HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by STANDRIDGE, M. Articles by MOUSTAID-MOUSSA, N. Search for Related Content PubMed PubMed Citation Articles by STANDRIDGE, M. Articles by MOUSTAID-MOUSSA, N.

Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats

MELISSA STANDRIDGE^*, RAMIN ALEMZADEH, MICHAEL ZEMEL^*, JOHN KOONTZ and NAIMA MOUSTAID-MOUSSA^*1

Departments of
^* Nutrition and
Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville Tennessee 37996, USA; and
Department of Pediatrics, University of Tennessee, Medical Center, Knoxville, Tennessee 37920, USA

¹Correspondence: University of Tennessee, 1215 Cumberland Ave., Department of Nutrition JHB 229, Knoxville, Tennessee 37996-1900, USA. E-mail:moustaid{at}utk.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have previously reported that attenuation of hyperinsulinemia by diazoxide (DZ), an inhibitor of glucose-mediated insulin secretion, increased insulin sensitivity and reduced body weight in obese Zucker rats. These findings prompted us to investigate the effects of DZ on key insulin-sensitive enzymes regulating adipose tissue metabolism, fatty acid synthase (FAS), and lipoprotein lipase (LPL), as well as on circulating levels of leptin. We also determined the direct effects of diazoxide on FAS in 3T3-L1 adipocytes. Seven-week-old female obese and lean Zucker rats were treated with DZ (150 mg/kg/d) or vehicle (C, control) for a period of 6 wk. Changes in plasma parameters by DZ include significant decreases in triglycerides, free fatty acids, glucose, and insulin, consistent with our previous reports. DZ obese rats exhibited lower plasma leptin levels (P<0.03) compared to their C animals. DZ significantly reduced adipose tissue FAS activity in both lean (P<0.0001) and obese (P<0.01) animals. LPL mRNA content was also decreased significantly in DZ-treated obese animals (P<0.009) as compared to their respective controls without a significant effect on lean animals. The possibility that DZ exerted a direct effect on adipocytes was further tested in cultured 3T3-L1 adipocytes. Although diazoxide (5 µM) alone did not change FAS activity in cultured 3T3-L1 adipocytes, it significantly attenuated insulin’s effect on FAS activity (P<0.001). We demonstrate that DZ regulates key insulin-sensitive enzymes involved in regulation of adipose tissue metabolism. These findings suggest that modification of insulin-sensitive pathways can be therapeutically beneficial in obesity management.—Standridge, M., Alemzadeh, R., Zemel, M., Koontz, J., Moustaid-Moussa, N. Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats

Key Words: lipoprotein lipase • fatty acid synthase • insulin • glucose • adipocytes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
HYPERINSULINEMIA, INSULIN RESISTANCE, and hyperlipidemia are characteristic features of obesity in humans and experimental animals (1 2 3 4) . Hyperinsulinemia and insulin resistance are believed to cause preferential shunting of substrates to adipose tissue and conversion of preadipocytes to adipocytes. This is associated with hypertrophy and hyperplasia of fat cells, inducing an unabated lipogenic state and obesity (5) . Further, it has been demonstrated that insulin plays a major role in modulation of key genes in lipid metabolism and triglyceride storage including fatty acid synthase (FAS) (6) , lipoprotein lipase (LPL) (7 , 8) , and leptin (9) . FAS catalyzes the synthesis of long chain fatty acids, palmitate from acetyl-CoA, and malonyl-CoA in the presence of NADPH (10) . FAS concentrations in hepatic and adipose tissues are highly sensitive to nutritional, hormonal, and developmental states (10 11) . Fasting in rats leads to diminished synthesis of FAS whereas refeeding a high-carbohydrate diet or insulin treatment increases FAS synthesis (12) . This gene is primarily regulated at the transcriptional level (13 , 14) . Another key enzyme involved in triglyceride accumulation is LPL, which hydrolyzes the triacylglycerol component of circulating lipoproteins and provides substrates for fatty acid uptake into adipose tissue. In obese Zucker (fa/fa) animals, LPL decreases in response to fasting (15) and is up-regulated after refeeding a high-carbohydrate diet or by insulin treatment (16) . LPL is regulated both at the transcriptional and protein level (15 16) . Leptin, the product of the ob gene primarily expressed in adipose tissue, is an adipocyte hormone that is secreted in response to food intake and acts centrally as a satiety factor (17 18 19) . The amount of circulating leptin is highly correlated with adiposity (20) .

