Stearoyl-CoA desaturase (SCD)1 catalyzes the rate-limiting reaction of monounsaturated fatty acid (MUFA) synthesis and plays an important role in the development of obesity. SCD1 is suppressed by leptin but induced by insulin. We have used animal models to dissect the effects of these hormones on SCD1. In the first model, leptin-deficient ob/ob mice were treated with either leptin alone or with both leptin and insulin to prevent the leptin-mediated fall in insulin. In the second model, mice with a liver-specific knockout of the insulin receptor (LIRKO) and their littermate controls (LOXs) were treated with leptin. As expected, leptin decreased SCD1 transcript, protein, and activity by >60% in ob/ob and LOX mice. However, the effects of leptin were not diminished by the continued presence of hyperinsulinemia in ob/ob mice treated with both leptin and insulin or the absence of insulin signaling in LIRKO mice. Furthermore, genetic knockout of sterol regulatory element–binding protein (SREBP)-1c, the lipogenic transcription factor that mediates the effects of insulin on SCD1, also had no effect on the ability of leptin to decrease either SCD1 transcript or activity. Thus, the effect of leptin on SCD1 in liver is independent of insulin and SREBP-1c, and leptin, rather than insulin, is the major regulator of hepatic MUFA synthesis in obesity-linked diabetes.
Stearoyl-CoA desaturases (SCDs) catalyze the rate-determining reaction in the synthesis of monounsaturated fatty acids (MUFAs), the introduction of a double bond in the Δ-9 position of acyl-CoAs (rev. in 1). There are four isoforms of SCD in the mouse, which are differentially regulated and vary in tissue distribution (2). SCD1 is the predominant isoform in liver.
SCD1 appears to be a major regulator of energy metabolism. Mouse models of obesity (leptin-deficient ob/ob mice), lipoatrophy (transgenic aP2-nSREBP-1c mice), and high-fat feeding have extremely high levels of SCD1, correlating with hepatic steatosis (3,4a). Mice deficient in SCD1, on the other hand, are lean, resistant to diet-induced obesity, and insulin sensitive (5).
Given its effects on energy metabolism, it would seem that SCD1 could be an important therapeutic target for the treatment of obesity, hepatic steatosis, or insulin resistance. In fact, ob/ob mice made deficient in SCD1 have decreased obesity, increased insulin sensitivity and energy expenditure, and reversal of their hepatic steatosis (3). Lipoatrophic mice made deficient in SCD1 also have dramatic improvement of their hepatic steatosis and hyperinsulinemia (4).
The mechanism by which SCD exerts its effects on energy metabolism is still not entirely understood. However, recent studies suggest several possibilities (rev. in 1). First, SCD1 activity could decrease lipid oxidation since MUFAs, unlike saturated fatty acids (SFAs), cannot inhibit acetyl-CoA carboxylase (ACC)-1. Disinhibition of ACC activity would lead to an accumulation of malonyl-CoA and repression of carnitine palmitoyl transferase-1, thereby preventing fatty acid oxidation in the mitochondria. Second, by altering the ratio of MUFAs to SFAs, SCD could regulate membrane fluidity; such changes have been linked to obesity, diabetes, and cardiovascular disease. Third, SCD1 might alter expression of the genes of lipid metabolism. SCD1-deficient mice have decreased transcription of sterol regulatory element–binding protein (SREBP)-1c and lipogenic genes but increased transcription of lipid oxidation genes (5). Finally, since SCD1 knockout mice are deficient in cholesteryl esters and triglycerides, even when their diets are supplemented by high levels of MUFA, it appears that decreasing SCD1 activity could decrease lipid accumulation by limiting the supply of endogenously synthesized MUFA (6).
SCD1 is subject to regulation at the levels of transcription, mRNA stability, and enzyme activity. Nutrients such as glucose, fructose, and cholesterol increase SCD expression, whereas polyunsaturated fatty acids decrease it (1). Hormones are also important regulators of SCD. It was discovered >20 years ago that insulin induces SCD activity in rats made diabetic with streptozotocin (7). This induction is due in part to activation of SCD1 transcription by insulin, which occurs in both animal and tissue culture models (8,9).
The effect of insulin on SCD1 appears to be largely mediated by SREBP-1c (10). SREBP-1c is a transcription factor positively regulated by insulin at the transcriptional level. It is upregulated in mouse models with hyperinsulinemia, like leptin deficiency and lipoatrophy, and downregulated in models with insulin deficiency, such as fasting and streptozotocin-induced diabetes (11–14). Mice expressing a constitutively active form of SREBP-1c have increased expression of SCD1 and increased synthesis of MUFAs, while knockout of SREBP-1c leads to a decrease in SCD1 expression (10,15).
Transcriptional profiling of livers of ob/ob mice has revealed that leptin also regulates SCD1 expression (3,16). Furthermore, the fact that making ob/ob mice deficient in SCD1 reverses much of the leptin-deficient phenotype suggests that SCD1 may be a pivotal mediator of leptin action (3).
