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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Objective:

Abnormal lipid metabolism and excess accumulation of lipid in non-adipose tissues are defining characteristics of obesity and its comorbidities. Expression and/or activity of stearoyl-CoA desaturase-1 (SCD1), a major regulator of lipid metabolism, is increased with obesity and the reduction/ablation of this enzyme is associated with an improved metabolic profile. Sterculic oil (SO), obtained from the seeds of the Sterculia feotida tree, contains a high concentration of cyclopropenoic fatty acids which are known inhibitors of SCD1. The purpose of this study was to determine the effects of SO supplementation on the development of obesity and insulin resistance in hyperphagic, obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Design & Methods:

Rats received either an AIN-93G diet (control) or an AIN-93G diet containing 0.5% SO for 10 weeks.

Results:

SO did not alter body weight or body composition. Importantly, the desaturase indices, a proxy for the activity of SCD1, were reduced in the liver and adipose tissue of SO supplemented animals. This reduction in SCD1 activity was associated with a reduction in fasting blood glucose concentrations and improved glucose tolerance. In addition, SO reduced intra-abdominal fat mass and adipocyte size and resulted in a ∼3-fold increase in GLUT1 gene expression in intra-abdominal fat. Liver triglyceride content and lipogenic gene expression were reduced by SO. Consistent with an improved metabolic phenotype, SO also improved plasma cholesterol, LDL-cholesterol, and triglyceride concentrations.

Conclusion:

Overall, our data demonstrate an improved metabolic phenotype with SO supplementation and suggest further studies are required to better understand the therapeutic potential of SO.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Obesity is one of the fastest growing public health problems caused by environmental and genetic factors. According to the CDC, in 2009, 26.7% of the American population was considered obese and this number is estimated to grow (1). With obesity, abnormal fatty acid metabolism occurs in which an increase in lipogenic enzymes promotes expansion of adipose tissue and deposition of ectopic fat. Increased accumulation of lipid in liver and muscle is associated with the development of insulin resistance and eventually type II diabetes (2, 3). While a range of therapies have been developed for the treatment of obesity-related type II diabetes, these pharmaceutical therapies often have adverse side effects or limited efficacy (4–7). Therefore, a continued interest exists in the identification of new drug targets and the development of novel therapeutic strategies for the treatment of obesity and insulin resistance/type II diabetes.

Stearoyl-CoA desaturase-1 (SCD1) is a major enzyme involved in the control of lipid metabolism and has emerged as a potential therapeutic target for reducing obesity and its associated metabolic complications including insulin resistance and hepatic steatosis (8–10). Initial studies using transgenic mice lacking SCD1 demonstrated that these mice are protected from diet-induced obesity as well as the development of insulin resistance (8, 10–12). In humans, increased SCD1 activity is associated with increased obesity and the metabolic syndrome (13, 14). For these reasons, there is increasing interest in the development of compounds targeting this enzyme for the treatment of obesity and its associated metabolic diseases (7, 15–17). In addition to pharmaceutical therapies, separate research efforts have focused in part on the identification and testing of nutraceuticals that will reduce the incidence of obesity and/or improve the metabolic state of obese individuals (18, 19).

A yet to be studied nutraceutical is sterculic oil, which is extracted from the seeds of the Sterculia feotida tree and has a novel fatty acid profile containing a high concentration of the cyclopropenoic fatty acids sterculic (∼55%) and malvalic (∼5%) acid (20). These fatty acids have been shown to selectively inhibit SCD1 in vitro and in vivo, suggesting they may be useful in the treatment of obesity and/or insulin resistance/type II diabetes (21, 22). The purpose of this study was to determine if sterculic oil supplementation could improve the metabolic state of hyperphagic, obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were supplemented with or without sterculic oil from 4 to 14 weeks of age, a glucose tolerance test was performed at week 13 and tissues were collected for analysis at week 14.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Experimental animals and protocols

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Missouri. OLETF and lean Long-Evans Tokushima (LETO) rats were generously provided by the Tokushima Research Institute (Tokushima, Japan). Animals were received at 4 weeks of age and maintained at a controlled temperature (22°C) and a 12-h light: 12-h dark cycle. OLETF animals were housed individually while LETO animals were housed in pairs and all animals were fed for ad libitum food intake.