We have previously demonstrated that attenuation of hyperinsulinemia in obese Zucker rats by diazoxide (DZ), an inhibitor of glucose-mediated insulin secretion, resulted in decreased rate of weight gain, enhanced adipocyte insulin receptor binding, and improved glucose tolerance (21 , 22) . This was associated with marked reduction of postabsorptive plasma triglyceride (TG) levels in DZ-treated obese animals. In these studies, food intake did not appear to be significantly changed by DZ. To identify potential cellular mechanisms accounting for the reduction in adiposity and plasma TG concentrations by DZ, we studied the effects of this drug on key markers of adiposity (namely, FAS, LPL, and leptin) in obese and lean Zucker rats. Furthermore, we tested the direct effects of DZ on adipocyte metabolism using cultured 3T3-L1 adipocytes. Our study demonstrates that DZ exerts direct effects on adipose tissue by decreasing the lipogenic effect of insulin, thus accounting in part for its weight-reducing effects.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals
Seven-week-old female Zucker obese (fa/fa) rats (190 to 246 g) and lean (Fa/?) rats (123 to 156 g) were used in this study. Animals were phenotyped at 4 wk of age on the basis of body weight and obtained at 6 wk of age from Charles River Laboratory (Wilmington, Mass.). The animals were housed in pairs in standard animal cages and were provided standard RMH 3000 rat chow (Agway, Syracuse, N.Y.) and water ad libitum. Obese and lean rats were divided into two subgroups: DZ-treated and control (C) subgroups. Diazoxide (150 mg/kg/d) was administered in two doses daily by gavage using Proglycem pediatric suspension (50 mg/ml; kindly provided by Baker-Norton Pharmaceuticals, Miami, Fla.). The control group was treated with an equivalent volume of vehicle suspension twice daily. Studies lasted for a period of 6 wk.

At the end of the 6 wk period and after an overnight fast (12 h), rats were anesthetized with an intramuscular (i.m.) injection of ketamine (65 to 100 mg/kg body weight). Blood was drawn into heparinized tubes by cardiac puncture and plasma was frozen. Omental fat was harvested for analysis. Animal procedures were reviewed and approved by the University of Tennessee Animal Care and Use Committee.

Plasma parameters
Glucose level was measured by the glucose oxidase method (Sigma Chemical, St. Louis, Mo.). Insulin concentration was determined by radioimmunoassay (RIA) using a double-antibody method (Linco Research, St. Louis, Mo.). Leptin was assayed in plasma with double antibody RIA using guinea pig anti-rat leptin, ¹²⁵I-labeled rat leptin as tracer, and rat leptin as standard (Linco Research). Triglyceride and cholesterol levels were measured by enzymatic methods using kits purchased from Sigma Diagnostics. Plasma free fatty acids (FFA) were determined by an enzymatic colorimetric method (Wako Chemicals, Richmond, Va.).

FAS activity
Adipose tissue was homogenized in 250 mM sucrose buffer. Fatty acid synthase activity was assayed spectrophotometrically in cytosolic extracts of adipose tissue by measuring the oxidation rate of NADPH, as previously described (23) . Data were expressed as nanomoles of NADPH oxidized/(min·mg) of cytosolic protein, which was assayed by the method of Bradford (24) .