While both insulin and leptin regulate SCD1, they also interact in a complex manner with one another. Insulin increases leptin secretion from adipocytes (17). Leptin, on the other hand, increases insulin sensitivity but decreases insulin secretion from pancreatic β-cells, leading to a fall in serum insulin levels (18). Cross talk between insulin and leptin signaling pathways has also been described (19,20). Thus, it is possible that leptin decreases SCD1 expression by decreasing serum insulin levels or by altering insulin signaling.
We use three in vivo model systems to better understand the roles of leptin and insulin in the regulation of SCD1. First, leptin-deficient ob/ob mice were treated with either leptin alone or both leptin and insulin, preventing the leptin-mediated fall in insulin. This allows us to compare the effects of leptin in the absence or presence of continued hyperinsulinemia. Second, liver insulin receptor knockout (LIRKO) mice, which completely lack hepatic insulin signaling, and their littermate controls (LOXs) were treated with leptin to determine the effects of leptin on SCD1 in the absence or presence of insulin signaling. Finally, SREBP-1c knockout mice and their control subjects were treated with leptin to assess the role of SREBP-1c in mediating SCD1 suppression by leptin.
RESEARCH DESIGN AND METHODS
Leptin treatment of ob/ob mice.
Male ob/ob mice (aged 6 weeks) and their lean ob control littermates were purchased from The Jackson Laboratories and acclimated for 2 weeks before study. Mice were implanted with osmotic pumps (1007D; Alzet, Cupertino, CA) containing either PBS or recombinant mouse leptin (24 μg/day; National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) for 4 days. Insulin-treated mice also received four subcutaneous bovine insulin pellets (Linbit; Linshin, Toronto, ON, Canada), a dose found to raise insulin levels in leptin-treated ob/ob mice to those found in untreated ob/ob mice. Fasting insulin and glucose levels were obtained in a cohort of ob/ob mice by subjecting them to a 6-h fast 2 days after initiating treatment with leptin or insulin.
Leptin treatment of LIRKO and SREBP-1c−/− mice.
Male LOX (cre−;IRlox/lox) and LIRKO (cre+;IRlox/lox) littermates (aged 6 weeks), generated as described previously (21), were treated with PBS or 30 μg/day leptin, as above. SREBP-1c−/− (15) mice were obtained from The Jackson Laboratories and backcrossed onto the 129SvEV background for six generations. Male knockout or wild-type control mice (aged 12 weeks) were implanted with pumps containing either PBS or leptin (24 μg/day; Amgen, Thousand Oaks, CA). After 4 days of treatment, mice were killed in the random-fed state and serum and liver samples were collected.
Assays.
Serum glucose was measured using a glucometer; insulin levels were measured using a radioimmunoassay (ob/ob experiment; Linco) or enzyme-linked immunosorbent assays (LIRKO and SREBP-1c knockout experiments; Crystal Chem); leptin levels were measured using an enzyme-linked immunosorbent assay (Crystal Chem).
Real-time PCR.
Total RNA was extracted and purified using the RNeasy kit (Qiagen) and used to direct cDNA synthesis using an RT-PCR kit (Clontech). RT-PCR was performed using SYBR green master mix (ABI) and 300 nmol/l of the relevant primers. Isoform-specific primers for SREBP-1c (22) have been described previously. SCD1 primers were 5′-CATCATTCTCATGGTCCTGCT-3′ and 5′-CCCAGTCGTACACGTCATTTT-3′. Expression was calculated as a function of 2-Ct and normalized to TATA-binding protein expression (5′-ACCCTTCACCAATGACTCCTATG-3′ and 5′-TGACTGCAGCAAATCGCTTGG-3′).
SCD enzymatic activity and immunoblotting.
Conversion of [1-14C]stearoyl-CoA to [1-14C]oleate was used to measure SCD enzyme activity from liver microsomes prepared from individual mice (3). These microsomes were also subjected to immunoblotting with antibodies against SCD that have been previously described (23). In the ob/ob experiment, SCD antibody was used at a 1:5,000 dilution; in the LIRKO experiment, it was used at a 1:500 dilution. Immunoblotting was performed per the Amersham ECL detection system kit protocol.
Hepatic lipid analysis.
Hepatic lipid analysis was performed by the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers. Lipids were extracted from liver (24). Phospholipids, triglycerides, and cholesteryl esters were scraped from the plates and methylated using BF3/methanol, as described by Morrison and Smith (25). The methylated fatty acids were extracted and analyzed by gas chromatography. All gas chromatographic analyses were carried out on an HP 5890 gas chromatograph equipped with flame ionization detectors and an HP 3365 Chemstation. Fatty acid methyl esters were identified by the computer by comparing the retention times to known standards. Inclusion of odd-chain fatty acids as standards permitted the quantitation of the amount of lipid in the sample.
Statistical analysis.
Statistical analysis of the data were performed using a two-tailed unpaired t test with unequal variance. Data are presented as the means ± SE unless otherwise indicated.
RESULTS
Leptin treatment of ob/ob mice
Characterization of leptin-treated ob/ob mice.