Four-week old, male OLETF rats were assigned to one of two dietary treatment groups: (a) a standard AIN-93G rodent diet (OLETF) or (b) an AIN-93G diet supplemented with 0.5% sterculic oil (OLETF SO; Supporting Information Table 1). Male LETO rats were used as a lean control and fed the AIN-93G diet (LETO). Food intake and body mass were measured weekly throughout the study.

Table 1. Fatty acid composition of control and sterculic oil diets
Fatty acid, %Control1SO2
  • 1

    AIN-93G purified rodent diet.

  • 2

    AIN-93G diet containing sterculic oil at 0.5 g/100 g.

  • 3

    Malvalic acid (2-octyl-1-cyclopropene-1-octanoic acid).

  • 4

    ND; not detected.

  • 5

    Sterculic acid (2-octyl-1-cyclopropene-1-heptanoic acid).

16:010.5910.83
18:03.943.99
18:1 (n − 9)20.9720.18
18:1 (n − 7)1.411.36
18:2 (n − 6)54.3349.95
18:3 (n − 3)6.926.12
18:CE3ND40.64
19:CE5ND3.81
Other1.843.14

Diet

Sterculic oil was extracted from Sterculic foetida seeds through a Soxhlet extraction (23). Sterculic seeds were finely ground, loosely packed into an extraction thimble, and then placed in a soxhlet apparatus for extraction. Petroleum ether was heated and cycled through the apparatus for 8 h and then collected and dried using a rotary evaporator to obtain the extracted oil. The AIN-93G control diet was purchased from Dyets (Bethlehem, PA) while we manufactured and pelleted the experimental diet containing sterculic oil using ingredients consistent with the AIN-93G diet. Sterculic oil was incorporated in the diet at 0.5 g/100 g diet in place of soybean oil (Supporting Information Table 1). Therefore, the caloric content of the two diets was similar and they maintained a constant ratio of fiber, vitamins, minerals, carbohydrates, and fat, and it differed only in fatty acid profile (Table 1).

Glucose tolerance test

At 13 weeks of age, animals were fasted overnight (10-12 h) and a baseline blood sample was taken from the tail vein at time 0. Then sterile glucose (2 g/kg body weight) was injected intraperitoneally, and blood was collected for measurement of glucose and insulin at 15, 30, 45, 60, and 120 min post-injection. Blood glucose concentrations were determined using a handheld glucometer, while plasma insulin concentrations were determined using an ELISA (ultrasensitive rat insulin ELISA Kit; Crystal Chem Inc., Downers Grove, IL). Glucose and insulin area under the curve (AUC) calculations were made using GraphPad Prism 4 software.

Body composition and tissue collection

At 13 weeks of age, body composition was measured using a Hologic Discover A QDR series DEXA machine calibrated for rats. At 14 weeks of age, the animals were fasted for 6 h and sacrificed by CO2 necrosis followed by exsanguination via cardiac puncture. Plasma was isolated via centrifugation, aliquoted, and snap frozen for future analysis. Tissues (intra-abdominal fat pads and liver) were dissected, weighed, and snap frozen for analysis of protein or gene expression. A separate portion of tissue was fixed, embedded in paraffin, and sectioned for histological analysis. A portion of subcutaneous fat was processed in a similar manner and total subcutaneous fat mass was calculated by subtracting the mass of the intra-abdominal fat from the total body fat quantified by DEXA analysis.

Plasma analysis

Plasma samples were analyzed for triglycerides, cholesterol, HDL-cholesterol, LDL-cholesterol, and non-esterified free fatty acids in a commercial laboratory (Comparative Clinical Pathology Services, Columbia, MO) on an automated clinical chemistry analyzer (AU680, Beckman-Coulter, Brea, CA) using manufacturer's defined assays. The same lab quantified plasma alanine aminotransferase (ALT) utilizing an Olympus AU680 Chemistry Immuno Analyzer (Olympus America Inc., Center Valley, PA). Plasma leptin, IL-6, glucagon, and MCP-1 were determined using a Milliplex mag rat metabolic magnetic bead panel kit (Cat # RMHMAG-84 Millipore; Billerica, MA) that was analyzed using a Luminex MAGPIX system (Luminex Corporation; Houston, TX) and Milliplex Analyst software (Millipore; St. Charles, MO).

