| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLE |
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and Department of Medicine, University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Robert V. Farese, Jr., M.D., Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, California 94141-9100. E-mail: . bfarese{at}gladstone.ucsf.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several questions remain unanswered regarding the effects of DGAT1 deficiency on leptin sensitivity. One aim of the current studies was to determine whether DGAT1 deficiency increases leptin sensitivity in murine models of obesity and diabetes associated with perturbations in the leptin pathway. To test this hypothesis, we infused leptin into AY/a and ob/ob mice and assessed whether DGAT1 deficiency enhanced their response to leptin infusion. We also sought to determine whether the effects of DGAT1 deficiency on leptin sensitivity are mediated through the hypothalamic leptin pathway. To test this, we performed intracerebroventricular leptin infusion studies in Dgat1-/- mice. We also measured the expression of key hypothalamic molecules involved in leptin signaling and in the regulation of food intake and energy expenditure. Our results provide additional evidence that DGAT1 deficiency enhances leptin sensitivity and suggest that some of this enhancement may occur directly in peripheral tissues.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
95% C57BL/6J, 5% 129/SvJae background) were generated previously (4). Wild-type (Dgat1+/+), ob/+, and AY/a mice (all in C57BL/6J background) were from The Jackson Laboratory (Bar Harbor, ME). AY/a mice are obese and insulin resistant, reflecting the antagonism of MSH in the hypothalamus (7, 8). Genotyping was performed as described (4, 9). Mice were housed in a pathogen-free barrier facility (12-h light/12-h dark cycle) and fed rodent chow (Ralston Purina Co., St. Louis, MO). For high-fat diet experiments, mice were fed a Western-type diet containing 21% fat by weight (Harlan Teklad, Madison, WI) for 4 wk unless stated otherwise. All experiments were approved by the Committee on Animal Research of the University of California, San Francisco.
Leptin infusion studies
For Northern blot studies, mice were injected with recombinant murine leptin (Linco Research, Inc., St. Charles, MO) ip for 3 d. For other studies, mice were infused with recombinant human leptin (gift from F. Chehab, University of California, San Francisco) with a microosmotic pump (Alza model 1002, DURECT Corp., Cupertino, CA) inserted sc into the interscapular region. For intracerebroventricular infusions, a cannula (Brain Infusion Kit II, DURECT Corp.) was attached to the microosmotic pump, and the needle was inserted into the lateral ventricle as described (6). Plasma leptin levels were measured by AniLytics, Inc. (Gaithersburg, MD).
Northern blots
Brown adipose tissue (BAT) was obtained from the interscapular region. Total RNA was isolated, and pooled RNA samples (10 µg) were subjected to electrophoresis and blot hybridization with 32P-labeled cDNA probes. Uncoupling protein 1 (UCP1) probe was a gift from M. Reitman (NIH, Bethesda, MD). Blots were rehybridized with a ß-actin probe (Ambion, Inc., Austin, TX) for loading normalization. Signals were quantified with a PhosphorImager (Bio-Rad Laboratories, Inc., Hercules, CA).
Immunoblots
BAT was homogenized, and pooled protein samples (75 µg) were loaded onto a 10% polyacrylamide gel, transferred to nitrocellulose, and incubated with an anti-UCP1 polyclonal primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Binding was detected by an anti-IgG antibody-peroxidase conjugate (Santa Cruz Biotechnology, Inc.) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were reprobed with an anti-low density lipoprotein receptor-related protein antibody (a gift from J. Herz, University of Texas, Southwestern, Dallas, TX) for loading normalization.
