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(The FASEB Journal. 2007;21:53-60.)
© 2007 FASEB

Resistance to leptin action is the major determinant of hepatic triglyceride accumulation in vivo

Sigal Fishman*, Radhika H. Muzumdar{dagger}, Gil Atzmon*, Xiaohui Ma*, Xiaoman Yang*, Francine H. Einstein{ddagger} and Nir Barzilai*,1

* Institute for Aging Research, and Diabetes Research and Training Center, Department of Medicine,

{dagger} Division of Pediatric Endocrinology, Children’s Hospital at Montefiore,

{ddagger} Department of Obstetrics and Gynecology and Women’s Health at Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA

1Correspondence: Institute for Aging Research, Department of Medicine, Belfer Bldg. #701, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. E-mail: barzilai{at}aecom.yu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Impairment of both insulin and leptin action has been implicated in the pathogenesis of nonalcoholic fatty liver disease. By assessing hepatic triglyceride (TG) stores in response to modulation of leptin action (by leptin infusion), we attempted to determine whether leptin has the major role in hepatic TG accumulation. TG were markedly decreased (by 63%, P<0.05) in young animals treated with leptin. However, this was also associated with improvement in hepatic insulin action (2-fold decrease in HGP during clamp, P<0.05). These effects on hepatic TG stores and insulin action were abolished in old rats who demonstrate leptin resistance. Since these experiments could not discern the role of leptin from the role of hepatic insulin action on hepatic TG stores, we further examined the effect of improvement of hepatic insulin action by visceral fat removal (VF-). Enhancement of hepatic insulin action in old VF-rats was associated with reduced hepatic TG stores (by 64% P<0.01). Because this manipulation may have induced an improvement in leptin action as well, we studied VF removal in a genetically leptin-resistant model (Zucker Diabetic Fatty rats, ZDF). Only in this mode was exclusive improvement of hepatic insulin action by VF removal not associated with reduced hepatic TG stores, suggesting that improved hepatic insulin action is not necessary for modulation of hepatic TG stores. By dissociating action of leptin from that of insulin, we suggest that the failure of leptin action is the major physiological mechanism for hepatic steatosis.—Fishman, S., Muzumdar, R. H., Atzmon, G., Ma, X., Yang, X., Einstein, F. H., Barzilai, N. Resistance to Leptin Action is the Major Determinant of Hepatic Triglyceride Accumulation in vivo.


Key Words: hepatic insulin action • steatosis • metabolic syndrome


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is the most common liver disease, with an estimated prevalence of 11–30% in the general adult population (1) . The hallmark feature, both histologically and metabolically, in the pathogenesis of NAFLD is the accumulation of triglycerides (TG) in the liver (2 , 3) . Excessive hepatic TG accumulation results from disruption in the balance between import and export of precursors and metabolites of TG. Suggested mechanisms include increased flux of free fatty acids (FFA) to the liver, increased de novo lipogenesis, and a decrease in clearance of TG, through impairment in ß-oxidation or very-low-density lipoproteins (VLDL) assembly (3 4 5) . Resistance to both insulin (IR) and leptin action have been implicated in accumulation of hepatic TG (6 7 8 9 10) , and it is of great importance from a therapeutic perspective to determine which component plays the major role in pathogenesis of steatosis.

Hepatic insulin resistance has been associated with steatosis in both rodents (11 , 12) and humans (13) . This resistance could result from the contribution of adipose tissue through increased flux of FFA to the liver (8 , 9) , or by secretion of numerous metabolically active fat-derived peptides and cytokines, such as adiponectin, leptin, and resistin, which may directly effect hepatic insulin action (14 , 15) . Visceral fat (VF) plays a key role in the pathogenesis of hepatic insulin resistance through its distinct adipokine profile (16) . The importance of this depot has been highlighted in studies that have demonstrated an improvement in insulin sensitivity with the removal of VF (17) .

Although the association between hepatic insulin resistance and hepatic TG accumulation is well established, a causative role for hepatic insulin resistance in the accumulation of TG has not yet been proven, and in fact, recent literature argues against a causative role. For example, certain diabetic animal models have demonstrated severe insulin resistance without the accumulation of TG in the liver (18) , and other physiological studies have shown that TG accumulation precedes the development of insulin resistance (12) .

