Carnitine Palmitoyltransferase 1b Deficiency Protects Mice from Diet-Induced Insulin Resistance

Background Carnitine Palmitoyl Transferase 1 (CPT1) is the rate-limiting enzyme governing long-chain fatty acid entry into mitochondria. CPT1 inhibitors have been developed and exhibited beneficial effects against type II diabetes in short-term preclinical animal studies. However, the long-term effects of treatment remain unclear and potential non-specific effects of these CPT1 inhibitors hamper in-depth understanding of the potential molecular mechanisms involved. Methods We investigated the effects of restricting the activity of the muscle isoform CPT1b in mice using heterozygous CPT1b deficient (Cpt1b+/−) and Wild Type (WT) mice fed with a High Fat Diet (HFD) for 22 weeks. Insulin sensitivity was assessed using Glucose Tolerance Test (GTT), insulin tolerance test and hyperinsulinemic euglycemic clamps. We also examined body weight/composition, tissue and systemic metabolism/energetic status, lipid profile, transcript analysis, and changes in insulin signaling pathways. Results We found that Cpt1b+/− mice were protected from HFD-induced insulin resistance compared to WT littermates. Cpt1b+/− mice exhibited elevated whole body glucose disposal rate and skeletal muscle glucose uptake. Furthermore, Cpt1b+/− skeletal muscle showed diminished ex vivo palmitate oxidative capacity by ~40% and augmented glucose oxidation capacity by ~50% without overt change in whole body energy metabolism. HFD feeding Cpt1b+/− but not WT mice exhibited well-maintained insulin signaling in skeletal muscle, heart, and liver. Conclusion The present study on a genetic model of CPT1b restriction supports the concept that partial CPT1b inhibition is a potential therapeutic strategy.


Introduction
Increased fatty acid availability and the concomitantly augmented mitochondrial fatty acid oxidation (FAO) capacity in skeletal muscle is a common feature in insulin resistant subjects [1,2]. Interfering with excessive fatty acid availability and oxidation hence may be a potential therapeutic strategy. Accumulated evidence has emerged that inhibiting FAO may sensitize insulin signaling in animals with insulin resistance [3][4][5][6][7] and in diabetic patients [7][8][9]. Carnitine Palmitoyl Transferase 1 (CPT1) on the mitochondrial outer membrane is a rate limiting enzyme that converts long-chain acyl CoA into long chain acylcarnitine, thus being one of the most studied therapeutic targets for FAO inhibition.
There are three isoforms of CPT1 (a, b and c). While CPT1a is expressed ubiquitously but most abundantly in the liver, CPT1b is expressed in skeletal muscle, heart, and adipose tissues, and CPT1c is expressed in the brain and testes (reviewed in [10]). HFD-fed mice show increased capacity of fatty acid oxidation in skeletal muscle homogenate and mitochondrial respiration with concomitantly increased CPT1 activity [11]. The expression of CPT1bis inversely related to insulin sensitivity among quartiles of metabolic syndrome patients [12]. Repressing the capacity of fatty acid oxidation may ameliorate insulin resistance. In supporting this hypothesis, several groups reported that CPT1 inhibitors improve insulin resistance in relatively short-term preclinical animal studies [5][6][7][8]13,14], even with increased intracellular lipid content. CPT1 inhibition in molecular genetic models due to the deletion of Malonyl-CoA-Decarboxylase (MCD) shows similar effects. The MCD null (Mcd −/− ) mice are protected from HFD-induced insulin resistance by substantially increased Malonyl-CoA-Mediated repression of CPT1, consequently decreasing fatty acid oxidation and increasing glucose oxidation [3]. On the other hand, a 20% electroporationmediated transient upregulation of CPT1b in the distal hind limb muscles is sufficient to improve HFD-induced insulin resistance in rats via attenuating triacylglycerol content, the membrane-to-cytosolic ratio of diacylglycerol, and protein kinase Cθactivation [15]. Therefore, further studies on the in vivo roles of specific CPT1 isoform using the more specific gene manipulating approach are essential to resolve these seemingly contradictory observations. The goal of the present study is to elucidate the effects of restricting fatty acid oxidation on diet-induced insulin resistancein the heterozygous Cpt1b knockout mice. This is the first study using a preclinical mouse model with Cpt1b-specific knockdown to investigate the long-term effects of CPT1b repression on HFD-induced insulin resistance and the underlying metabolic mechanisms.