LPL Northern analysis
RNA was isolated by centrifugation of adipose tissue homogenates using the cesium chloride density gradient method and analyzed by Northern blotting, as we previously described (25) . Membranes were hybridized with ³²P-labeled cDNA probes for LPL (kindly provided by Dr. S. Fried, Rutgers University, N.J.) and 18S (Promega, Madison, Wis.). Unbound probe was removed by washing membranes in 2x saline-sodium phosphate-EDTA (SSPE) for 45 min at 25°C and then in 0.1x SSPE-0.1% sodium dodecyl sulfate for 60 min at 65°C. After washing, membranes were exposed to X-ray film (Dupont, Wilmington, Del.). Autoradiograms were analyzed by densitometric scanning, and data were expressed as a ratio of LPL to 18S.

3T3-LI cell culture
3T3-L1 cells were grown and differentiated as described previously (23) . Briefly, cells were grown to confluence in standard medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum). At confluency, cells were induced to differentiate by the addition of dexamethasone (250 nM) and iso-butylmethlyxanthine (0.5 mM) to standard medium for 72 h. Cells were maintained for three additional days in standard medium, then changed to serum-free medium (containing 1% bovine serum albumin), followed by treatment with diazoxide (5 µM) and/or insulin (10 nM) as indicated in the figure legends.

Statistics
The reported values represent the mean ± SD. Statistical analysis of subgroup was preformed by one-way analysis of variance, with significant differences between means determined by post hoc analysis using Dunnett’s mutiple range test (see Tables 1 and 2 ) and General linear model univarate analysis of variance (see Figs. 1 2 3 4B ) at P<0.05.

View larger version (12K): [in this window] [in a new window]   Figure 1. Effects of DZ on plasma leptin levels. Lean (C, control, n=14; DZ, diazoxide, n=12) and obese (C, n=22; DZ, n=16) Zucker rats were treated for 6 wk. Data are the mean ±SD. Lean control vs. obese control *(P<0.001) and control obese vs. diazoxide-treated obese **(P<0.03).

View larger version (9K): [in this window] [in a new window]   Figure 2. Effects of DZ on FAS enzyme activity. Adipose tissue was harvested from control (C, n=3) and diazoxide (DZ, n=3) -treated obese and lean Zucker rats. FAS was assayed in cytosolic extracts and enzymatic activity is expressed as nanomoles of NADPH oxidized/(min·mg protein). Data are the mean ±SD. *Lean control vs. DZ lean (P<0.0001), **lean control vs. obese control (P<0.0001), and ***control obese vs. DZ obese (P<0.01).

View larger version (9K): [in this window] [in a new window]   Figure 3. Effects of DZ on FAS enzyme activity in 3T3-L1 adipocytes. Adipocytes were harvested from control (C) (n=3), insulin (10 nM) (n=3), diazoxide (DZ) (5 µM) (n=3), or a combination of insulin (10 nM)+ diazoxide (5 µM) -treated (n=3) 3T3-L1 adipocytes. FAS was assayed in cytosolic extracts, and enzymatic activity is expressed as nanomoles of NADPH oxidized/(min·mg protein). Data are the mean ±SD. *(P<0.001) vs. all other treatments and control vs. insulin-treated**(P<0.001).

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of diazoxide (DZ) on LPL mRNA from control (C, n=3) and DZ (n=3) lean and obese. A) RNA was isolated and analyzed by Northern blot, which was normalized to 18S. A representative Northern blot is shown. B) Densitometric scanning of autoradiograms from obese control (C, n=3) and DZ-treated obese (n=3) *(P<0.01). Due to the low expression of LPL mRNA in the lean animals, the values from densitometric scanning are not reported in panel B.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effects of diazoxide on weight
Table 1 shows initial, final, and changes in animal weights over the period of this study. As seen in our previous studies, control lean and control obese show a significant difference (P<0.001) in body weight gain. In final weight measurements, DZ obese animals show significant differences when compared to strain control (P<0.001).