Tissue fatty acid and triglyceride analysis

Tissue fatty acids were extracted using a modified version of Folch and Bligh & Dyer. Briefly, tissues were homogenized in a 50 mM Trizma hydrochloride and 1 mM EDTA-disodium salt solution and chloroform:methanol:acetic acid (2:1:0.015, v/v/v) was added to create a phase separation. The bottom, organic layer was isolated, dried under N2, and methylated using 0.5 N sodium methoxide. Fatty acid methyl esters were analyzed using a gas chromatograph (Varian Star 3400) equipped with a 100 m, 0.25 mm I.D., and 0.20 μm film column (Supelco, Bellefonte, PA). Gas chromatograph conditions were a flow rate of 1 ml/min. with an initial temperature of 140°C held for 5 min. The column temperature was then increased to 250°C at a rate of 2°C/min, and then held at 250°C for 15 min. Fatty acid peaks were identified using pure methyl ester standards (Supelco). Liver triglyceride content was determined using a modified protocol described by Schwartz and Wolins (24). Briefly, frozen liver was extracted in PBS-10 mM EDTA buffer and protein concentration determined by a BCA protein assay (Pierce; Rockford, IL). Samples were assayed in duplicate and 25 μg of protein extract was subjected to organic extraction. TG was then quantified using a colorimetric enzyme-linked kit (Sigma; St. Louis, MO).

Real-time quantitative PCR

Adipose tissue mRNA was extracted using Qiagen RNeasy Lipid Mini kits and liver RNA was isolated using Qiagen RNeasy Mini kits according to manufacturer's instructions. mRNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from 1 μg of RNA, and real-time quantitative PCR was performed using PWR SYBR Green (StepOne plus; Applied Biosystems). Fold changes were calculated as 2-ΔΔCT with B2M used as the endogenous control.

Western analysis

Antibodies recognizing SCD1 (Alpha Diagnostic; Cat. # SCD11-A; San Antonio, TX), GLUT1 (Abcam Cat. # ab652 Cambridge, MA), and B-actin (Cell signaling; Cat. #4970; Danvers, MA) were obtained for Western blot analysis. Tissue samples were homogenized in 5% SDS-Lysis buffer and total protein quantified using a BCA assay (Pierce). Equal concentrations of protein (40 μg) were loaded per sample, separated using gel electrophoresis, and transferred to a PVDF membrane (Invitrogen, California). Membranes were incubated with the indicated antibody and horseradish-peroxidase coupled anti-species antibodies. Proteins were visualized by enhanced chemiluminescence and quantified by densitometry using AlphaView SA software version 3.2.1.0 (Cell Biosciences Inc.). Total SCD1 and GLUT1 were normalized to B-actin to verify uniform protein loading.

Statistics

Treatment differences were analyzed by one-way analysis of variance (ANOVA) with main effect significance set at p < 0.05. Significant main effects were followed by a Newman-Keuls Multiple comparison test. Values are reported as mean ± standard error.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Body weight, food intake, and body composition

Similar to previous reports (25, 26), body weight gain and food intake across the experimental period were significantly increased in the OLETF group as compared to the LETO group (Figure 1; Table 2). However, sterculic oil supplementation did not alter body weight gain or food consumption between OLETF and OLETF SO groups (Figure 1; Table 2). In addition, total body composition among the three groups was determined by DEXA analysis. We observed that fat mass was greater in the OLETF and OLETF SO groups as compared to the LETO animals and sterculic oil supplementation did not alter total body composition (Supporting Information Table 2).

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Figure 1. Sterculic oil supplementation did not alter food intake or body weight gain in OLETF rats. A: Food intake and (B) body weight gain over a 10-week experimental period. n = 8-9 per group; values are reported as mean ± SE.