Leptin may have a central role in accumulation of hepatic TG (10 , 19) through the regulation of fat and its distribution (10 , 20) and by modulating hepatic ß-oxidation (21) . This hormone is secreted exclusively by adipocytes and functions as a sensor of fat mass. Through its hypothalamic receptor, it exerts anorectic and thermogenic effects (22 , 23) . The identification of variants of the OB-receptor in several other tissues (24) , including liver and adipose tissue (25) , and marked improvements in glucose (Glc) tolerance following leptin administration to obese animals (26) suggest that leptin may play a more complex role in the regulation of intermediate metabolism than originally recognized.

Since insulin and leptin resistance develop in parallel (27) and efforts to change one induce a change in the other (28 , 29) , it has been difficult to assess the independent role of leptin on hepatic insulin action and TG storage. Here, through a series of experiments utilizing a novel in vivo technology, the unique role of leptin resistance in the development of steatosis is delineated. First, we define the correlation between hepatic insulin action and hepatic TG content using young lean and old obese control rats. As the older rats are characterized by increased fat mass, decreased insulin sensitivity, and resistance to the beneficial effect of leptin on body composition (30 31 32 33) , they serve as an ideal model for studying the natural course of hepatic fat accumulation in conditions associated with both insulin and leptin resistance. Subsequently, by using a variety of interventions, both surgical and pharmacological, we dissect the effects of improved insulin action from the role of leptin on hepatic TG storage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
A total of 56 F1 hybrid of Brown Norway (FNB) (purchased from NIA) and 12 ZDF rats (Charles River Laboratories, Wilmington, MA, USA) were studied. The animals were housed in individual cages and subjected to a standard light (6:00 acetoxymethyl ester (AM) to 6:00 PM)–dark (6:00 PM to 6:00 AM) cycle. All rats were fed standard chow (catalog no. 5001; Purina Mills, St. Louis, MO, USA) provided 59% calories from carbohydrates, 20% from protein, and 21% from fat. For all groups of rats described below, one week before the in vivo studies, rats were anesthetized by inhalation of methoxyflurane, and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery. The venous catheter extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. All the animals were studied after 6 h of fasting, while awake, unstressed under hyperinsulinemic euglycemic clamp conditions (as described below).

Rats were assigned to four experiments.

The relationship between hepatic TG and hepatic insulin action
As rats age, the combination of wt gain and aging results in significant increase in TG content in the liver. We used young and old rats (3–20 mo old, n=20, body wt from 260 to 573 g) to establish the normal association between hepatic TG and hepatic insulin action in nonmanipulated rodents across the life span. This data were then used as a reference to demonstrate the impact of other interventions (pharmacologic or surgical) on hepatic TG and insulin action.

Leptin action on hepatic TG and hepatic insulin action
To study the effect of leptin on hepatic insulin action and hepatic TG content total of 24 FBN male young and old rats, we infused leptin or saline using subcutaneous (s.c.) osmotic pumps as described previously (32) . Rats in the leptin group were fed ad libitum and studied at 4 mo of age (n=12) or 21 mo of age (n=12). Because leptin decreases food intake, the saline group was pair-fed to the leptin group. All rats were implanted with s.c. minipumps that delivered either recombinant mouse leptin (~0.5 mg/kg/ day) or a similar vol of normal saline (NS) for 7 d and were studied by euglycemic hyperinsulinemic clamp after approximately 6 h of fasting while awake and unstressed.

Removal of visceral fat (VF-) on hepatic TG and hepatic insulin action
To test the effect of modulation of hepatic insulin action (by visceral fat removal) on hepatic TG stores, 12 FBN rats were studied. Rats were anesthetized [pentobarbital 50 mg/kg body wt intraperitoneally (i.p.)] and randomly assigned to one of the two surgical procedures at 15 mo of age: 1) Visceral fat removal (VF-) (n=6), in which all visible epididymal (Epi) and the perinephric (Peri) fat pads were removed through lower abdominal incision, weighed, and immediately frozen in liquid nitrogen. A total amount of 15.7 ± 1.5 g of visceral fat was removed in VF- group. 2) Sham-operated (SO) (n=6), in which the abdominal cavity was incised and visceral fat was mobilized but not removed. At 20 mo old, the rats were anesthetized and indwelling catheters were placed. Recovery was continued until body wt and daily food intake were within 5% of preoperative levels. Clamp studies were performed in chronically catheterized rats after 6 h of fasting, while awake and unstressed.