Effects of diazoxide on plasma glucose, lipids, and insulin
Table 2 shows postabsorptive plasma levels of glucose, FFA, cholesterol, TG, and insulin after 6 wk of C (control) or DZ (diazoxide) treatment. As expected, postabsorptive plasma glucose and insulin concentrations were significantly higher among C obese animals compared with C lean rats. Postabsorptive plasma levels of TG (P<0.001), FFA (P<0.001), glucose (P<0.001), and insulin (P<0.001) were significantly decreased in DZ obese as compared with C obese. In lean animals, however, only plasma FFA (P<0.001) and insulin (P<0.001) were significantly decreased by DZ treatment (P<0.001). Plasma cholesterol concentrations were not significantly affected by DZ treatment in either obese or lean animals.

Effects of diazoxide on plasma leptin
Plasma leptin levels were dramatically elevated in obese vs. lean (P<0.001). There were no significant differences in lean animals treated with DZ when compared to lean control animals. However, DZ did significantly decrease plasma leptin levels of obese treated animals compared to obese control animals (P<0.03) (Fig. 1).

Effects of diazoxide on FAS enzyme activity in lean and obese animals
Adipose tissue FAS enzyme activity was significantly higher in C obese rats than C lean animals (P<0.0001). DZ treatment decreased adipose tissue FAS enzyme activity in both lean (P<0.0001) and obese (P<0.01) rats as compared with their respective control animals (Fig. 2 ).

Effects of diazoxide on FAS enzyme activity in 3T3-L1 adipocytes
To test whether diazoxide exerts a direct effect on FAS activity, cultured 3T3-L1 cells were treated with 5 µM diazoxide. In cultured 3T3-L1 cells, no direct effects of diazoxide (5 µM) on FAS activity were observed. DZ significantly attenuated insulin’s effect on FAS activity (P<0.001) (Fig. 3 ).

LPL mRNA content
As previously reported, the control lean animals expressed dramatically less LPL mRNA than did their C obese counterparts (Fig. 4 ). DZ obese rats showed a markedly lower LPL mRNA content than C obese animals (P<0.01) (Fig. 4B ). However, DZ treatment did not affect LPL mRNA in lean animals as compared with their controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Consistent with our previous reports, DZ treatment resulted in a significant reduction of plasma FFA in both obese and lean rats, but decreased triglyceride storage in adipose tissue and plasma TG concentration only in obese animals. Further, DZ treatment caused a significant improvement in postabsorptive plasma glucose concentration in obese rats without a significant effect in the lean animals (21 , 22) and suppressed insulin secretion in both strains.

It has been suggested that in young obese Zucker rats, the development of hyperinsulinemia leads to an enhanced lipogenic state (26) . Insulin hypersecretion is believed to precede the development of hyperphagia, although this does not appear to be necessary for the early increase in weight found in preweaning obese rats (27) . This is supported by previous observations that food restriction in obese rats results in increased energy efficiency (28) . Insulin can act as a satiety signal, and brain insensitivity to the effect of insulin in hyperinsulinemic obese animals may lead to the development of hyperphagia (29) . In our previous studies, we found that DZ reduces weight gain, improve insulin sensitivity, and reduces the rate of fat production, yet may not produce a major effect on feeding behavior in either obese or lean rats (21 , 22) . We have recently reported differences in feeding behavior between DZ obese and control obese groups during the first 2 wk of DZ treatment, when body weight differences were emerging (30) . In the current and earlier studies, however, food intake was measured during the final week of drug treatment when the shift in metabolic and behavioral controls may already have occurred. Our recent studies demonstrate that food intake of DZ obese rats was markedly decreased as compared with control obese between 7 and 11 wk of age (30) . Maggio and Vasselli (31) demonstrated that suppression of hyperinsulinemia by DZ in obese Zucker rats resulted in decreased food intake and rate of weight gain and suggested that hyperinsulinemia contributed to both obesity and hyperphagia in the Zucker rat.