Real-time PCR
Total RNA was isolated from white adipose tissue (WAT) and hypothalamic blocks and reverse-transcribed into cDNA (SuperScript II, Invitrogen, Carlsbad, CA). Real-time PCR was performed as described (10). Briefly, primer and probe sequences (Table 1
) were selected with Primer Express (PE Applied Biosystems, Foster City, CA). Real-time PCR was performed with the ABI Prism 7700 System (Perkin-Elmer). Expression levels were calculated by the comparative cycle of threshold detection method (Perkin-Elmer technical bulletin). Expression of ß- actin was used for loading normalization.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Similar findings were observed in weight-matched, 12- to 14-wk-old AY/a mice (weight reduction of 5.3 ± 1.7% for Dgat1-/- AY/a mice vs. 1.3 ± 1.4% for Dgat1+/+ AY/a mice on d 6, P < 0.05).
|
). These findings suggest that the increased weight loss in leptin-treated Dgat1-/- AY/a mice resulted from increased energy expenditure rather than reduced food intake.
Increased weight loss in response to peripheral leptin infusion in Dgat1-/- ob/ob mice
To test the hypothesis that DGAT1 deficiency increases leptin sensitivity in a leptin-deficient obesity model, we performed low-dose (3 µg/d) peripheral leptin infusions in Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice. In contrast to higher rates of infusion, 3 µg/d of sc leptin infusion causes only a temporary drop in food intake and does not result in continuous weight loss in ob/ob mice (12). Leptin infusion sc achieved comparable increases in plasma leptin levels in Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice (0.5 ± 0.1 vs. 0.6 ± 0.2 ng/ml, P > 0.05). Leptin infusion suppressed the normal weight gain of young (10- to 14-wk-old) Dgat1+/+ ob/ob mice (Fig. 1C). The same dose of leptin caused an additional 8% weight loss in age-matched Dgat1-/- ob/ob mice, indicating increased leptin sensitivity.
Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice ate similar amounts of food at baseline (15.0 ± 4.2 vs. 14.7 ± 0.4% of body weight, P > 0.05). After 1 d of leptin infusion, Dgat1-/- ob/ob mice showed a greater reduction in food intake than Dgat1+/+ ob/ob mice (73 ± 20% vs. 33 ± 5%, P < 0.05). On d 2-6, however, Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice had similar changes in food intake (Fig. 1D). These findings imply that the increased weight loss in leptin-treated Dgat1-/- ob/ob mice primarily resulted from increased energy expenditure rather than reduced food intake.
Increased fasting-induced weight loss in leptin-treated Dgat1-/- ob/ob mice
Because DGAT1 deficiency increases energy expenditure in mice with an intact leptin pathway (4), we hypothesized that leptin infusion would result in a higher energy expenditure in Dgat1-/- ob/ob mice than in Dgat1+/+ ob/ob mice. To estimate energy expenditure, we fasted mice for 16 h and measured the amount of weight lost during the fast. Because energy intake is eliminated, the amount of fasting-induced weight loss provides a simple approximation of energy expenditure. Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice lost similar amounts of weight after a 16-h fast (Fig. 2), suggesting a lack of difference in energy expenditure in the absence of leptin. Leptin infusion increased the fasting-induced weight loss by approximately 15% in Dgat1+/+ ob/ob mice but by approximately 70% in Dgat1-/- ob/ob mice. As a result, leptin-treated Dgat1-/- ob/ob mice lost significantly more weight than leptin-treated Dgat1+/+ ob/ob mice after fasting. These results are similar to those observed in fasted Dgat1-/- and Dgat1+/+ mice (not shown) and provide additional evidence that leptin infusion caused a greater increase in energy expenditure in Dgat1-/- ob/ob mice than in Dgat1+/+ ob/ob mice.
|
(PPAR) in WAT, and measured their expression in Dgat1-/- AY/a and Dgat1-/- ob/ob mice.
DGAT1 deficiency was associated with increased UCP1 expression during high-fat feeding and in AY/a mice, models in which DGAT1 deficiency confers protection against obesity (Fig. 3A). In contrast, DGAT1 deficiency had no effect on the low level of UCP1 expression in ob/ob mice. Leptin treatment, however, significantly increased UCP1 expression in Dgat1-/- ob/ob mice but had minimal effects on UCP1 expression in Dgat1+/+ ob/ob mice. These results support the notion that DGAT1 deficiency enhanced the activation of the leptin pathway and that this occurred only in the presence of leptin.
|
expression in WAT during high-fat feeding and in AY/a mice (Fig. 3B). In contrast, Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice had similarly low levels of PPAR expression, supporting the hypothesis that the effects of DGAT1 deficiency on gene expression required the presence of leptin.