Hepatic TG and hepatic insulin action in a genetic model of leptin resistance
To study the effect of improvement in hepatic insulin action (by VF removal) on hepatic TG content in leptin-resistant state, we used ZDF rats (n=12; Charles River Laboratories). These rats are leptin-resistant due to a mutation in the leptin receptor. Rats were anesthetized at 2 mo of age and randomly assigned to one of two groups: 1) ZDVF- (n=6). Rats in which all visible epididymal (Epi) and the perinephric (Peri) fat pads were surgically removed, weighed, and immediately frozen in liquid nitrogen. An average amount of 10.5 ± 0.3 g of visceral fat was removed in the ZDVF group; 2) ZDVF+ (n=6), Sham operations were performed at 2 mo of age. Eight weeks after the surgeries for VF removal, rats were anesthetized, indwelling catheters were placed, and studies were performed following recovery. The technique of visceral fat removal and part of the basic characteristics were previously published (34) .

Body composition
Lean body mass (LBM) and fat mass (FM) were calculated as described elsewhere (20) . Briefly, rats received an intra-arterial bolus injection of 20 µCi of tritiated-labeled water (3H2O; New England Nuclear, Boston, MA, USA), and plasma samples were obtained at 30-min intervals. Steady-state conditions for plasma 3H2O-specific activity were achieved within 45 min in all studies. Five plasma samples obtained between 1 and 3 h were used to calculate the total body distribution of water. At the completion of each experiment Epi, Peri, and mesenteric fat (or their remnant) were dissected and weighed.

Hepatic TG content measurement
Triglycerides was measured by triglyceride kit (GPO- Trinder Sigma Diagnostics, St. Louis, MO, USA). Frozen liver tissue (200 mg) was homogenized in 4 ml of 2:1 chloroform methanol. The solution was vortexed and filtered through Sharkskin. After adding 0.2 ml of 0.58% NaCl solution, the filtrate was centrifuged at 1000 rpm for 5 min. The upper phase was aspirated. The chloroform phase was further analyzed. The sample was evaporated in chloroform to a vol of less than 1 ml. A final vol of 1 ml was made up by adding chloroform. Chloroform-containing samples, water, or standard solution of glycerol (10–20 µl)were added to the cuvettes. Reagent (1 ml) was added. After incubation at 30 degrees for 10 min, the samples was read in spectrophotometer at 540 nm.

Hyperinsulinemic euglycemic clamp
The major two effects of insulin in vivo, stimulation of disposal of Glc into the peripheral tissues (peripheral insulin sensitivity), and diminishing production of Glc by the liver (hepatic insulin sensitivity), were estimated in conscious rats using a combination of insulin (3 mU/kg per min) clamp and tracer dilution techniques. All rats received a primed-continuous (15–40 µCi bolus, 0.4 µCi/min) infusion of HPLC purified [3H-3]-glucose (New England Nuclear, Boston, MA, USA) throughout the study. After 120 min (to allow for the measurement of basal HGP), a primed continuous infusion of insulin (3 mU/kg/min) and a variable infusion of a 25% Glc solution were started and periodically adjusted, to clamp the plasma Glc concentration at the basal level for the remaining 120 min of the clamp. Somatostatin (1.5 µg/kg/min) was infused to suppress endogenous insulin secretion. Plasma samples for determination of 3H-glucose-specific activity were obtained at 10-min intervals throughout the insulin infusion. Samples were also obtained for determination of plasma insulin, leptin, and FFA concentrations at 30-min intervals throughout the study. At the end of the insulin infusion, rats were anesthetized, the abdomen was quickly opened, and the rectus muscle was freeze-clamped in situ with aluminum tongs precooled in liquid nitrogen. The time from the injection until freeze clamping of the muscle was less than 1 min. Epididymal, mesenteric, and perinephric fat pads were dissected and weighed at the end of each experiment. All tissue samples were stored at –80°C for subsequent analysis (35) . The study protocol was reviewed and approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine.

Biochemical analyses
Plasma Glc was measured by the Glc oxidase method (Glucose Analyzer II; Beckman Instruments, Palo Alto, CA, USA), and plasma insulin was measured by RIA using rat insulin standards. Plasma leptin was assayed using the Linco leptin assay kit (Linco Research, St. Charles, MO, USA). Plasma nonesterified fatty acid concentrations were determined by an enzymatic method with an automated kit according to the manufacturer’s specification (Waco Pure Chemical, Osaka, Japan).

Expression of leptin
Total RNA from fat depots was extracted following Clontech’s protocol with some modifications as described previously (16) . First-strand cDNA was synthesized with random primers, and total RNA as a template using the SuperScript preamplification system (Life Technologies, Inc.). Primers for leptin and for ß-actin were designed with the assistance of the computer program Oligo 4.0 (National Biosciences, Plymouth, MN, USA) (16) . Quantification of leptin and its signal was performed by a light cycle (Roche, Mannheim, Germany) and normalized for ß-actin signal to correct for loading irregularities.