Animals
The heterozygous Cpt1b +/− knockout mice [16] and wild-type (WT) littermates in C57BL/6J background were used. While the homozygous CPT1b knockout is lethal, Cpt1b +/− mice show no overt abnormal phenotype and whole body metabolic changes [16]. All mice were kept on 12-hour/12-hour light/dark cycle (light on at 06:00 hours) at 22.0 ±1.0°C and had ad libitum access to water and standard rodent diet (Harlan Laboratories 7017 NIH-31 Mouse/Rat Sterilizable Diet, 14% kcal% fat). Mice (4 -5 week old, male) of high fat diet (HFD) feeding groups were given ad libitumaccess to HFD (60% kcal% fat) (Research DIETS D12492) and water. Body weights were recorded every other week. All experimental procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham (UAB).

Hyperinsulinemiceuglycemic clamp
The procedures of hyperinsulinemiceuglycemic clamp (insulin clamp) in mice were adapted from Dr. Pessin's group [17] with minor modifications based on reports from other groups [18,19]. Briefly, mice were anesthetized by Isoflurane via a Vaporizer-MiniVentmouse ventilator system (HUGO SACHS ELECTRONIK, Harvard Apparatus GmbH, Hugstetten, Germany). A catheter was surgically implanted into the right jugular vein and threaded under the dorsal skin of mice. Three days after surgery, the mouse was fasted 5 hrs (08:00-13:00) and then placed in a rat-size restrainer with its tail taped. The catheter was connected to a CMA 402 syringe pump (CMA Microdialysis, Stockholm, Sweden). [6-3 H]-glucose was infused at 0.5 μCi/min for 2 hrs without insulin and then infused at 1μCi/min with insulin (Humulin R, Eli Lilly 2.5 mU kg −1 min −1 ) for 2 hrs, by which time the blood glucose was maintained at 7.8 -8.9 mmol/L by adjusting 20% glucose infusion rate in the mouse under the conscious condition. 10 μCi 2-[ 14 C]-deoxy-D-glucose was infused 40 min before the end of the 120 min euglycemic clamp. The blood glucose level was measured from tail tip snipped blood samples using a Contour glucometer (Bayer). At the end of the clamp study, tissues were harvested and snapped frozen in liquid nitrogen after the mouse was euthanized. The plasma glucose level was measured using an Analox GM7 Micro-Stat Analyzer (Analox Instruments, London, UK). The specific activity of plasma glucose, the glucose infusion rate (GIR), the whole body glucose disposal rate (Gd), and the tissuespecific glucose uptake were measured and calculated as previously described [20].

Lipid measurements
Frozen gastrocnemius muscles were pulverized using a pulverizor (Bio Spec Products Inc.) in liquid nitrogen and weighed in small tubes as previously described [21]. For the nonesterified fatty acids (NEFA), lipids were extracted using the Bligh & Dyer method [22]. NEFA and TAG were measured using a NEFA-HR Kit (Wako) and a Triglyceride Quantification Kit (BioVision K622-100). For the acylcarnitine assay, 6 volume of 80 % acetonitrile was added to pulverized tissue weight (about 50 mg). Tissue mixtures were sonicated 10 times, centrifuged at 12,000 rpm 10 min at 4 °C, and supernatants were transferred to new tubes. The supernatants were dried under a stream of nitrogen at 40 °C and resuspended in 100 μl of 50% acetonitrile. The acylcarnitine content was measured by using Electrospray Ionization Tandem Mass Spectrometry [23].

Ex vivo oxidation assay
Intact muscle oxidation assay was performed as previously described [24]. Extensor digitorumlongus (EDL) muscles were excised from euthanized mice and incubated with 700 μl of Krebs-Ringer Phosphate buffer containing 0.1 μCi/ml of BSA-conjugated [ 14 C]palmitateor [ 14 C]-glucose in sealed 14 ml tubes with center wells containing 1N NaOH at 37°C for 1 hour with 200 rpm shaking. After incubation, 400 μl of 3.5 M HClO 4 was injected into the media and incubated at 50°C for 3 hours to capture oxidized substrates to NaOH and the radioactivity was measured by scintillation counter [24].