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Table 2. Animal characteristics at 14 weeks of age1
VariableLETOOLETFOLETF SOp value
  • 1

    n = 8-9 per group; data are presented as means ± SEM; means with different superscripts differ.

  • 2

    AT; adipose tissue.

  • 3

    Sum of gonadal, perirenal, and mesenteric adipose tissue depots.

  • 4

    Calculated as total fat mass measured by DEXA minus total intra-abdominal AT mass.

Body weight, g391.5 ± 7.7b515.9 ± 11.7a501.2 ± 9.3a< 0.01
Food intake, g/day17.2 ± 1.3b25.0 ± 1.7a25.0 ± 1.6a< 0.01
Tissue weight, g    
 Liver12.9 ± 0.4c18.8 ± 0.7a16.4 ± 0.5b< 0.05
 Gonadal AT25.4 ± 0.3c12.8 ± 1.1a9.5 ± 0.4b< 0.05
 Mesenteric AT3.0 ± 0.2c8.8 ± 0.6a7.0 ± 0.2b< 0.05
 Perirenal AT7.3 ± 0.5b21.6 ± 0.8a19.5 ± 0.8a< 0.01
 Intra-abdominal AT315.8 ± 0.9c43.2 ± 2.4a36.0 ± 1.5b< 0.05
 Subcutaneous AT438.9 ± 2.9b124.5 ± 9.3a106.5 ± 6.3a< 0.01
Blood glucose, mg/dl78.2 ± 2.5c93.4 ± 2.2a83.6 ± 2.9b< 0.05
Insulin, ng/ml2.0 ± 0.1b3.8 ± 0.8a3.9 ± 0.3a< 0.01

Sterculic oil improves fasting glucose and glucose clearance

Consistent with an obese phenotype and previous reports (25, 26), fasting blood glucose and plasma insulin concentrations were elevated in OLETF rats as compared to LETO controls (Table 2). Sterculic oil supplementation attenuated the increase in blood glucose levels but did not prevent the increase in plasma insulin (Table 2). Significantly, despite a lack of difference in total body weight, sterculic oil supplementation resulted in a 50% improvement in glucose clearance during a glucose tolerance test (Figure 2A,C). This improvement occurred independent of a change in plasma insulin response during the tolerance test (Figure 2B,D). As anticipated, glucose clearance and insulin response during the tolerance test were most sensitive in the lean LETO control animals (Figure 2).

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Figure 2. Sterculic oil supplementation improved glucose tolerance. A: Blood glucose and (B) Serum insulin levels during an intraperitoneal glucose tolerance test. C: Glucose and (D) Insulin AUC post challenge. OLETF SO rats had a 50% reduction in glucose AUC indicating an improved ability to clear glucose from circulation. n = 8-9 per group; values are reported as mean ± SE; means with different superscripts differ by p < 0.01.

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Sterculic oil influences regional body fat distribution and metabolism

The desaturase indices are a ratio of the product and substrate fatty acids for the SCD1 enzyme (16:1/16:0 and 18:1/18:0) and can be used as a proxy for the activity of the enzyme. As mentioned above, an increase in SCD1 expression and activity, as well as the accompanying desaturase indices have previously been associated with obesity and the metabolic syndrome (14, 27). Our data are in agreement with these previous reports and demonstrate an increase in the desaturase indices in the adipose tissue of the OLETF group as compared to LETO (Figure 3). Consistent with our hypothesis that sterculic oil inhibits the activity of SCD1, we observed a reduction in the desaturation indices in both intra-abdominal and subcutaneous adipose depots (Figure 3). Furthermore, additional changes occurred to the fatty acid profile of these adipose depots including the incorporation of the cyclopropenoic fatty acids, sterculic and malvalic acid (Supporting Information Tables 3–6).