Leptin has been shown to suppress SCD1 expression in ob/ob mice (3). However, leptin treatment also decreases serum insulin levels, raising the possibility that the effects of leptin on SCD1 are secondary to the decrease in serum insulin. To test this hypothesis, we treated ob/ob mice subcutaneously through an osmotic pump for 4 days with 24 μg/day recombinant leptin (Leptin), implantation of insulin pellets (Insulin), or both (Leptin + Insulin) to maintain hyperinsulinemia in the face of leptin treatment. Untreated ob/ob mice (−) and mice treated only with insulin were implanted with vehicle-containing pumps; lean control mice (Control, ob/+ or +/+) were included for comparison.
Table 1 shows that aside from raising insulin levels approximately twofold, insulin treatment alone did not have much effect. In this experiment, insulin-treated mice ate slightly less than untreated ob/ob mice, but this was not a reproducible finding. Leptin treatment, on the other hand, caused dramatic weight loss (∼6 vs. ∼0 g in untreated mice) and decreased food intake by almost 90% compared with untreated ob/ob mice. Treatment with leptin also led to complete resolution of hyperinsulinemia with a decrease in serum insulin levels from 7.7 to 1.1 ng/ml, the same level found in lean controls.
When mice were treated with insulin, in addition to leptin, they remained hyperinsulinemic with fasting insulin levels similar to those found in untreated ob/ob mice (7.4 vs. 7.7 ng/ml in untreated mice). Interestingly, the same dose of insulin that produced a serum insulin level of 17.6 ng/ml in the absence of leptin produced a serum insulin level of only 7.4 ng/ml in the presence of leptin. Similarly, leptin levels were more than threefold lower in mice treated with both leptin and insulin than in mice treated with leptin alone. In both experiments, insulin and leptin were derived largely, or completely, from an exogenous source at a fixed rate. Thus, insulin appeared to stimulate the clearance of leptin, and, conversely, leptin appeared to stimulate the clearance of insulin. The basis of these changes in clearance is not known but is currently under investigation. As a correlate to these hormone levels, ob/ob mice treated with insulin, in addition to leptin, appeared to lose less weight and eat more than mice treated with leptin alone, although they had a more dramatic improvement in blood glucose control.
SCD1 mRNA, protein, and activity in leptin-treated ob/ob mice.
Using real-time RT-PCR, we measured SCD1 transcript in the livers of these mice (Fig. 1A). We found that leptin treatment of ob/ob mice suppressed SCD1 mRNA, and the degree of suppression was the same in either the presence or absence of exogenous insulin treatment. SCD protein paralleled mRNA levels and was undetectable in leptin-treated mice, even when they were simultaneously treated with insulin (Fig. 1C). SCD activity, which was measured as conversion of [1-14C]stearoyl-CoA to [1-14C]oleoyl-CoA by liver microsomes, also paralleled mRNA levels with a similar 10-fold suppression by leptin in the absence or presence of continued hyperinsulinemia (Fig. 1B). Insulin treatment, on the other hand, did not significantly alter SCD1 transcript or activity by itself, or in conjunction with, leptin treatment.
Changes in fatty acid composition in leptin-treated ob/ob mice.
To determine whether the changes in SCD activity were reflected in the proportion of MUFAs and SFAs, hepatic lipids were extracted and the relative contribution of the major fatty acids was quantitated using gas chromatography. The ratio of palmitoleate to palmitate (16:1 to 16:0) and oleate to stearate (18:1 to 18:0) could then be calculated for each of the major lipid fractions (Fig. 2).
Treatment with insulin alone had no significant effect on the ratio of MUFAs to SFAs in the phospholipid and triglyceride fractions. Leptin, however, decreased the ratios of palmitoleate to palmitate and oleate to stearate by 30–50% in the phospholipid and triglyceride fractions with more variable effects in the cholesteryl ester fraction. Additional treatment with insulin did not diminish this effect. While leptin decreased the ratio of MUFAs to SFAs, the degree to which it restored these ratios to the normal values seen in the lean controls varied among the lipid fractions. After leptin treatment, the ratio of MUFAs to SFAs dropped below normal in the phospholipid fraction but remained high in the triglyceride and cholesteryl ester fractions.
Leptin treatment of LIRKO mice
Characterization of leptin-treated LIRKO mice.
LIRKO mice lack hepatic insulin signaling entirely. This leads to unsuppressed hepatic gluconeogenesis, elevated serum insulin levels (due to both an increase in insulin secretion and a decrease in insulin clearance), and mild hyperglycemia (21). Surprisingly, they are also hyperleptinemic, with a dramatic increase in leptin-binding protein (soluble receptor) in the serum (S.E. Cohen, E. Kokkotou, S.B.B., T. Kondo, J. Kratzsch, C.S. Mantzoros, C.R.K., unpublished observations). Thus, untreated LIRKO mice had slightly higher blood glucose values, almost 15-fold higher insulin levels, and 5-fold higher leptin levels (Table 2). Despite their hyperleptinemia, LIRKO mice responded in a manner similar to LOX mice when treated with 30 μg/day leptin through an osmotic subcutaneous pump. LOX and LIRKO mice had a similar decrease in weight gain, a 40% decrease in food intake, a 50% decrease in nonfasted insulin levels, and a 7- to 10-fold increase in serum leptin levels. However, the leptin levels achieved in the LIRKO mice were almost four times higher than those achieved in LOX mice treated with the same dose of leptin, consistent with a change in leptin clearance in the LIRKO mouse.