Oxidation assays in isolated mitochondria
The procedures of mitochondrial oxidation assay were adapted from Dr. Kove's group as previously described [3]. Gastrocnemius muscle (one hind limb from one mouse) was homogenized by using hand-held drill with Potter-Elvehjem homogenizer. Supernatants were kept following a low speed (1,000 xg) centrifugation, and subsequently centrifuged again at 12,000 xg to isolate mitochondria. Mitochondrial protein concentration was measured by Lowry method and 100 μg of mitochondria was incubated with 0.1 μCi/ml of [ 14 C]-palmitate including reaction buffer for 2 hrs. The oxidation reaction was stopped by injecting perchloric acid. Oxidized substrates were metabolized as 14 CO 2 and captured by NaOH in the central well and the radioactivity was measured by scintillation counter. Incompletely oxidized substrates (acid soluble metabolites-ASM) remained in the reaction mixture were centrifuged. Then the supernatants were used to detect 14 C by liquid scintillation counter.

Real-time qPCR
Total RNA extraction and cDNA synthesis were done by using RNeasy ® Mini kit (QIAGEN), Advantage ® RT-for PCR kit (Clontech), Power SYBR ® Green PCR master mix. The real-time PCR was performed by using Step One Real-Time PCR System (Applied Biosystems) with standard curve for the transcript quantification. The primer sequences are listed in Supplementary Table 1.

Body composition analysis
Fat and lean mass were measured in vivo using a quantitative magnetic resonance imaging system (QMR, EchoMRI ™ 3-in-1, Echo Medical System, Houston, TX, USA) at UAB Small Animal Physiology Core as previously described and validated [25].

Comprehensive Lab Animal Monitoring System (CLAMS) analysis
Respiration rate, food intake, energy expenditure, and physical activity were accurately quantified as previously described using the CLAMS (Columbus Instruments Inc., OH, USA) [26]. Mice were individually kept in CLAMS chambers with ad libitum access to food and water for 6 days and the data sets for the last 3 days were averaged for the analysis. Respiratory exchange ratio (RER) was calculated as CO 2 generation/O 2 consumption. Energy expenditure was expressed as (kcal/hr).

Indirect calorimetry analysis
Energy expenditure, RER, and activity were also measured with another set of mice using an 8-cage indirect calorimetry system (CaloSys, TSE Systems, Bad Homburg, Germany) as previously described [27]. Mice were individually kept in airtight plastic cages with ad libitum access to food and water and a continuous flow of air was maintained through all cages. Mice were acclimated to the cages for 48 hours prior to the measurement period of 22 hours. Total energy expenditure was calculated over the 22 hours, and presented as per 24hours. Resting energy expenditure was calculated as the average of the 3 lowest 18 minute periods during the measurement.

Rectal temperature measurement
Body temperatures of mice were measured using rectal probe provided from Echocardiogram (Vevo770, Visualsonics Inc., Toronto, Ontario, Canada) under isofluraneinduced anesthesia at around 13:00.

Statistical analysis
GraphPad Prism5 software was used to conduct Two-tailed Student's t-test and One-Way ANOVA with Tukey post hoc test. The ANCOVA was done using SAS9.1 software. Differences between groups were regarded significant at p<0.05 probability level. Data are expressed as means ± SE.