The synthesis and storage of fat involves the interaction of nutrients, hormonal factors, and key regulatory enzymes such as fatty acid synthase. Hyperinsulinemic animals are characterized by enhanced adipose tissue FAS activity (32) contributing to fat accretion in an obesity state. Similarly, insulin increases FAS activity and gene transcription in human adipocytes (6) . Therefore, attenuation of circulating insulin by diazoxide can potentially reduce adipose tissue FAS activity and lipid storage in obese subjects. In our study, parallel to the decreased insulinemia, DZ treatment resulted in decreased adipose tissue FAS activity and plasma FFA levels in both lean and obese animals. Further, it is possible that the anti-obesity effect of DZ may be at least partly due to its extrapancreatic effects in peripheral tissue, and therefore may be in part independent of its insulin-lowering action. In evaluating the direct effect of DZ on adipocytes, we recently demonstrated that DZ-induced membrane hyperpolarization resulted in indirect inhibition of Ca²⁺ influx, thereby causing decreased lipogenesis and increased lipolysis in primary cultures of human adipocytes (33) . We have previously shown that the product of the obesity gene, agouti, regulates adipocyte intracellular Ca²⁺ and stimulates FAS activity via a Ca²⁺-dependent process (23 , 34) . This was supported by studies demonstrating that treatment of obese yellow (A^vy/a) mice with nifedipine, a Ca²⁺ channel blocker, resulted in a significant decrease in fat pad weights and adipose FAS activity (35) . In the present study, although DZ alone failed to decrease FAS activity in 3T3-L1 adipocytes, it completely inhibited insulin-induced FAS activity. Our recent report that adipocytes express sulfonylurea receptors and adipocyte energy storage may be modulated by this receptor (33) supports this mechanism. Moreover, the sulfonylurea K⁺[ATP] antagonist glibenclamide, which depolarizes ß cells and thereby stimulates insulin release, also increased intracellular Ca²⁺ in adipocytes (33) . Glibenclamide also caused a comparable stimulation of FAS activity, which was inhibited by DZ and the calcium channel antagonist nifedipine (33) . Thus, antagonism of K⁺[ATP] channels stimulates Ca²⁺ influx and, consequently, lipogenesis, whereas the K⁺[ATP] channel agonist DZ antagonizes these effects and presumably inhibits lipogenesis. These data strongly support diazoxide-induced antagonism of insulin-stimulated adipocyte lipogenesis and suppression of lipolysis, possibly coupled with suppression of insulin release as a likely mechanism for the anti-obesity effects of diazoxide (33) .

Fat cell hypertrophy and increased adipose LPL activity are the earliest manifestations of obesity in hyperinsulinemic obese Zucker rats (36) . Insulin regulation of LPL activity is well documented (7 , 8) . In our study, the suppression of plasma insulin resulted in significant reduction of adipose LPL mRNA in obese but not lean animals. When given with a meal, diazoxide has been shown to inhibit insulin secretion (37) . Studies by Picard et al. (38) have demonstrated that insulin secretion is necessary for the full expression of the response of LPL in adipose tissue and that when food intake is controlled, DZ is still capable of reducing LPL in adipose tissue (38) . Our study shows that decreasing insulin levels by treatment with DZ causes a reduction in LPL in obese animals. This decreased insulinemia was also associated with significant reduction of plasma triglyceride concentration only in DZ obese animals. The lack of treatment effect on LPL mRNA in lean rats may be due to the absence of significant changes in the rate of weight gain, triglycerides, and adiposity in these animals.

In our study, plasma leptin concentrations were significantly higher in obese than in lean rats, as documented in other obesity models (19 , 20) . DZ treatment resulted in a significant suppression of circulating leptin in obese rats presumably as a result of decreased adiposity. Since ob gene expression has previously been shown to be increased by insulin, it is also likely that marked reduction in circulating leptin levels in DZ obese is a consequence of decreased ob gene transcription after decreased insulinemia and decreased rate of weight gain and adiposity.