Statistical analysis
The regression line with 95% CI was created with data from normal, unmanipulated rats with age ranges of 3–20 mo, and the average of the treated groups was plotted on the curve. The significance of differences between the groups was evaluated by the two-sample t test. Pearson correlation coefficients were calculated to estimate the relationship between variables. All values are presented as means ± SE. A P value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relationship between hepatic TG content and hepatic insulin action
The correlation between hepatic TG content and hepatic insulin sensitivity was studied in a total of 20 FBN rats with age ranges of 3–20 mo or 260–573 g. With increased age, accumulation of hepatic TG increased. Both hepatic insulin sensitivity and peripheral Glc uptake were impaired with aging during clamp study (data not shown). The inverse relationship between hepatic TG content and hepatic insulin sensitivity was demonstrated (as evidenced by the decreased ability of insulin to suppress hepatic Glc production during clamp) (–r=0.57; P=0.008). This relationship was used as a reference in subsequent experiment protocols to demonstrate the effects of various surgical and pharmacological manipulations on both hepatic TG accumulation and hepatic insulin resistance. (Data are provided in supplement)

Leptin reduces hepatic TG content and improves hepatic insulin sensitivity in young lean rats (Table 1 , Fig. 1 A)
Subcutaneous leptin administration to 3-mo-old rats for 7 d significantly affected body composition and the metabolic characteristics compared to pair-fed saline-infused animals. Table 1 (Young) demonstrates that, with comparable food intake, body wt was reduced to the same extent in the leptin-treated group and the pair-fed group. However, leptin infusion induced changes in fat distribution and resulted in significant depletion of visceral fat compared with the pair-fed group. Basal metabolic characteristics of the leptin-treated group were similar to the pair-fed animals. However, as depicted in Fig. 1A , leptin infusion exerted a profound beneficial effect beyond its anorectic effect by decreasing hepatic TG content by 64% (P<0.05). In parallel, an improvement in hepatic insulin action was noted (2-fold decrease in HGP during clamp, compared to pair-fed controls, P=0.05). No significant differences were found in peripheral Glc uptake (Rd) and FFA levels (Table 1) during the clamp. In light of the effects of leptin on both hepatic TG stores and hepatic insulin action, the independent role of leptin in hepatic TG stores could not be delineated.


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  		Table 1. Effect of chronic leptin on body composition and metabolic characteristics


Figure 1
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  		Figure 1. Changes in hepatic TG content and in hepatic insulin sensitivity (i.e., reduction in HGP during hyperinsulinemic clamp) after leptin infusion in young lean (1A) and old obese (1B) rats. Young lean rats (12) and old obese rats (12) were implanted with miniosmotic pump to infuse either leptin or saline for 7 d. At the end of leptin infusion, hyperinsulinemic clamp study (3 mU/Kg/min) was employed. Hepatic TG stores were assessed. Mean values ± SE. of young (1A) and old (1B) leptin treated rats and pair- fed were then imposed on the reference curve obtained by plotting the TG level against hepatic insulin sensitivity in young and old nonmanipulated rats (see Materials and Methods, and Results). *P < 0.05 vs. young leptin, #P < 0.05 vs. young leptin, **P = NS vs. old leptin, ##P = NS vs. old leptin.

Leptin fails to modulate hepatic TG content or hepatic insulin sensitivity in old obese rats (Table 1 , Fig. 1B )
Basal leptin levels in old rats were increased by over 25-fold compared with young rats, suggesting severe leptin resistance. Although leptin administration doubled the plasma leptin levels, leptin failed to induce changes in body composition and fat distribution (Table 1) . As depicted in Fig. 1B , leptin did not modulate hepatic TG content or hepatic insulin action, suggesting a key role for leptin in modulating hepatic TG stores. However, as leptin failed to modulate both TG and hepatic insulin action, the independent role of leptin in modulation of hepatic TG stores could not be established.