Mice with CPT1b deficiency are protected against HFD-induced insulin resistance
We investigated whether partial CPT1b deficiency determines insulin sensitivity in mice under CHD and HFD feeding conditions using OGTT and ITT. At baseline (CHD 4 weeks), CHD-fed Cpt1b +/− and WT littermates showed no difference in OGTT and ITT ( Figure 1A and 1B). After 8 weeks of HFD feeding, Cpt1b +/− mice showed attenuated blood glucose levels at 60 minutes time point during OGTT ( Figure 1C). The blood glucose levels during ITT were not changed at the 30 minute time point, but were lower in Cpt1b +/− than in WT mice at 60 and 120 minute time points ( Figure 1D). At 22 weeks after HFD feeding, Cpt1b +/− mice showed substantially improved glucose tolerance compared with WT littermates. The area under curve (AUC) of OGTT in Cpt1b +/− mice was decreased about 45 % compared with WT ( Figure 1E). The blood glucose levels during ITT were not changed at the 30 minute time point but lower in Cpt1b +/− than in WT mice at the later time points ( Figure 1F). These results indicate that CPT1b deficiency improves glucose and insulin tolerance under a HFD condition. Cpt1b +/− mice showed no difference in ITT and OGTT until 32 weeks of their age (Supplemental Figure 1) suggesting that CPT1b deficiency does not alter insulin sensitivity and glucose tolerance under a normal diet condition.
To gain insights into the tissue-specific insulin sensitivities, we further assessed HFD feeding mice using the hyperinsulinemiceuglycemic clamp at 22 weeks after HFD feeding. A much higher glucose infusion rate (GIR) in Cpt1b +/− mice than in WT mice was required to maintain blood glucose levels (7.8 -8.9 mmol/L) in response to the constant insulin infusion ( Figure 1G). Consistently, whole body glucose disposal rate (Gd) was higher in Cpt1b +/− than in WT mice ( Figure 1H), indicating improved systemic insulin sensitivity in the Cpt1b +/− mice compared with WT mice. Insulin-stimulated glucose uptake was increased by about 2-fold in Cpt1b +/− relative to WT skeletal muscle (Figure 1), whereas there was no difference between two groups in Gonadal White Adipose Tissue (GWAT)( Figure 1H). The plasma glucose level was significantly lower in Cpt1b +/− mice than in WT littermates at 8 weeks and 22 weeks of HFD feeding. No changes could be detected in plasma NEFA throughout the observation period in both groups. On the other hand, plasma insulin (p<0.01) and leptin levels surged after 22 weeks of HFD in WT from 8 weeks, while insulin levels in Cpt1b +/− mice were substantially lower than their WT controls at 22 weeks. The plasma insulin level during insulin clamp was not different between two groups, while plasma NEFA level was substantially decreased in Cpt1b +/− mice (Table 1).

CPT1b deficiency in mice leads to repressed fatty acid oxidation and enhanced glucose oxidation in the skeletal muscle under a HFD condition
We further investigated whether changes in substrate metabolism correlated with different insulin sensitivity in Cpt1b +/− vs. WT mice. CPT1b deficient muscle did not show decreased palmitate oxidation capacity compared to WT muscle under a CHD condition (Figure 2A). The rate of ex vivo palmitate oxidation in skeletal muscle of Cpt1b +/− mice after 22 weeks of HFD feeding was 40 % lower than in WT mice (p<0.05) (Figure 2A), whereas the rate of ex vivo glucose oxidation was 50 % higher in Cpt1b +/− than in WT muscles ( Figure 2B). Other tissues such as heart, brown adipose tissue, and liver showed no difference in the ex vivo oxidation capacity between two groups (data not shown) under a HFD condition. The palmitate oxidation rate in isolated mitochondria was also decreased in CPT1b +/− compared with WT muscle ( Figure 2C) with no change in incomplete oxidation ( Figure 2D). To assess whether the decreased fatty acid oxidation capacity would result in intramyocellular lipid accumulation, skeletal muscle lipid contents were assessed after 22 weeks of HFD feeding. There was no difference in TAG and NEFA levels between Cpt1b +/− and WT muscle ( Figure 2E and 2F). Notably, acylcarnitine profile in skeletal muscle of HFD-feeding Cpt1b +/− mice was also not different from WT ( Figure 2G). In the transcript analysis, Cpt2 (p<0.01), Cpt1a, Acs (acyl-CoA synthase long-chain family member 1), Dgat (diacylglycerol O-acyltransferase 2), Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), and Mlycd (malonyl-CoA decarboxylase) were significantly decreased in Cpt1b+/− muscle (P<0.05) (Supplemental Figure 2) supporting that lipid was not accumulated in Cpt1b+/− muscle. Therefore, the above results implicate that modest repression of FAO in skeletal muscle could increase glucose oxidation without overt intramuscular lipid accumulation.

