOX, liver CPT, and VLCAD were still elevated in the livers of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ , and Fig. 5B shows that the mRNA levels for AOX, muscle CPT, UCP1, UCP2, and UCP3 remained higher in the BAT of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ relative to the PPAR␣ Ϫ/Ϫ mice. The expression of the PPAR␣ mRNA in liver was not altered between the wild-type and SCD1 Ϫ/Ϫ mice. These results suggest that the increased expression of PPAR␣ target genes due to SCD1 deficiency occurs through a PPAR␣-independent process. SCD1 Mutation Increases Fatty Acid Oxidation in PPAR␣ Ϫ/Ϫ Mice-To determine whether the SCD1 mutation increases the rate of fatty acid ␤-oxidation in PPAR␣ Ϫ/Ϫ mice, we assayed the oxidation of [ 14 -C]palmitoyl-CoA in liver mitochondria isolated from PPAR␣ Ϫ/Ϫ and SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. The oxidation of [ 14 -C]palmitoyl-CoA was 54% higher (n ϭ 5 mice/ group, p Ͻ 0.05) in the liver (Fig. 6A) of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice compared with PPAR␣ Ϫ/Ϫ mice. Oxidation of [ 14 -C]palmitoyl-CoA was also increased in the BAT of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice relative to PPAR␣ Ϫ/Ϫ mice (data not shown). CPT-1 is the enzyme that is responsible for the transport of activated fatty acid into mitochondria for ␤-oxidation, and its activity would be expected to increase during increased fatty acid oxidation. Fig. 6B shows that the CPT-1 protein level and enzyme activity are 35 and 60% higher, respec-tively, in the liver of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice than in the liver of PPAR␣ Ϫ/Ϫ mice (n ϭ 6 mice/group, p Ͻ 0.001). The increase in CPT-1 protein level is consistent with the increase in CPT-1 mRNA shown in Fig. 5A.
The increase in CPT-1 activity could be attributed to a decrease in malonyl-CoA, the substrate for lipogenesis, and inhibitor of fatty acid oxidation. The content of malonyl-CoA is 35% less (n ϭ 6 mice/group, p Ͻ 0.001) in the SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ than in the PPAR␣ Ϫ/Ϫ mice (Fig. 6C). Malonyl-CoA levels were also reduced in the BAT of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice (data not shown). Because malonyl-CoA production can be blocked by serine 79 phosphorylation of ACC (50, 54 -57), we measured phospho-ACC (␣ plus ␤) in the livers of PPAR␣ Ϫ/Ϫ and SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. The level of Ser-79 phosphorylation of ACC was 75% higher in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice relative to the PPAR␣ Ϫ/Ϫ mice (Fig. 6D), whereas the total amount of ACC1 and ACC2 protein was decreased by 60 and 50%, respectively, in SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. Because ACC Ser-79 phosphorylation is catalyzed by AMPK, which is activated by phosphorylation (56 -59), we also measured phospho-AMPK. The amount of phosphorylated AMPK was 50% higher in the liver of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice relative to the PPAR␣ Ϫ/Ϫ mice, whereas the total amounts of ␣ 1 -AMPK and ␣ 2 -AMPK were not significantly changed. These results confirm that the increased fatty acid oxidation observed in the liver of SCD1 Ϫ/Ϫ -deficient mice is independent of PPAR␣ pathway and involves other mechanisms including the activation of AMPK (50). DISCUSSION We showed previously that mice with a deficiency of SCD1 are protected against increased adiposity, have increased energy expenditure, and show up-regulation of PPAR␣ target genes in the liver. Liver and plasma triglycerides were decreased, and the genes of lipogenesis were down-regulated. These observations led to the hypothesis that one of the consequences of SCD1 deficiency is to partition fat toward increased oxidation while decreasing lipogenesis. PPAR␣ is the predominant PPAR subtype expressed in mouse liver and BAT, and it plays a central role in the transcriptional activation of peroxi- somal, mitochondrial, and microsomal fatty acid oxidation genes in these tissues (15,16). To test the hypothesis that SCD1 deficiency activates the PPAR␣ pathway, we generated SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice and studied them with respect to adiposity, energy expenditure, and expression of PPAR␣ target genes in the liver and BAT tissue. The study shows that SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice are still protected against adiposity, show increased energy expenditure, have reduced liver steatosis, and show increased fatty acid oxidation. The expression of ␤-oxidation genes in the liver and BAT remains elevated. Taken together, these results suggest that the phenotypes generated by SCD1 deficiency are independent of the PPAR␣ pathway.
The mechanism of increased expression of PPAR␣ target genes in the liver and BAT of SCD1 Ϫ/Ϫ mice is still unknown. However, as recently reported by Kertsen et al. (30), the results in BAT are consistent with the PPAR␣-independent mechanism of the induction of the UCP-1 gene expression upon fasting. In BAT and under fasting conditions, other transcription factors such as PPAR␦, PPAR␥, and thyroid hormone receptor have been shown to control the expression of UCP1 (60). These transcription factors are co-activated by binding with a coldinducible co-activator PPAR␥ coactivator 1␣, which is induced through the ␤ 3 -adrenergic receptor signaling (60). We previously found that SCD1 Ϫ/Ϫ mice exhibited increased expression of the ␤ 3 -adrenergic receptor isoform without alteration in the expression of the ␤ 1 and ␤ 2 isoforms. The expression of PPAR␥ coactivator 1␣ levels is increased in BAT (61) and in the liver. 2 Therefore, the increased expression of UCPs and fatty acid oxidation genes in BAT of SCD1 Ϫ/Ϫ may be due to activation of either PPAR␥ or thyroid hormone receptor with the binding of PPAR␥ coactivator 1␣.