SCD1 transcript, protein, and activity in leptin-treated LIRKO mice.
Real-time RT-PCR analysis showed that in the absence of leptin treatment, LIRKO mice had an ∼80% reduction in the level of SCD1 expression compared with LOX mice (Fig. 3A). Nonetheless, leptin treatment decreased SCD1 mRNA by 60% in both LIRKO and LOX mice. This decrease was reflected in SCD protein levels in LOX mice. In LIRKO mice, SCD protein levels were below the threshold of detection, even in the absence of leptin, precluding us from determining whether leptin treatment of these mice resulted in further suppression (Fig. 3C). SCD activity in liver microsomes from LIRKO mice paralleled SCD1 transcript levels and was only 10% of that observed in LOX mice (Fig. 3B). As with SCD1 mRNA, both LOX and LIRKO mice had a 70–80% suppression of SCD activity by leptin. Thus, leptin can effectively suppress SCD transcript and activity in the liver, even in the complete absence of insulin signaling.
Changes in fatty acid composition in leptin-treated LIRKO mice.
Since leptin treatment decreased SCD transcript and activity in LOX and LIRKO mice, we predicted that it would also decrease the ratio of MUFAs to SFAs in the liver. In LOX mice, leptin decreased the ratios of palmitoleate to palmitate (16:0 to 16:1) by 50–75% and oleate to stearate (18:1 to 18:0) by 30% in the phospholipid and triglyceride fractions (Fig. 4). In contrast, despite lower levels of SCD activity, untreated LIRKO mice did not have significantly lower ratios of MUFAs to SFAs when compared with LOX mice. Moreover, leptin treatment of LIRKO mice had no effect on the ratio of MUFA to SFA in any of the lipid fractions. Thus, insulin signaling, in addition to leptin, may be required to alter the composition of hepatic lipids.
Expression of SREBP-1c mRNA in leptin-treated ob/ob and LIRKO mice.
The SREBPs are a family of nuclear transcription factors that are known to regulate lipid and cholesterol synthesis (rev. in 26). SREBP-1c normally regulates SCD1 and is therefore important in the synthesis of MUFAs (10). SREBP-1c is upregulated in ob/ob mice, lipoatrophic mice, and insulin receptor substrate (IRS)-2 knockout mice and is decreased by leptin treatment in each model (12,27). In the ob/ob liver, leptin effectively suppressed SREBP-1c even in the presence of insulin treatment (Fig. 5A). In contrast, leptin did not change SREBP-1c in LOX mice and increased SREBP-1c in LIRKO mice (Fig. 5B).
Leptin treatment of SREBP-1c knockout mice.
To further assess the role of SREBP-1c in leptin-mediated suppression of SCD1, we infused leptin into SREBP-1c knockout mice. We found that leptin decreased body weight and food intake to the same extent in SREBP-1c knockout mice as in wild-type mice (online appendix [available at http://diabetes.diabetesjournals.org]). Leptin decreased SCD1 transcript by 40% and SCD1 activity by 65% in the knockout mice, which was comparable to the changes seen in wild-type mice (Fig. 6A and B). Leptin did not alter SREBP-1c or SREBP-1a mRNA in either wild-type or knockout mice (data not shown). Therefore, leptin can reduce SCD1 expression and activity even in absence of the transcription factor SREBP-1c.
DISCUSSION
The fact that knockout of SCD1 in mice causes increased energy expenditure, increased insulin sensitivity, and resistance to diet-induced obesity suggests that SCD1 may be a global regulator of energy metabolism (5). Since leptin suppresses SCD1 transcript and activity, it has been proposed that SCD1 may mediate many of leptin’s effects on energy metabolism (3). However, leptin decreases insulin secretion (18) and may also interfere with insulin action (20), raising the possibility that some of the effects of leptin on SCD1 may be secondary to changes in insulin levels or insulin action. In the current study, we used three different genetic models in which individual components of the system had been deleted, showing that leptin suppresses SCD1 through an insulin-independent pathway that does not require SREBP-1c.
In one experimental paradigm, we treated leptin-deficient ob/ob mice with leptin alone or leptin and insulin. We found that leptin suppresses SCD1 transcript, SCD protein, SCD activity, and the ratio of MUFAs to SFAs in ob/ob mice. The presence of continued hyperinsulinemia using exogenous insulin does not impair these effects. Thus, leptin suppression of SCD1 does not require a fall in insulin levels.
In the second experiment, we treated LOX and LIRKO mice with leptin to assess the effects of leptin in the complete absence of insulin signaling. We found that leptin suppresses SCD1 transcript, protein, and activity to similar levels in the presence or absence of insulin signaling. Interestingly, however, the effect of leptin on hepatic fatty acid composition appeared to require insulin signaling, as leptin decreased the ratio of MUFAs to SFAs in the LOX mice but not in the LIRKO mice, which lack hepatic insulin action. Although the exact role of insulin in determining the fatty acid composition of the liver is not known, insulin may act by altering fatty acid uptake, synthesis, and/or clearance.