Insulin signal transduction is well maintained in Cpt1b +/− mice skeletal muscles
To confirm the improved insulin sensitivity of Cpt1b +/− mice under a long-term HFD condition on insulin signaling, we subjected tissue samples from hyperinsulinemiceuglycemic clamp study to Western blot analysis of key insulin signaling proteins. The phosphorylation of IRS1 at Ser302 was substantially lower in Cpt1b +/− than in WT muscles (p<0.01) ( Figure 3A), but there was no difference in the heart ( Figure 3B). No signals of phosphorylation of IRS1 at Ser302 were detected in liver and GWAT (data not shown). The phosphorylation of AKT at Ser473 was substantially elevated in muscle (p<0.01) ( Figure 3A), heart (p<0.01) ( Figure 3B), and liver (p<0.01) ( Figure 3C), but decreased in GWAT ( Figure 3D) (p<0.05) of Cpt1b +/− mice. The phosphorylation of ERK1/2 at Thr202/Tyr204 was maintained in muscle (p<0.05) ( Figure 3A), liver (p<0.01) ( Figure 3C), but had no difference in heart ( Figure 3B) and decreased in GWAT ( Figure 3D) of Cpt1b +/− mice. These results demonstrate that Cpt1b +/− mice are protected from HFDinduced impaired insulin signaling in muscle, heart, and liver, but not in GWAT.

Mice with CPT1b deficiency are smaller than control mice subjected to HFD
We found no difference in body weight and body composition between Cpt1b +/− and WT mice under a CHD condition throughout life-time (data not shown). However, Cpt1b +/− mice showed a smaller increase of body weight than WT mice after 4 weeks of HFD feeding ( Figure 4A and 4B). At 8 weeks after HFD feeding, Cpt1b +/− mice exhibited 50 % less body fat mass and 14% less lean mass than WT mice ( Figure 4C). However, ANCOVA analysis revealed that these changes were proportional to the total body mass, suggesting that the improved insulin sensitivity of Cpt1b +/− mice is independent of changes in body fat mass.

CPT1b deficiency does not influence whole body energy balance in response to HFD feeding
We next determined whether the altered substrate oxidation capacity of skeletal muscle in Cpt1b +/− mice under a HFD condition affects the whole body energy balance. We measured respiratory exchange rate (RER), energy expenditure, activity, and food intake using CLAMS at 22 weeks after HFD feeding. Interestingly, none of these parameters were different between two groups ( Figure 5A-5D). We confirmed the RER assessment of CLAMS analysis by using indirect calorimeter with another set of mice at 22 weeks after HFD feeding. Cpt1b +/− mice showed no difference in energy expenditure ( Figure 5E) with very subtle decreased RER ( Figure 5F), accompanied with weight loss compared to WT during the 2 day measurement period ( Figure 5G). Consistently, the HFD-fed WT, but not Cpt1b +/− mice, showed higher rectal temperature ( Figure 5H), suggesting that the whole body energy metabolism of Cpt1b +/− mice was not substantially altered. Analysis of fecal energy content revealed no differences between the two groups (data not shown). Therefore, reduced fatty acid oxidation and increased glucose oxidation in skeletal muscle appear to be in a balanced state that does not substantially alter whole body metabolism and energy balance in mice subjected to HFD.