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Figure 3. Sterculic oil supplementation reduced adipose tissues desaturase indices. Desaturase indices (A) 16:1/16:0 and (B) 18:1/18:0 are the product to substrate ratio for SCD1, and serve as a proxy of SCD1 activity. n = 4 per group; values reported as mean ± SE; means with different superscripts differ by p < 0.01

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As expected, the adipose tissue depots from the lean LETO group were smaller than the corresponding depots of the OLETF and OLETF SO groups (Table 2). Interestingly, despite a lack of observed difference in total body weight or body composition, sterculic oil reduced the mass of the gonadal adipose depot by ∼25% and the mesenteric adipose depot by ∼20% when compared to the OLETF group (Table 2). We did not observe a difference in the mass of perirenal or subcutaneous adipose tissue between OLETF and OLETF SO groups. The reduction in gonadal fat pad mass was accompanied by a reduction (∼25%) in average adipocyte size within the depot and this difference was not observed in the subcutaneous adipose depot (Supporting Information Figure 1). Overall, the combined mass of the intra-abdominal fat pads was significantly less (∼17%) in OLETF SO animals as compared to the OLETF group (Table 2). Given this apparent discrepancy between the observed reduction in intra-abdominal fat and lack of difference in total body composition (Table 2; Supporting Information Table 2), it is quite possible that this minor reduction in intra-abdominal fat was not within the sensitivity range of the DEXA or perhaps this fat may have been stored elsewhere within the body. Regardless, this reduction in intra-abdominal fat mass is consistent with our observation of improved glucose tolerance (Figure 2).

The sterculic oil induced reduction in intra-abdominal fat was accompanied by a reduction in the relative gene expression of the adipokine leptin, whose expression was increased in the OLETF group but unchanged between the LETO and OLETF SO groups (Figure 4). We observed an increase in the relative gene expression of the insulin-independent glucose transporter GLUT1 in the sterculic oil supplemented animals, although total adipose tissue GLUT1 protein was not different among groups (Figure 4). Furthermore, our observation of reduced desaturase indices in OLETF SO adipose depots occurred independent of a reduction in the gene or protein expression of SCD1 (Figures 3 and 4). Relative expression of these three gene targets was unchanged in subcutaneous adipose tissue (Figure 4D).

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Figure 4. Sterculic oil supplementation altered intraabdominal adipose tissue gene expression without affecting subcutaneous adipose tissue gene expression. Sterculic oil supplementation decreased leptin gene expression and increased GLUT1 gene expression in the (A) gonadal, (B) perirenal, and (C) mesenteric intraabdominal adipose tissues depots. Relative mRNA expression of leptin, SCD1, and Glut1 were not altered in the (D) subcutaneous adipose depot. Sterculic oil supplementation did not alter Glut1 or SCD1 protein expression in (E) gonadal or (F) subcutaneous adipose depots. n = 6-9 per group; values are reported as mean ± SE; means with different superscripts differ by p < 0.05.

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Sterculic oil reduces liver triglyceride accumulation and lipogenic gene expression

Consistent with a lesser body mass, the livers of the LETO group were significantly smaller than the OLETF and OLETF SO groups (Table 2). However, when liver mass was normalized to body weight, liver weight was only increased in the OLETF group (Figure 5A). These results may be explained in part by an increase in the triglyceride content of the OLETF livers (Figure 5B). Once again, the desaturase indices indicate that SCD1 activity was reduced in the livers of the OLETF SO animals (Figure 5C). These differences in liver triglyceride content and desaturation indices were also accompanied by changes in the liver fatty acid profile among all groups (Supporting Information Table 7). Interestingly, these changes occurred independent of any changes in hepatic SCD1 gene or protein expression (Figure 5D,E). Although, sterculic oil did prevent the increase in the lipogenic genes SREBP-1c and FAS in the livers of the OLETF SO group as compared to the OLETF group (Figure 5D). Hepatic IL-6 mRNA expression did not differ among groups, while TNFα mRNA expression was increased with obesity and unaffected by sterculic oil (Figure 5D). However, plasma TNFα concentrations were below the limits of detection for all three experimental groups (data not shown). In addition, mRNA expression of the gluconeogenic genes phosphoenyl pyruvate carboxykinase (PEPCK) and glucose-6 phosphatase (G-6 Pase) was unchanged among the LETO, OLETF, and OLETF SO groups (Figure 5D). These results are consistent with previous reports demonstrating elevated blood glucose concentrations in the OLETF rat are associated with impaired glucose uptake rather than uncontrolled hepatic glucose production (25, 26).