In contrast to ob/ob mice, leptin did not decrease SREBP-1c mRNA in either LIRKO or LOX mice, suggesting that leptin may act through an SREBP-1c–independent pathway to suppress SCD1. This is confirmed by the finding that leptin treatment decreases SCD1 transcript and activity to a similar extent in both SREBP-1c knockout and wild-type mice. Thus, leptin does not require SREBP-1c to suppress SCD1.
In these experiments, the role of insulin in regulating SCD1 is clearly subordinate to that of leptin. However, it is possible that in states of low or absent leptin, insulin becomes an important regulator of SCD1 transcription. Thus, in ob/ob mice, hyperinsulinemia in the absence of leptin drives high expression of SCD1. Reducing serum insulin in these mice, by pair feeding, for example, decreases SCD1 levels (3). Similarly, streptozotocin-induced diabetic animals, which have low levels of leptin, also have very low levels of SCD1 that are restored with insulin treatment (8).
Both insulin and leptin, in addition to other factors such as glucose, alter the expression of SREBP-1c (10,28). Insulin activates the liver X receptor (LXR), which interacts with LXR binding sites in the SREBP-1c promoter and increases expression of SREBP-1c (29), which in turn upregulates transcription of SCD1 (30). Therefore, in the refed state, the induction of SCD1 expression by insulin is abolished by knockout of SREBP-1c (15). Leptin, on the other hand, has been shown to decrease SREBP-1c in ob/ob mice, IRS-2 knockout mice, and lipoatrophic mice, as well as wild-type rats made hyperleptinemic by adenoviral gene transfer (12,27,31). In contrast to insulin, the effects of leptin on SCD1 are not abolished by knockout of SREBP-1c. Interestingly, fructose and, to a lesser extent, LXR agonists are also able to regulate SCD1 independently of SREBP-1c (15,32).
Several studies suggest that the central nervous system (CNS), rather than the liver, is the primary site of leptin action. First, leptin treatment of hepatoma cell lines and primary hepatocytes does not suppress SCD1 mRNA (data not shown). Second, liver-specific knockout of the leptin receptor does not produce the hepatic steatosis, hyperinsulinemia, or obesity characteristic of ob/ob mice (33). Knockout of the leptin receptor in neurons, on the other hand, recapitulates the phenotype of the global leptin receptor deficiency of db/db mice, with hyperphagia, obesity, and hepatic steatosis (33). Finally, intracerebroventricular infusion of a very small amount of leptin is able to suppress SCD1 transcription and activity without increasing serum leptin levels (4).
In the CNS, leptin impacts several neurotransmitters, including neuropeptide Y, agouti-related protein, cocaine- and amphetamine-regulated transcript, proopiomelanocortin, and melanin-concentrating hormone (MCH). Recent studies have suggested that manipulation of the neural circuitry can alter hepatic lipogenesis. Intracerebroventricular infusions of neuropeptide Y can decrease hepatic ACC activity and lipogenic gene transcription (34). Intracerebroventricular injection of a melanocortin receptor agonist decreases hepatic SCD1 expression (35). On an ob/ob background, knockout of MCH lowers hepatic SCD1 transcript (36).
There are several mechanisms by which leptin signaling in the CNS might be transmitted to the liver. First, leptin may decrease hepatic SCD by acting through other hormones such as corticosterone, which is known to enhance Δ-9 desaturase activity in hepatoma cells (37). Interestingly, both corticosterone and SCD1 are high in ob/ob mice but decreased by knockout of MCH (36). Alternatively, leptin may act through autonomic innervation of the liver to alter SCD activity. Finally, leptin may act by altering the availability of nutrients such as cholesterol or polyunsaturated fatty acids. In this regard, it is worth noting that the proportion of linoleic acid (18:2), arachidonic acid (20:4), or both were increased by leptin treatment of LOX and ob/ob mice (data not shown).
In summary, SCD1 is a promising target for therapeutic interventions but its regulation is complex. While insulin and leptin are important regulators of SCD1 expression and activity, we show here that the role of insulin is subordinate to that of leptin. However, insulin may have effects on SCD1 under some circumstances and play a role in the accumulation of hepatic MUFAs. Decreasing SCD1 activity in insulin-resistant states may be beneficial in a number of diseases. Our data suggest that increasing leptin action rather than decreasing insulin action may be more important in regulating SCD1.