The muscle CPT-1 and UCP-3 mRNA levels are higher in the muscle of SCD1 Ϫ/Ϫ and SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice (data not shown). The major PPAR isoform in muscle is ␦/␤. Thus the increased fatty acid oxidation caused by SCD1 deficiency may not be confined to the liver and BAT but may be global and regulated by different PPAR isoforms expressed in different tissues. The SCD1 deficiency could be generating ligands for other PPAR isoforms and not PPAR␣ as previously proposed. PPAR␦-transgenic mice and mice treated with PPAR␦-specific agonists like SCD1 Ϫ/Ϫ mice are protected from high fat dietinduced adiposity and insulin resistance because of the induc-  , n ϭ 6). B, food intake of male mice fed a high fat diet (n ϭ 6). C, the weight of reproductive fat pad from mice fed a high fat diet for 8 weeks (n ϭ 4, female mice; n ϭ 6, male mice). D, white adipose tissue sections from male mice fed a high fat diet. Magnification, ϫ100. *, p Ͻ 0.05 versus wildtype (WT) mice; #, p Ͻ 0.05 versus PPAR␣ Ϫ/Ϫ mice. wks, weeks.
tion of genes of fatty acid oxidation and thermogenesis in the muscle and BAT (28,29). Also, similar to SCD1 Ϫ/Ϫ mice, PPAR␥-deficient mice and mice administrated with PPAR␥ antagonists ameliorate the development of adiposity and insulin resistance induced by high fat diet feeding (27,62). The studies to ascertain the involvement of other PPAR isoforms in SCD1 deficiency-mediated increase in fatty acid oxidation, reduced adiposity, and increased energy expenditure will require the crossing of the SCD1 Ϫ/Ϫ mice with PPAR␤/␦ Ϫ/Ϫ or PPAR␥ Ϫ/Ϫ mice, and these studies are currently underway.
We hypothesized previously that the reduction in hepatic triglycerides in SCD1 Ϫ/Ϫ mice was due to increased PPAR␣mediated oxidation of the fatty acids. We found in this study that the hepatic triglyceride levels were lower and that fatty acid oxidation was higher in SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ relative to the PPAR␣ Ϫ/Ϫ mice. Moreover, male PPAR␣ Ϫ/Ϫ mice exhibited a severe liver steatosis after high fat feeding, whereas little fat deposition was observed in the liver of SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ mice. These observations suggested that the reduction of hepatic triglycerides and increased fatty acid oxidation by SCD1 deficiency were independent of PPAR␣ activation. Therefore, several alternative mechanisms for increased fatty acid oxidation by SCD1 deficiency are considered. First, although the PPAR␣ target genes were still elevated in the SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ mice, lipogenesis was still down-regulated. The protein levels of ACC1 and ACC2 in the liver of SCD1 Ϫ/Ϫ / PPAR␣ Ϫ/Ϫ mice were lower than those in PPAR␣ Ϫ/Ϫ mice (Fig.  6D), consistent with a decrease in ACC gene expression. The reduction in fatty acid synthesis and AMPK-mediated inactivation of acetyl-CoA carboxylase (Fig. 6D) can concomitantly enhance fatty acid oxidation by reducing malonyl-CoA levels and thereby removing the inhibition on CPT-1. However, the mechanisms of inhibition of lipogenesis and activation of AMPK by SCD1 deficiency are presently unknown. In summary, it has long been known that PPAR␣ controls the expression of a number of genes involved in mitochondrial and perixosomal ␤-oxidation and plays an important role in maintaining energy homeostasis. The results presented here support the hypothesis that there are other mechanisms induced by SCD1 deficiency that lead to activation of genes of fatty acid oxidation independent of PPAR␣ activation. Also, as we showed earlier (50) and in the results shown here, posttranscriptional mechanisms such as the activation of AMPK induced by SCD1 deficiency can be partly responsible for the increased fatty acid oxidation in the SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ double knock-out mice. Overall, the studies show that the SCD1 deficiency overrides the PPAR␣ deficiency and continues to protect mice against adiposity through increased thermogenesis and increased fatty acid oxidation. A further understanding of the regulation of SCD and the development of inhibitors for its activity will be important steps in the treatment of obesity and its associated risk factors.  6. A, palmitoyl-CoA oxidation in liver mitochondria from PPAR␣ Ϫ/Ϫ and SCD1 Ϫ/Ϫ /PPAR␣ Ϫ/Ϫ male mice fed a chow diet (n ϭ 6 each). B, CPT-1 protein level and activity in the liver (n ϭ 6). C, malonyl-CoA level in the liver (n ϭ 6). D, phospho-ACC, ACC1, and ACC2 protein levels in liver, phospho-AMPK, and ␣ 1 and ␣ 2 AMPK protein levels in the liver. *, p Ͼ 0.01 versus PPAR␣ Ϫ1Ϫ mice.