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Figure 5. Sterculic oil reduced liver triglyceride content, desaturase indices, and lipogenic gene expression. A: Liver weight as a percentage of body weight was increased in the OLETF group and sterculic oil supplementation prevented this increase. B: Triglyceride content of the liver was increased ∼30% in the OLETF group. C: Sterculic oil reduced liver desaturase indices, a proxy of SCD1 activity. D: Sterculic oil prevented the increase in hepatic lipogenic mRNA expression but had no effect on the expression of inflammatory or gluconeogenic genes. E: Protein expression of SCD1 remained unchanged between the OLETF and OLETF SO groups. Data are presented as means ± SE; n = 8-9 per group; means with different superscripts differ p < 0.05.

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Sterculic oil improves the plasma lipid and cytokine profile

The plasma lipoprotein profile was analyzed at 14 weeks of age. Obesity was associated with an increased concentration of total cholesterol, LDL-cholesterol, and triglycerides in the OLETF group (Table 3). Sterculic oil supplementation prevented the increase in total and LDL-cholesterol while partially reducing plasma triglyceride concentrations (Table 3). The concentrations of HDL-cholesterol and nonesterified fatty acids were not different among groups (Table 3). Consistent with increased adipose tissue mass, plasma Leptin concentrations were increased in both OLETF and OLETF SO groups (Table 3). Circulating concentrations of the pro-inflammatory cytokine IL-6 were elevated with obesity in the OLETF group and sterculic oil supplementation prevented this increase (Table 3). However, concentrations of the chemoattractant MCP-1 did not differ among groups (Table 3). We also observed a small but significant increase in plasma ALT associated with obesity and unchanged by sterculic oil supplementation (Table 3). Overall, the improved plasma lipoprotein profile and reduced IL-6 concentrations are consistent with our previous data demonstrating an improved phenotype with sterculic oil consumption.

Table 3. Plasma characteristics at 14 weeks of age1
 LETOOLETFOLETF SOp value
  • 1

    n = 8-9 per group; data are presented as means ± SE; means with different superscripts differ.

Plasma proteins, ng/ml    
 Leptin5.6 ± 0.83b18.6 ± 2.7a16.6 ± 3.5a< 0.01
 MCP-10.5 ± 0.00.6 ± 0.10.4 ± 0.00.07
 IL-60.4 ± 0.0b1.0 ± 0.2a0.3 ± 0.1b< 0.01
 ALT (U/L)26.0 ± 4.8b35.1 ± 4.6a34.1 ± 2.8a< 0.05
Plasma lipids, mg/dl    
 Triglyceride138.2 ± 16.3c240.9 ± 66.0a167.3 ± 11.3b< 0.05
 Total cholesterol148.0 ± 8.0b183.9 ± 22.1a135.3 ± 7.1b< 0.01
 HDL-C35.9 ± 1.837.5 ± 3.334.1 ± 1.80.15
 LDL-C36.9 ± 3.6b54.0 ± 6.7a42.8 ± 1.7b< 0.01
 NEFA (mmol/l)0.7 ± 0.10.9 ± 0.20.9 ± 0.10.13

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Previously, it has been demonstrated that mice lacking SCD1 are protected from obesity, hepatic steatosis, and insulin resistance (8, 10, 11). More recent studies utilizing SCD1-targeted antisense oligonucleotides or pharmaceutical inhibitors of SCD1 have also reported an improved metabolic profile (16, 28, 29). Consequently, there is an increasing interest in targeting SCD1 to reduce obesity and its associated metabolic complications (7). Due to its high concentration of cyclopropenoic fatty acids, sterculic oil is a known natural inhibitor of SCD1 (21). However, the ability of sterculic oil to regulate SCD1 and treat obesity and its associated metabolic complications has not been previously investigated. Therefore, the purpose of this study was to examine the effects of sterculic oil supplementation on the development of obesity and insulin resistance in the hyperphagic, obese OLETF rat.