In conclusion, we postulate that diazoxide-induced antagonism of adipocyte lipogenesis, and possibly coupled with partial normalization of insulin levels, resulted in a reversal of increased FFA and triglyceride synthesis. DZ suppression of insulin release (37) , its early anorectic effects (30) , and its direct anti-insulin effects on adipocytes via recently documented sulfonylurea receptors (33) are likely mechanisms for the anti-obesity effects of diazoxide. The combined effect resulted in decreased rate of weight gain.

	FOOTNOTES

 
Received for publication June 4, 1999. Revised for publication October 13, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Jeanrenaud, B. (1978) Hyperinsulinemia in obesity syndromes: its metabolic consequences and possible etiology. Metabolism 12,1881-1892
2. Bray, G. A., York, D. A. (1979) Hypothalamic and genetic obesity in experimental animals. An autonomic endocrine hypothesis. Physiol. Rev. 59,719-809[Free Full Text]
3. Penicaud, L., Kinebanyan, M. F., Ferre, P., Morin, J., Kande, J., Smadja, C., Marfaing-Jallat, P., Picon, L. (1989) Developmental of VMH obesity: in vivo insulin secretion and tissue insulin sensitivity. Am. J. Physiol. 257,E255-E260[Abstract/Free Full Text]
4. Le Stunff, C, Bougneres, P. (1994) Early changes in postprandial insulin secretion, not in insulin sensitivity, characterize juvenile obesity. Diabetes 43,696-702[Abstract]
5. Caro, J. F., Dohm, L. G., Pories, W. J., Sinha, M. K. (1989) Cellular alteration in liver, skeletal muscle, adipose tissue responsible for insulin resistance in obesity and type II diabetes. Diabetes Metab. Rev 5,665-689[Medline]
6. Claycombe, K. J., Jones, B. H., Standridge, M. K., Guo, Y., Chun, J. T., Taylor, J. W., Moustaid-Moussa, N. (1998) Insulin increases fatty acid synthase gene transcription in human adipocytes. Am. J. Physiol. 274,R1253-R1259[Abstract/Free Full Text]
7. Ewart, S. H., Carroll, R., Severson, D. L. (1997) Lipoprotein lipase activity in rat cardiomyocytes is stimulated by insulin and dexamthasone. Biochem. J. 327,439-442
8. Appel, B., Fried, S. K. (1992) Effects of insulin and dexamethasone on lipoprotein lipase in human adipose tissue. Am. J. Physiol. 262,E695-E699[Abstract/Free Full Text]
9. Kim, J. B., Wright, M., Yao, K. M., Mueller, E., Solanes, G., Lowell, B. B., Spiegelman, B. M. (1998) Nutritional and insulin regulation of fatty acid synthase and leptin gene expression through ADD1/SREBP1. J. Clin. Invest. 101,1-9[Medline]
10. Wakil, S. J., Stoops, J. K., Joshi, V. C. (1983) Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52,579-586
11. Volpe, J. J., Vagelos, P. R. (1976) Mechanisms and regulation of biosynthesis of saturated fat. Physiol. Rev. 56,339-417[Free Full Text]
12. Lakshmanan, M. R., Nepokroeff, C. M., Porter, J. W. (1972) Control of the synthesis of fatty acid synthetase in rat liver by insulin, glucagon, and adenosine 3:5' cyclic monophosphate. Proc. Natl. Acad. Sci. USA 69,3516-3519[Abstract/Free Full Text]
13. Paulauskis, J. D., Sul, H. S. (1988) Cloning and expression of mouse fatty acid synthase and other specific mRNAs. Developmental and hormonal regulation in 3T3–L1 cells. J. Biol. Chem. 263,7049-7054[Abstract/Free Full Text]
14. Paulauski, J. D., Sul, H. S. (1989) Regulation of mouse fatty acid synthase in liver. J. Biol. Chem. 264,574-577[Abstract/Free Full Text]
15. Cryer, A. (1981) Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Int. J. Biochem. 13,525-541[Medline]
16. Kraemer, F. B., Takeda, D., Natu, V., Sztalryd, C. (1998) Insulin regulates lipoprotein lipase activity in rat adipose cells via wortmannin- and rapamycin-sensitive pathways. Metabolism 47,555-559[Medline]
17. Caro, J. F., Sinha, M. K., Kolaczynski, J. W., Zhang, P. L., Considine, R. V. (1996) Leptin: the tale of an obesity gene. Diabetes 45,1455-1462[Medline]
18. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J. M. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature (London) 372,425-432[Medline]
19. Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hetch, R., Winters, D., Boone, T., Collins, F. (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269,540-543[Abstract/Free Full Text]
20. Halaas, J. L., Boozer, C., Blair-West, J., Fidahusein, N., Denton, D. A., Friedman, J. M. (1997) Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Natl. Acad. Sci. USA 16,8878-8883
21. Alemzadeh, R., Slonim, A. E., Zdanowicz, M. M., Maturo, J. (1993) Modification of insulin resistance by diazoxide in obese Zucker rats. Endocrinology 133,705-712[Abstract]
22. Alemzadeh, R., Jacobs, W., Pitukcheewanont, P. (1996) Antiobesity effects of diazoxide in obese Zucker rats. Metabolism 45,334-341[Medline]
23. Jones, B. H., Kim, J. H., Zemel, M. B., Woychik, R. P., Michaud, E. J., Wilkison, W. O., Moustaid, N. (1996) Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity. Am. J. Physiol. 270,E192-E196[Abstract/Free Full Text]
24. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem. 72,248-254[Medline]
25. Moustaid, N., Sul, H. S. (1991) Regulation of expression of the fatty acid synthase gene in 3T3–L1 cells by differentiation and triiodothyronine. J. Biol. Chem. 266,18550-18554[Abstract/Free Full Text]
26. Jeanrenaud, B. (1979) An overview of experimental models of obesity. Recent Adv. Obes. Res. 2,111-122
27. Tenenbaum, R., Ochoa, F. Y., Sassano, M. L., Chua, S. C., Leibel, R. L., Campfield, L. A. (1994) Altered insulin secretion from the pancreas of pro-obese Zucker rat pups: evidence for an early and persistent genetic defect. Int. J. Obes. 18 (abstr.),67
28. Zucker, L. M. (1975) Efficiency of energy utilization by the Zucker hereditarily obese rat ‘fatty’. Proc. Soc. Exp. Biol. Med. 148,498-500[Abstract]
29. Woods, S. C., Porte, D., Jr (1983) The role of insulin as a satiety factor in the central nervous system. Adv. Metab. Disord. 10,457-468[Medline]
30. Alemzadeh, R., Holshouser, S. (1999) Effects of diazoxide on brain capillary insulin receptor binding and food intake in hyperphagic obese Zucker rats. Endocrinology
31. Maggio, C. A., Vasselli, J. R. (1990) Insulin hypersecretion contributes to hyperphagia and obesity in the Zucker rat. FASEB J 4 (abstr.),A511
32. Guichard, C., Dugail, I., Le liepve, X., Lavau, M. (1992) Genetic regulation of fatty acid synthase expression in adipose tissue: overexpression of the gene in genetically obese rats. J. Lipid Res. 33,679-687[Abstract]
33. Shi, H., Moustaid-Moussa, N., Wilkison, W. O., Zemel, M. B. (1999) Role of the sulfonylurea receptor (SUR) in regulating human adipocyte metabolism. FASEB J 13,1833-1838[Abstract/Free Full Text]
34. Zemel, M. B., Kim, J. H., Woychik, R. P., Michaud, E. J., Kadwell, S. H., Patel, I. R., Wilkison, W. O. (1995) Agouti regulation of intracellular calcium: role in insulin resistance of viable mice. Proc. Natl. Acad. Sci. USA 92,4733-4737[Abstract/Free Full Text]
35. Kim, J. H., Mynatt, R. L., Moore, J. W., Woychik, R., Moustaid, N., Zemel, M. B. (1996) The effects of calcium channel blockade on agouti-induced obesity. FASEB J 10,1646-1652[Abstract]
36. Gruen, R. K., Greenwood, M. R. (1981) Adipose tissue lipoprotein lipase and glycerol release in fasted Zucker (fa/fa) rats. Am. J. Physiol. 241,E76-E83[Abstract/Free Full Text]
37. Mariot, P., Gilon, P., Nenquin, M., Henquin, J. C. (1998) Tolbutamide and diazoxide influence insulin secretion by changing the concentration but not the action of cytoplasmic Ca²⁺ in ß-cells. Diabetes 47,365-373[Abstract]
38. Picard, F., Naimi, N., Richard, D., Deshaies, Y. (1999) Response of adipose tissue lipoprotein lipase to the cephalic phase of insulin secretion. Diabetes 48,452-459[Abstract]