Discussion
In the current study, we provide evidence supporting the beneficial effects of CPT1b ablation related fatty acid oxidation restriction against insulin resistance induced by HFD. We investigated a genetic mouse model with Cpt1b +/− deficiency subjected to 22 weeks of HFD feeding. We illustrate that CPT1b mediated fatty acid oxidation in skeletal muscle is a crucial site for the maintenance of insulin sensitivity via adjusting the balance of local fatty acid and glucose oxidation rates.
Pharmacological CPT1 inhibition has been suggested as an effective therapy to improve insulin sensitivity in type 2 diabetes patients for nearly two decades [28][29][30]. A CPT1 inhibitor, etomoxir, improves glucose homeostasis in patients by repressing fatty acid oxidation and elevating glucose oxidation, presumably by the Randle cycle mechanism, despite the subsequent intramyocellular lipid accumulation [7]. Nevertheless, opposite results have also been reported, in which etomoxir is associated with exacerbated insulin resistance with concomitantly increased intracellular lipid accumulation in rodents [31]. Oxfenicine, another CPT1 inhibitor, increases glucose oxidation in HFD-fed mice with increased respiratory exchange ratio (RER), but no intramyocellular lipid accumulation [4]. The inconsistent outcomes from the CPT1 inhibitor studies are most likely derived from the different experimental approaches and the different degrees/subtype specificities of these CPT1 inhibitors. While pharmacological studies provide highly clinical relevant insights into the effects of a specific CPT1 inhibitor, this approach does not target specific CPT1 isoforms with inherent non-CPT1 inhibition effects (e.g., etomoxir has been shown to be a PPARα activator [32]). Moreover, most of previous etomoxir studies were performed with high dosages and relatively short-term assessments on the acute effects of etomoxir, which mostly acts on CPT1a, the liver isoform of CPT1 [33]. Oxfenicine, which inhibits more CPT1b than CPT1a [34], seems to be a promising anti-diabetic drug, since it improves insulin sensitivity without intramuscular lipid accumulation in mice [4]. It remains challenging to exclude the potential off-target effects in pharmacological studies. Additional studies on animal models with genetic manipulations should help overcome the related difficulties. Our current study using the genetic mouse model with CPT1b deficiency provides specific insights and clarifies the effect of CPT1b deficiency on HFD-induced insulin resistance.
Despite the encouraging support of CPT1b inhibition as a potential therapy for insulin resistance as reported here, many other studies using various CPT1 inhibitors have shown adverse effects [31,35] due to intramyocellular fatty acid-related metabolites (e.g., DAGs, acylcarnitines, ceramides) accumulation. In this study we prove that repressed fatty acid oxidation due to heterozygous CPT1b deficiency in skeletal muscle did not lead to accumulation of lipid metabolites, which is consistent with the Oxfenicine study [34]. Previous study showed increased fatty acid oxidation in ex vivo muscle tissues of HFD-fed WT mice [11]. Zucker diabetic fatty (ZDF) rats showed increased fatty acid oxidationrelated gene expression in their muscle with increased muscle acylcarnitine accumulation [3]. In contrast, a human study showed that palmitate oxidation was significantly decreased, but only in extremely obese patients [36]. Additional studies also demonstrated opposite results either with reduced or elevated fatty acid oxidation in obese human skeletal muscle [37,38]. However, the current study did not detect substantial change at least in the EDL muscle before and after HFD. These different results may be derived from many factors, such as the degrees of obesity and the measurement methods. Decreased palmitate oxidation of the HFD-fed Mcd −/− mice improved insulin sensitivity with reduced incomplete fatty acid oxidation [3], supporting the hypothesis that increased incomplete fatty acid oxidation is one of the potential mechanisms of HFD-induced insulin resistance. Unexpectedly, our experiment using the same protocol did not detected any difference of incomplete fatty acid oxidation in isolated mitochondria between Cpt1b +/− and WT muscle under a HFD condition. While the reasons for this discrepancy remain incompletely understood, we suspect that blocking the entry of long chain fatty acids into the mitochondria of Cpt1b +/− muscle may not be robust enough to cause incomplete fatty acid oxidation, yet is sufficient to repress fatty acid oxidation and upregulating glucose oxidation in skeletal muscle. Therefore, it is probable that partial CPT1b repression protects mice from HFD-induced insulin resistance by attenuating HFD-induced upregulation of fatty acid oxidation and upregulating glucose oxidation in skeletal muscle. Since it has been extensively proposed that intramyocellular lipid accumulation may be one of the main causes of insulin resistance, a potential detrimental effect after prolonged intramyocellular lipid accumulation is still likely. It is possible that the suppressed FAO in Cpt1b +/− mice did not reach the threshold that leads to the mismatch of lipid supply and degradation and subsequent intramyocellular lipid accumulation.