Lack of enhanced response to intracerebroventricular leptin infusion in Dgat1-/- mice
In both previous (5) and current studies, DGAT1 deficiency predominantly enhanced the effects of leptin on increasing energy expenditure without a comparable effect on reducing food intake. These results suggest that DGAT1 deficiency may directly modulate peripheral leptin sensitivity. To test this hypothesis, we infused leptin into the central nervous system at doses (5-20 ng/d) that do not increase peripheral circulating levels of leptin (6). In agreement with published data (6), 5 ng/d of leptin had no effect on the body weight of Dgat1-/- and Dgat1+/+ mice (Fig. 4, A and B). Higher doses of leptin suppressed the normal growth curves of young (10- to 14-wk-old) Dgat1-/- and Dgat1+/+ mice to a similar extent. Dgat1-/- and Dgat1+/+ mice also had comparable levels of reduction in food intake in response to central leptin infusions (Fig. 4, C and D). Similar experiments in AY/a and ob/ob mice showed that DGAT1 deficiency did not enhance the response to intracerebroventricular leptin infusion in these two obesity models (not shown).
|
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The enhanced weight loss in response to peripheral leptin infusion in Dgat1-/- AY/a and Dgat1-/- ob/ob mice provides additional evidence that DGAT1 deficiency increases leptin sensitivity. The results in Dgat1-/- AY/a mice are especially striking. Because of their impaired melanocortin-4 receptor signaling, both young and adult AY/a mice normally are highly resistant to leptin and do not respond to peripheral leptin administration (6, 11). The increased leptin sensitivity in adult Dgat1-/- AY/a mice may reflect their decreased adiposity (5) or decreased plasma leptin levels at baseline. However, we observed a similar, enhanced response to leptin infusion in young Dgat1-/- AY/a mice, which had a mean body weight similar to that of age-matched Dgat1+/+ AY/a mice. Leptin sensitivity was also increased in Dgat1-/- ob/ob mice, whose mean body weight and total fat pad content are similar to those of age-matched Dgat1+/+ ob/ob mice (5). Furthermore, the results in Dgat1-/- ob/ob and Dgat1+/+ ob/ob mice are not confounded by a difference in baseline plasma leptin levels. Thus, these results suggest that the increased leptin sensitivity associated with DGAT1 deficiency did not result from decreased adiposity or decreased plasma leptin levels per se but may be due to DGAT1 deficiency itself.
The lack of an enhanced response to intracerebroventricular leptin infusion in Dgat1-/- mice was somewhat surprising and contrary to the traditional model of leptin action. Leptin, an adipocyte-derived peptide hormone, regulates energy expenditure and food intake primarily through receptors in the hypothalamus (13, 14). If DGAT1 deficiency increased leptin sensitivity by up-regulating the hypothalamic leptin pathway, central leptin infusion should have resulted in an enhanced response in Dgat1-/- mice. One possible explanation for this lack of effect is that central leptin infusion, unlike peripheral leptin infusion, results in an all-or-none response (6). Thus, an increase in leptin sensitivity (i.e. a shift to the right in the dose-response curve) in Dgat1-/- mice may not be detected. Another possibility is that intracerebroventricular and peripheral leptin infusions activate different populations of neurons in the hypothalamus and that only neurons activated by peripheral infusion are modulated by DGAT1 deficiency.