![FIG. 1. Leptin suppresses SCD1 in both the presence and absence of insulin treatment. As described in research design and methods, RNA and microsomes were prepared from the livers of ob/ob mice treated with leptin and/or insulin. Data are presented as means ± SE for A and B (n = 4–8). A: SCD1 mRNA was measured using real-time RT-PCR and primers specific for SCD1. *P ≤ 7 × 10−3 vs. untreated ob/ob mice. B: SCD activity was measured from liver microsome preparations as the production of oleoyl-CoA from [1-C14]stearoyl CoA. *P < 7 × 10−5 vs. untreated ob/ob mice. C: Liver microsomes (27 μg) were subjected to immunoblotting with an antibody against SCD as described in research design and methods.](https://ada.silverchair-cdn.com/ada/content_public/journal/diabetes/55/7/10.2337_db05-0742/2/m_zdb0070644200001.jpeg?Expires=1716316406&Signature=omPyGAoFQVIl4gYMbJvdU0ug84obdSiPwmqY6L6~SjSwwFSodlSxczkG3ei3mXRygMz671gXGmrwcOSun2kbn7fvGT80rHivuesfgLuBMLFzzTxvboQHy8lkW5Zu5jnCMS~blvHYqNurCGvLLmBn8k-rtblIzI4hwOJfP90Joe0NRBta49Fh5uFRjyauJ-AQJfGLQia-DOfQiE~Tb8WetRIImRCLecrj1Y17cLBmZKmJAKmf3U6PLL7jpKvI9~-0rV2AXoHDgn1km0cqrPtaHQ21OhF4Za87dxB4BFenEb99E79NlbGxQkuvkVXD3UoP6mU7wKSdg5G5lRvB3vf5RA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Leptin suppresses SCD1 in both the presence and absence of insulin treatment. As described in research design and methods, RNA and microsomes were prepared from the livers of ob/ob mice treated with leptin and/or insulin. Data are presented as means ± SE for A and B (n = 4–8). A: SCD1 mRNA was measured using real-time RT-PCR and primers specific for SCD1. *P ≤ 7 × 10−3 vs. untreated ob/ob mice. B: SCD activity was measured from liver microsome preparations as the production of oleoyl-CoA from [1-C14]stearoyl CoA. *P < 7 × 10−5 vs. untreated ob/ob mice. C: Liver microsomes (27 μg) were subjected to immunoblotting with an antibody against SCD as described in research design and methods.
Leptin suppresses SCD1 in both the presence and absence of insulin treatment. As described in research design and methods, RNA and microsomes were prepared from the livers of ob/ob mice treated with leptin and/or insulin. Data are presented as means ± SE for A and B (n = 4–8). A: SCD1 mRNA was measured using real-time RT-PCR and primers specific for SCD1. *P ≤ 7 × 10−3 vs. untreated ob/ob mice. B: SCD activity was measured from liver microsome preparations as the production of oleoyl-CoA from [1-C14]stearoyl CoA. *P < 7 × 10−5 vs. untreated ob/ob mice. C: Liver microsomes (27 μg) were subjected to immunoblotting with an antibody against SCD as described in research design and methods.
Leptin decreases the proportion of hepatic MUFAs in ob/ob mice. Liver samples from ob/ob mice and their lean controls were homogenized in chloroform. Lipids were extracted, separated by thin-layer chromatography, methylated, and analyzed by gas chromatography. The ratios of palmitoleate to palmitate (16:1 to 16:0, left panels) and oleate to stearate (18:1 to 18:0, right panels) in the three major lipid fractions were calculated for each mouse and expressed as means ± SE (n = 4 for each group). *P < 0.05 compared with untreated ob/ob; no significant differences were found between mice treated with leptin alone compared with those treated with both leptin and insulin.
Leptin decreases the proportion of hepatic MUFAs in ob/ob mice. Liver samples from ob/ob mice and their lean controls were homogenized in chloroform. Lipids were extracted, separated by thin-layer chromatography, methylated, and analyzed by gas chromatography. The ratios of palmitoleate to palmitate (16:1 to 16:0, left panels) and oleate to stearate (18:1 to 18:0, right panels) in the three major lipid fractions were calculated for each mouse and expressed as means ± SE (n = 4 for each group). *P < 0.05 compared with untreated ob/ob; no significant differences were found between mice treated with leptin alone compared with those treated with both leptin and insulin.
![FIG. 3. Leptin decreases SCD1 in LIRKO livers. Livers were harvested from the mice (described in Table 2) and used to prepare RNA for real-time RT-PCR analysis and microsomes for assay of SCD activity and immunoblotting. Data are presented as means ± SE for A and B (n = 4–8). A: Real-time PCR analysis using primers specific for SCD1. *P ≤ 0.01 vs. untreated LOX mice; +P ≤ 0.01 for leptin treatment of LIRKO mice. B: SCD1 activity was measured as the production of oleoyl-CoA from [1-14]stearoyl CoA. *P < 0.03 vs. untreated LOX mice; +P < 0.03 for leptin treatment of LIRKO mice. C: Liver microsomes (150 μg) were subjected to immunoblotting with antibodies against SCD. SCD protein was not detectable in LIRKO mice even after prolonged exposure.](https://ada.silverchair-cdn.com/ada/content_public/journal/diabetes/55/7/10.2337_db05-0742/2/m_zdb0070644200003.jpeg?Expires=1716316406&Signature=fT-VB5fU3x5zjLvQBW6R0qn28Ei0RSDcg2rU3BCX7IXhfFC~p1RILOa-g4nPuj-hYxgtNZ-vRdJr078CpWC1HTrsS2Y4mCtgrtnE5c9twBpm~w3BB51neo~5ZItCsIvea2NYSKnrgdH8dS5V1Z5l9hDIYhxc-WVyQI32lTXRwoPeu4GaLpLuy7lbwswbIofUBOGUNf83VI3dmWVm0wJwO869dTVrDCfYUWbjoa3BC5LuZGIpdoz8U0JtpbjfDa1tomXjSRcew5mWil8oYU6fDrF-uy6D5YOgeR22LOMCpgEkYMmQAQg1oDMGE5J4EGpRXCkMtCyrNbY5alspU3dpLg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Leptin decreases SCD1 in LIRKO livers. Livers were harvested from the mice (described in Table 2) and used to prepare RNA for real-time RT-PCR analysis and microsomes for assay of SCD activity and immunoblotting. Data are presented as means ± SE for A and B (n = 4–8). A: Real-time PCR analysis using primers specific for SCD1. *P ≤ 0.01 vs. untreated LOX mice; +P ≤ 0.01 for leptin treatment of LIRKO mice. B: SCD1 activity was measured as the production of oleoyl-CoA from [1-14]stearoyl CoA. *P < 0.03 vs. untreated LOX mice; +P < 0.03 for leptin treatment of LIRKO mice. C: Liver microsomes (150 μg) were subjected to immunoblotting with antibodies against SCD. SCD protein was not detectable in LIRKO mice even after prolonged exposure.