Consistent with previous studies reporting an association between SCD1 expression/activity and obesity (13, 27, 30), we observed an increase in the tissue SCD1 expression and desaturation indices in the obese OLETF animals (Figures 3–5). Sterculic oil supplementation prevented the increase in desaturation indices demonstrating that a partial inhibition of SCD1 was achieved in the OLETF SO rats (Figure 3 and 5). Interestingly, inclusion of sterculic oil in the diet did not alter food intake, body weight gain, or total body composition between OLETF and OLETF SO animals over the experimental period (Table 2; Supporting Information Table 2). These data differ from SCD1 null mice which consume ∼25% more food than their wild type counterparts and have significant reductions in adiposity and body weight (8, 11). Partial inhibition of SCD1 through pharmacological and antisense oligonucleotide inhibitors also had no effect on food consumption, while changes in body weight were dependent on the extent of inhibition (31, 32).

Despite a lack of difference in body weight or total body composition, sterculic oil prevented the modest increase in fasting blood glucose concentrations that occurred in the OLETF group (Table 2). Furthermore, glucose tolerance testing revealed a 50% improvement in glucose clearance in the OLETF SO group as compared to the OLETF group (Figure 2). At the same time, sterculic oil did not affect fasting plasma insulin concentrations or insulin response to the glucose challenge (Figure 2 and Table 2). Given the adverse consequences associated with chronic hyperinsulinemia, an inability of SO to reduce insulin concentrations may temper enthusiasm regarding the other metabolic improvements associated with SO supplementation. While previous studies have reported reduced SCD1 activity is accompanied by a reduction in circulating insulin concentrations, this improvement often occurs along with significant reductions in body weight and/or adiposity (8, 10, 31) which we also did not observe in the present study. It remains to be determined what effect long-term supplementation of SO will have on blood glucose and insulin concentrations as the severity of the disease state progresses.

In an attempt to better understand other metabolic changes that may have occurred with sterculic oil supplementation, we investigated several intra-abdominal fat pads along with subcutaneous adipose tissue. While we were unable to detect any changes in total body composition, sterculic oil did reduce intra-abdominal fat mass and adipocyte size, but had no effect on subcutaneous fat (Table 2; Supporting Information Figure 1). Consistent with a reduction in fat mass, sterculic oil prevented the increase in Leptin expression observed in the intra-abdominal fat of OLETF rats (Figure 4A–C). Reductions in intra-abdominal fat and Leptin expression are associated with an improved metabolic state and may help explain the improvements in fasting glucose and glucose tolerance. However, plasma concentrations of Leptin were elevated in the OLETF group and unchanged by sterculic oil supplementation (Table 3). Given the lack of difference in total adiposity between OLETF and OLETF SO animals, potential differences in Leptin production by smaller intra-abdominal fat depots may not be great enough to cause a significant reduction in circulating Leptin concentrations.

On further analysis of the intra-abdominal adipose tissue, the OLETF SO group had a significant increase (∼3-fold) in GLUT1 mRNA expression, but not protein expression, as compared to OLETF and LETO rats (Figure 4A–C, E). Inhibition of SCD1 has previously been reported to have varied effects on glucose uptake and/or glucose transporter expression. For example, Hyun et al. (16) reported that white adipose tissue specific deletion of SCD1 in mice and pharmacological inhibition of SCD1 in 3T3-L1 adipocytes caused increased glucose uptake and GLUT1 expression. However, SCD1 null mice have increased expression of GLUT4 and no changes in GLUT1 suggesting that both the tissue and extent of SCD1 inhibition may influence regional glucose transporter expression and glucose metabolism (16). In agreement with this hypothesis, partial inhibition of SCD1 in liver and adipose tissue increases glucose uptake (29), while complete inhibition of SCD1 in the liver does not protect mice from diet induced obesity or the resulting insulin resistance (32). A limitation of the current study is a lack of data related to muscle, which is a major site of glucose uptake as well as lipid oxidation/storage; future studies utilizing hyperinsulinemic euglycemic clamps and insulin signaling experiments will be required to definitively identify the target tissue(s) and mechanism(s) by which sterculic oil is improving glucose tolerance.