Nonetheless, this lack of enhanced response to intracerebroventricular leptin infusion suggests that DGAT1 deficiency may enhance leptin action at a site other than the hypothalamus, perhaps directly in peripheral tissues. Leptin has direct, peripheral effects on lipolysis (15), adipogenesis (16), and fatty acid oxidation (17, 18). Because DGAT1 is expressed in peripheral tissues that have important roles in energy and lipid metabolism (e.g. skeletal muscle, liver, WAT) (2), DGAT1 deficiency may enhance peripheral leptin sensitivity. This hypothesis is also supported by our findings that Dgat1-/- and Dgat1+/+ mice had similar levels of expression of key hypothalamic genes involved in leptin signaling and in the regulation of food intake and energy expenditure. Although these findings do not exclude small changes in gene expression within specific hypothalamic nuclei, they provide additional evidence that DGAT1 deficiency enhances leptin sensitivity in a location other than the hypothalamus. An increase in peripheral leptin sensitivity may also help to explain how DGAT1 deficiency primarily enhances the effect of leptin on increasing energy expenditure without a comparable effect on reducing food intake.
In summary, DGAT1 deficiency enhanced the response to peripheral leptin infusion in Agouti yellow and leptin-deficient mice, two genetic models of obesity and insulin resistance. These results provide additional evidence that DGAT1 deficiency increases leptin sensitivity. Further, our results suggest that DGAT1 deficiency does so in part through a peripheral mechanism. Because most human obesity is associated with leptin resistance, the ability of DGAT1 deficiency to increase leptin sensitivity in a murine model of leptin resistance supports the idea that pharmacological inhibition of DGAT1 may be an effective therapy for human obesity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: BAT, Brown adipose tissue; DGAT1, acyl coenzyme A:diacylglycerol acyltransferase; PPAR, peroxisome proliferator-activated receptor ; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Received February 20, 2002.
Accepted for publication April 10, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. J. Stone, M. C. Levin, and R. V. Farese Jr. Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2 J. Biol. Chem., December 29, 2006; 281(52): 40273 - 40282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Streeper, S. K. Koliwad, C. J. Villanueva, and R. V. Farese Jr. Effects of DGAT1 deficiency on energy and glucose metabolism are independent of adiponectin Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E388 - E394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. E. Yen, M. Monetti, B. J. Burri, and R. V. Farese Jr. The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters J. Lipid Res., July 1, 2005; 46(7): 1502 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yamazaki, E. Sasaki, C. Kakinuma, T. Yano, S. Miura, and O. Ezaki Increased Very Low Density Lipoprotein Secretion and Gonadal Fat Mass in Mice Overexpressing Liver DGAT1 J. Biol. Chem., June 3, 2005; 280(22): 21506 - 21514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Turkish, A. L. Henneberry, D. Cromley, M. Padamsee, P. Oelkers, H. Bazzi, A. M. Christiano, J. T. Billheimer, and S. L. Sturley Identification of Two Novel Human Acyl-CoA Wax Alcohol Acyltransferases: MEMBERS OF THE DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) GENE SUPERFAMILY J. Biol. Chem., April 15, 2005; 280(15): 14755 - 14764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Chen and R. V. Farese Jr Inhibition of Triglyceride Synthesis as a Treatment Strategy for Obesity: Lessons From DGAT1-Deficient Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 482 - 486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Stone, H. M. Myers, S. M. Watkins, B. E. Brown, K. R. Feingold, P. M. Elias, and R. V. Farese Jr. Lipopenia and Skin Barrier Abnormalities in DGAT2-deficient Mice J. Biol. Chem., March 19, 2004; 279(12): 11767 - 11776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Cao, J. Lockwood, P. Burn, and Y. Shi Cloning and Functional Characterization of a Mouse Intestinal Acyl-CoA:Monoacylglycerol Acyltransferase, MGAT2 J. Biol. Chem., April 11, 2003; 278(16): 13860 - 13866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. C. Chen, Z. Ladha, S. J. Smith, and R. V. Farese Jr. Analysis of energy expenditure at different ambient temperatures in mice lacking DGAT1 Am J Physiol Endocrinol Metab, January 1, 2003; 284(1): E213 - E218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Chen, S. J. Stone, P. Zhou, K. K. Buhman, and R. V. Farese Jr Dissociation of Obesity and Impaired Glucose Disposal in Mice Overexpressing Acyl Coenzyme A:Diacylglycerol Acyltransferase 1 in White Adipose Tissue Diabetes, November 1, 2002; 51(11): 3189 - 3195. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