Leptin decreases SCD1 in LIRKO livers. Livers were harvested from the mice (described in Table 2) and used to prepare RNA for real-time RT-PCR analysis and microsomes for assay of SCD activity and immunoblotting. Data are presented as means ± SE for A and B (n = 4–8). A: Real-time PCR analysis using primers specific for SCD1. *P ≤ 0.01 vs. untreated LOX mice; +P ≤ 0.01 for leptin treatment of LIRKO mice. B: SCD1 activity was measured as the production of oleoyl-CoA from [1-14]stearoyl CoA. *P < 0.03 vs. untreated LOX mice; +P < 0.03 for leptin treatment of LIRKO mice. C: Liver microsomes (150 μg) were subjected to immunoblotting with antibodies against SCD. SCD protein was not detectable in LIRKO mice even after prolonged exposure.
Leptin does not decrease MUFA content in LIRKO mice. Lipids derived from the mice (described in Table 2) were analyzed by gas chromatography, as in Fig. 2. The ratios of palmitoleate to palmitate (16:1 to 16:0, left panels) and oleate to stearate (18:1 to 18:0, right panels) in the three major lipid fractions were calculated for each mouse and expressed as means ± SE (n = 4 for each group). *P < 0.05 vs. untreated LOX mice.
Leptin does not decrease MUFA content in LIRKO mice. Lipids derived from the mice (described in Table 2) were analyzed by gas chromatography, as in Fig. 2. The ratios of palmitoleate to palmitate (16:1 to 16:0, left panels) and oleate to stearate (18:1 to 18:0, right panels) in the three major lipid fractions were calculated for each mouse and expressed as means ± SE (n = 4 for each group). *P < 0.05 vs. untreated LOX mice.
Leptin decreases SREBP-1c in ob/ob mice but not LOX or LIRKO mice. Livers from the mice (described in Tables 1 and 2) were used to generate RNA for real-time RT-PCR analysis with primers specific for SREBP-1c or IRS-2. Data are presented as means ± SE (n = 6–8 for each group). A: Leptin (LEP) treatment of ob/ob mice decreases SREBP-1c in the presence or absence of insulin (INS). *P < 10−4 compared with untreated ob/ob mice. B: Leptin treatment does not decrease SREBP-1c in LOX or LIRKO mice. *P = 0.002 vs. untreated LOX mice; #P < 0.05 for leptin treatment of LIRKO mice.
Leptin decreases SREBP-1c in ob/ob mice but not LOX or LIRKO mice. Livers from the mice (described in Tables 1 and 2) were used to generate RNA for real-time RT-PCR analysis with primers specific for SREBP-1c or IRS-2. Data are presented as means ± SE (n = 6–8 for each group). A: Leptin (LEP) treatment of ob/ob mice decreases SREBP-1c in the presence or absence of insulin (INS). *P < 10−4 compared with untreated ob/ob mice. B: Leptin treatment does not decrease SREBP-1c in LOX or LIRKO mice. *P = 0.002 vs. untreated LOX mice; #P < 0.05 for leptin treatment of LIRKO mice.
Leptin decreases SCD1 in SREBP-1c−/− mice. Livers were used to prepare RNA for real-time RT-PCR analysis (A) and microsomes to assay SCD activity (B). Data are presented as means ± SE (n = 6–8 for each group). *P < 0.01 vs. untreated wild-type (WT) mice; #P < 0.001 vs. untreated SREBP-1c−/− mice.
Leptin decreases SCD1 in SREBP-1c−/− mice. Livers were used to prepare RNA for real-time RT-PCR analysis (A) and microsomes to assay SCD activity (B). Data are presented as means ± SE (n = 6–8 for each group). *P < 0.01 vs. untreated wild-type (WT) mice; #P < 0.001 vs. untreated SREBP-1c−/− mice.