The insulin sensitizing effect of CPT1b deficiency is also evident by the remarkable difference in the insulin-stimulated phosphorylation of Ser302 of insulin receptor substrate 1 (IRS1) in the skeletal muscle. Ser302 in rat/mouse IRS-1 (corresponding to Ser307 of human IRS-1) is one of the molecular mechanisms proposed as an indicator of cellular energy status underpinning the development of insulin resistance [39,40]. Transgenic mice with muscle-specific IRS-1 serine to alanine are protected from fat-induced insulin resistance in skeletal muscle [41]. Since the phosphorylation of IRS1 at Ser302 is substantially induced by hyperinsulinemia [40], the lower insulin level (Table 1) may also contribute to the less phosphorylation of IRS1 at Ser302 in Cpt1b +/− mouse muscle. The change in IRS1 phosphorylation could only be detected in skeletal muscle but not in other tissues, supporting that skeletal muscle is the main tissue responsible for the improved insulin sensitivity in Cpt1b +/− mice under a HFD condition. Therefore, our results support that CPT1b repression sensitizes insulin action via increased glucose oxidation and insulin signaling in skeletal muscle. It is not clear why the insulin signal transduction in GWAT was suppressed at this point, but it appears that was not sufficient to offset the insulin sensitizing effect of partial CPT1b repression in skeletal muscle.
The insulin sensitizing effect of partial CPT1b deficiency appears to be unrelated to a decrease of relative body fat, since the fat mass was not correlated with mouse genotype assessed by ANCOVA. Despite the matched fat/lean mass ratio between the Cpt1b +/− and WT mice, serum level of leptin was markedly higher in WT than in Cpt1b +/− mice (Table 1). It is plausible that the greater total body fat in the HFD-fed WT mice may contribute to the higher serum level of leptin. It is intriguing that the Cpt1b +/− mice showed a slower growth rate than their WT counterparts in response to HFD with proportional decreases of fat and lean mass. Albeit the exact mechanism underpinning this phenomenon is not clear, it is plausible that a potentially less energy production rate from glucose oxidation may play certain roles. Because of the lack of neurohumoral responses, the ex vivo oxidation assays using isolated muscle, isolated mitochondria, and homogenate may not reflect what exactly happens under the in vivo condition. This may explain why the CPT1b deficient mice showed repressed fatty acid oxidation and increased glucose oxidation in skeletal muscle, yet without major shift in whole body metabolism [42,43].
Interestingly, the effects of CPT1 deficiencies on growth and insulin resistance are subtypedependent. One study found that Cpt1c +/− mice are smaller than WT mice even under a CHD condition and they are susceptible to insulin resistance, obesity, and hepatosteatosis under a HFD condition [44]. Previously we reported that the Cpt1a +/− mice were resistant against HFD-induced insulin resistance, but showed hepatosteatosis with increased hepatic CPT1b expression [45]. We also found that Cpt1b +/− mice showed 40% decreased CPT1 activity in muscle and there were no significant changes in body weight, tissue histology, serum free fatty acid [16], and normal cardiac function and heart weight compared to WT littermates [46]. In this study we found that Cpt1b +/− mice showed no difference in glucose and insulin tolerance under a CHD condition at least up to 32 weeks of age. Insulin sensitivity of Cpt1b +/− mice is well maintained under a HFD condition. Therefore, it appears that CPT1b is a better specific CPT1 target for the treatment of insulin resistance.
Taken together, the current investigation confirmed the beneficial effects of CPT1b repression on diet-induced insulin resistance. Further studies will be required to identify CPT1b-specific inhibitors, and to confirm the effectiveness and safety of prolonged inhibition of CPT1b in animals and in patients with insulin resistance and type II diabetes.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
The technical services of the UAB Metabolism Core Laboratory were supported by grants P30DK56336, UL 1RR025777, and P60DK079626. Small Animal Phenotyping Core was supported by UAB Diabetes Research Center P60DK079626 and the UAB Nutrition Obesity Research Center P30DK056336. The Targeted Metabolomics and Proteomics Laboratory were supported partly by P30DK079337 and P30AR50948. The opinions expressed in this article are not those of the NIH or any other organization with which the authors are affiliated.

Grants
This work was supported by grants from National Institutes of Health (1RO1 HL085499 and 1RO1 HL084456 to Dr. Yang, 1RO1 RR02599 to Dr. Wood, and T32 HL007457 to Dr. Kim).       Plasma lipid and hormone profile after 8 weeks, 22 weeks of HFD feeding, and insulin clamp (n=7-8).