Another reported metabolic affect of SCD1 inhibition is protection from hepatic steatosis (11, 31, 32). In this study, liver weight was significantly different among all the three experimental groups (Table 2). However, when liver mass was normalized by body weight, there was no difference in liver mass between LETO and OLETF SO animals while OLETF liver mass was increased (Figure 5A). This increased liver mass was associated with a 30% increase in liver triglyceride content of the OLETF rats as compared to both LETO and OLETF SO rats (Figure 5B). Recently, lipid metabolites such as diacylglcerols and ceramides have been implicated in the pathogenesis of insulin resistance (33) and while not assessed in the current study, it is possible that these bioactive lipids were also reduced with sterculic oil treatment. Nonetheless, the reduced liver triglyceride content of OLETF SO animals is consistent with a partial inhibition of SCD1 (32). The desaturase indices demonstrate sterculic oil reduced the activity of SCD1 in the liver and this occurred independent of any changes in SCD1 gene or protein expression (Figure 5C–E). These data demonstrate the ability of SCD1 inhibition via sterculic oil to prevent the increase in liver triglyceride accumulation that occurs with obesity. Moreover, this reduction was associated with a reduction in the expression of the lipogenic genes SREBP-1c and FAS (Figure 5B and D). Previous groups have suggested that liver damage may occur in certain situations when SCD1 is inhibited (22). In the present plasma ALT concentrations were not elevated by sterculic oil supplementation suggesting hepatotoxicity was not a concern (Table 3). Congruent with our observations of improved glucose tolerance, reduced intra-abdominal adipose tissue and decreased hepatic steatosis, sterculic oil supplementation also improved the plasma lipid profile of OLETF SO rats. Specifically, sterculic oil prevented the increase in plasma total cholesterol and LDL cholesterol and partially reduced plasma triglyceride concentrations (Table 3). These data are consistent with previous reports of SCD1 inhibition (32, 34). However, there are also reports where unique dietary conditions in conjunction with SCD1 inhibition can cause increases in plasma total cholesterol, LDL cholesterol, and triglycerides (35, 36). In addition, we observed no effect of sterculic oil on circulating concentrations of free fatty acids or HDL cholesterol (Table 3). Previous reports have speculated that SCD1 inhibition may result in an inflammatory response (37). However, in this study, sterculic oil prevented the increase in plasma IL-6 concentrations observed in OLETF rats and MCP-1 concentrations were not different among any of the experimental groups (Table 3). Furthermore, plasma TNF-α concentrations were below the limit of detection for all the three experimental groups (data not shown).

Overall, our results provide proof of concept, that sterculic oil can be used to effectively inhibit SCD1 and improve a number of obesity-associated metabolic factors. Specifically, sterculic oil improved glucose uptake and the plasma lipid profile while reducing intra-abdominal fat mass and hepatic lipid accumulation. When calculated, the human equivalent dose of sterculic oil used in this study was 40.5 mg/kg BW (38), or just under 5 g/d for a 120 kg person. Additional studies will be required to identify the minimal effective dose, but even at this dose, human application appears feasible. We acknowledge that 14 weeks of age represents an early stage in the development of obesity-associated metabolic complications in the OLETF rat and future studies are required to determine the ability of sterculic oil to maintain an improved metabolic phenotype over an extended period. In summary, our data suggest sterculic oil may provide a novel therapeutic approach for the treatment of obesity-associated metabolic complications.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Dr. Dale Bauman at Cornell University for providing the seeds from the Sterculia feotida tree and Jim Browning at the University of Missouri for his assistance manufacturing the sterculic oil diet. We would also like to thank the following individuals at the University of Missouri for their assistance working with the animals and in the laboratory: Dr. R. Scott Rector, Dr. John Thyfault, Dr. Heather Leidy, Dr. Ingolf Gruen, Dr. E. Matthew Morris, Grace Meers, Lakdas Fernando, Anthony Belenchia, and Tim Heden.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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