This article has been cited by other articles:

				P. A. Baldock, S. J. Allison, P. Lundberg, N. J. Lee, K. Slack, E.-J. D. Lin, R. F. Enriquez, M. M. McDonald, L. Zhang, M. J. During, et al. Novel Role of Y1 Receptors in the Coordinated Regulation of Bone and Energy Homeostasis J. Biol. Chem., June 29, 2007; 282(26): 19092 - 19102. [Abstract] [Full Text] [PDF]

				A. Sainsbury, H. T. Bergen, D. Boey, D. Bamming, G. J. Cooney, S. Lin, M. Couzens, N. Stroth, N. J. Lee, D. Lindner, et al. Y2Y4 Receptor Double Knockout Protects Against Obesity Due to a High-Fat Diet or Y1 Receptor Deficiency in Mice Diabetes, January 1, 2006; 55(1): 19 - 26. [Abstract] [Full Text] [PDF]

				M. B. Zemel The Role of Dairy Foods in Weight Management J. Am. Coll. Nutr., December 1, 2005; 24(suppl_6): 537S - 546S. [Abstract] [Full Text] [PDF]

				R. Alemzadeh and K. M. Tushaus Modulation of Adipoinsular Axis in Prediabetic Zucker Diabetic Fatty Rats by Diazoxide Endocrinology, December 1, 2004; 145(12): 5476 - 5484. [Abstract] [Full Text] [PDF]

				J. M. Harkins, N. Moustaid-Moussa, Y.-J. Chung, K. M. Penner, J. J. Pestka, C. M. North, and K. J. Claycombe Expression of Interleukin-6 Is Greater in Preadipocytes than in Adipocytes of 3T3-L1 Cells and C57BL/6J and ob/ob Mice J. Nutr., October 1, 2004; 134(10): 2673 - 2677. [Abstract] [Full Text] [PDF]

				M. B. Zemel Mechanisms of Dairy Modulation of Adiposity J. Nutr., January 1, 2003; 133(1): 252S - 256. [Abstract] [Full Text] [PDF]

				K. Baran, E. Preston, D. Wilks, G. J. Cooney, E. W. Kraegen, and A. Sainsbury Chronic Central Melanocortin-4 Receptor Antagonism and Central Neuropeptide-Y Infusion in Rats Produce Increased Adiposity by Divergent Pathways Diabetes, January 1, 2002; 51(1): 152 - 158. [Abstract] [Full Text] [PDF]

				S. Bassilian, S. Ahmed, S. K. Lim, L. G. Boros, C. S. Mao, and W.-N. P. Lee Loss of regulation of lipogenesis in the Zucker diabetic rat. II. Changes in stearate and oleate synthesis Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E507 - E513. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by STANDRIDGE, M. Articles by MOUSTAID-MOUSSA, N. Search for Related Content PubMed PubMed Citation Articles by STANDRIDGE, M. Articles by MOUSTAID-MOUSSA, N.