Effects of leptin treatment on ob/ob mice
| ob/ob | ||||||||
|---|---|---|---|---|---|---|---|---|
| Control | Vehicle | Leptin | Insulin | Leptin + insulin | ||||
| Change in weight (g) | 0 ± 0.3 | −6.4 ± 1.4* | 0.6 ± 0.3 | −4.3 ± 0.3† | ||||
| Food intake (g · day−1 · mouse−1) | 6.2 ± 0.4† | 11.3 ± 0.7 | 1.2 ± 0.3† | 9.2 ± 0.4* | 2.1 ± 0.4† | |||
| Fasting blood glucose (mg/dl) | 143 ± 9 | 268 ± 72 | 165 ± 21 | 162 ± 28 | 92 ± 11‡ | |||
| Fasting insulin (ng/ml) | 1.1 ± 0.4† | 7.7 ± 0.4 | 1.1 ± 0.2† | 17.6 ± 2.4* | 7.4 ± 1.1‡ | |||
| Leptin (ng/ml) | 3.2 ± 0.5† | 0 | 27.4 ± 4.5* | 0 | 8.4 ± 1.4†‡ | |||
| ob/ob | ||||||||
|---|---|---|---|---|---|---|---|---|
| Control | Vehicle | Leptin | Insulin | Leptin + insulin | ||||
| Change in weight (g) | 0 ± 0.3 | −6.4 ± 1.4* | 0.6 ± 0.3 | −4.3 ± 0.3† | ||||
| Food intake (g · day−1 · mouse−1) | 6.2 ± 0.4† | 11.3 ± 0.7 | 1.2 ± 0.3† | 9.2 ± 0.4* | 2.1 ± 0.4† | |||
| Fasting blood glucose (mg/dl) | 143 ± 9 | 268 ± 72 | 165 ± 21 | 162 ± 28 | 92 ± 11‡ | |||
| Fasting insulin (ng/ml) | 1.1 ± 0.4† | 7.7 ± 0.4 | 1.1 ± 0.2† | 17.6 ± 2.4* | 7.4 ± 1.1‡ | |||
| Leptin (ng/ml) | 3.2 ± 0.5† | 0 | 27.4 ± 4.5* | 0 | 8.4 ± 1.4†‡ | |||
Data are means ± SE (n = 4–6). Vehicle-treated ob/ob mice and their untreated lean control littermates (Control) were compared with ob/ob mice treated with leptin (24 μg/day), insulin, or both for 4 days. The weight change and food intake were measured over the 4 days and the last 24 h, respectively. After 2 days of treatment, mice were fasted for 4 h and insulin and blood glucose were measured. Leptin was measured in the nonfasted state at the end of the experiment.
P < 0.05,
P < 0.005 vs. untreated ob/ob;
P < 0.05 vs. treatment with leptin alone.
Effects of leptin treatment on LIRKO mice
| LOX | LOX + leptin | LIRKO | LIRKO + leptin | |
|---|---|---|---|---|
| Change in weight (g) | 0.8 ± 0.2 | −0.2 ± 0.2* | 1.4 ± 0.3 | 0.3 ± 0.3† |
| Food intake (g · day−1 · mouse−1) | 3.5 ± 0.1 | 2.4 ± 0.1* | 4.0 ± 0.3 | 2.3 ± 0.1† |
| Blood glucose (mg/dl) | 146 ± 7 | 128 ± 8 | 179 ± 17 | 125 ± 10† |
| Insulin (ng/ml) | 0.30 ± 0.06 | 0.17 ± 0.17 | 4.3 ± 0.85‡ | 2.0 ± 0.44† |
| Leptin (ng/ml) | 6.6 ± 0.9 | 59 ± 12* | 34 ± 7.7‡ | 229 ± 21† |
| LOX | LOX + leptin | LIRKO | LIRKO + leptin | |
|---|---|---|---|---|
| Change in weight (g) | 0.8 ± 0.2 | −0.2 ± 0.2* | 1.4 ± 0.3 | 0.3 ± 0.3† |
| Food intake (g · day−1 · mouse−1) | 3.5 ± 0.1 | 2.4 ± 0.1* | 4.0 ± 0.3 | 2.3 ± 0.1† |
| Blood glucose (mg/dl) | 146 ± 7 | 128 ± 8 | 179 ± 17 | 125 ± 10† |
| Insulin (ng/ml) | 0.30 ± 0.06 | 0.17 ± 0.17 | 4.3 ± 0.85‡ | 2.0 ± 0.44† |
| Leptin (ng/ml) | 6.6 ± 0.9 | 59 ± 12* | 34 ± 7.7‡ | 229 ± 21† |
Data are means ± SE (n = 4–8). LOX and LIRKO mice were treated with vehicle or leptin (30 μg/day) for 4 days. The change in weight and food intake were measured over the 4 days. Blood glucose, insulin, and leptin were measured in the nonfasted state at the end of the experiment, just prior to killing.
P < 0.05, leptin treatment of LOX;
P < 0.05, leptin treatment of LIRKO;
P < 0.05, LOX vs. LIRKO (untreated).
S.B.B. and M.M. contributed equally to this work.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
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Article Information
The work was funded in part by grants DK31036 and DK45935 (to C.R.K.), T32 DK63702-01 (to S.B.B.), and DK62388 (to J.M.N.). Additionally, this work was supported by the Affymetrix Core of the Joslin Diabetes Center (Diabetes Genome Anatomy Project DK60837-02) and the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers (National Institutes of Health DK59637-01).
The authors thank Dr. Juris Ozols for the antibody to SCD1, Dr. Jeffrey Friedman for recombinant leptin, and Laureen Mazzola for excellent technical assistance.