Improvement in hepatic insulin and leptin sensitivity is associated with reduction in hepatic TG content in old obese rats (Table 2 , Fig. 2 A)
To assess the effects of enhancing hepatic insulin action on hepatic TG content, we subjected old obese rats to visceral fat removal, a surgical manipulation that was previously shown to increase insulin sensitivity (17) . At the end of the study, food intake, body wt, and lean body mass were comparable between SO and the VF- group, in light of a 5-fold decrease in VF (Table 2) . Basal insulin was decreased in the VF- group, indicating enhanced insulin sensitivity. During insulin clamp, the peripheral Glc uptake (Rd) was significantly increased (Table 2) . Interestingly, the ability of insulin to suppress FFA was not improved significantly by VF extraction, (27.11±6% reduction in VF- vs. 21.4±3.5% in SO). As depicted in Fig. 2A , VF extraction improved both hepatic insulin sensitivity (by 2.8-fold, P=0.05) and hepatic TG content (by 64%, P<0.01), resulting in a shift in the old rats toward the "young" portion of the reference curve. Moreover, hepatic TG content was reduced to a larger extent than would be expected by the improvement of hepatic insulin action alone (Fig. 2A ). Of note, VF extraction also resulted in significant reduction in plasma leptin levels (Table 2) . To exclude the possibility that leptin reduction emanated simply from reduction in amount of fat tissue, we examined leptin gene expression in the different fat depots. While SO rats had similar expression of these peptides in s.c. (SC) fat and mesenteric (visceral) fat, leptin expression was decreased by ~75% in SC fat compared with mesenteric fat in VF- rats. In the face of unchanged food intake, we interpret these findings to suggest that leptin sensitivity was restored in these animals in parallel to the improvement in hepatic insulin action (Table 2) . Thus, though we demonstrated the centrality of leptin resistance in TG storage, we could not differentiate its role from the role of hepatic insulin action.


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  		Table 2. Body composition and metabolic characteristic in FBN and ZDF rats


Figure 2
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  		Figure 2. Changes in hepatic TG content and in hepatic insulin sensitivity (i.e., reduction in HGP during hyperinsulinemic clamp) after VF removal in old rats (A) and in ZDF rats (B). Fifteen-month-old rats (12) and 2-mo-old ZDF rats (12) were subjected to visceral fat removal or sham operation. Five months and 8 wk later, respectively, both groups were studied under hyperinsulinemic clamp (3 mU/Kg/min). Mean values ± SE of data from all studied groups were then imposed on the reference curve obtained by plotting the TG level against hepatic insulin sensitivity in young and old nonmanipulated rats (see Materials and Methods and Results). *P < 0.01 vs. old VF-, #P = 0.05 vs. old VF-, {dagger}P < 0.05 vs. reference curve, **P < NS vs. ZDF VF-, ##P = 0.05 vs. ZDF VF-.

Improvement in hepatic insulin sensitivity does not improve hepatic TG storage in leptin-resistant ZDF rats (Table 2 , Fig. 2B )
By removing visceral fat in leptin resistant rats, we induced improvement in insulin resistance without affecting leptin action. Food intake remained similar between the groups, and their body weights were comparable at the end of the study (Table 2) . Metabolic characteristic of the groups are presented in Table 2 . During insulin and somatostatin infusions, ZDFV- plasma Glc level was clamped at ~8.4 mmol/l, with a variable rate of infusion of 25% Glc. ZDVF+ under similar insulin infusion, developed hyperglycemia due to severe insulin resistance, so no Glc infusion was needed during the study. Because hyperglycemia suppresses the rates of hepatic Glc production independent of insulin (36) , these rates were adjusted for its additive effect. As shown in Fig. 2B , hepatic TG content was not significantly reduced despite an improvement in hepatic insulin sensitivity (~8-fold decrease in HGP during clamp compared to ZDF+, P=0.05) as would be expected by the standard curve. Leptin level was reduced in ZDVF- (Table 2) probably due to a reduction in total amount of fat. Leptin gene expression remained proportional between SC and mesenteric fat after VF removal (1.1 in ZDVF+ vs. 1.09 in ZDVF-). This model highlights the specific effect of leptin on hepatic TG independent of hepatic insulin action.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many patho-physiological processes are associated with events that lead to NAFLD, and an integrative approach demands experimental procedures to identify the primary event from associative events that may not play a major role leading to this condition. Realizing this challenge, we employed several animal models and novel physiological procedures to determine the relative role of leptin resistance vs. hepatic insulin resistance in the pathogenesis of TG accumulation in the liver. Since their actions are interrelated, it is difficult to differentiate their unique independent roles in hepatic TG storage. By using three sets of experiments to modulate hepatic TG content through alteration in insulin and/or leptin action, we provide evidence that leptin failure is the underlying mechanism for hepatic TG accumulation and is independent of hepatic insulin action, thereby challenging the common notion that insulin resistance is the major determinant in the process. The correlation between hepatic TG content and hepatic insulin sensitivity determined from normal young lean and old obese rats served as a reference for the assessment of the effects of modulating hepatic insulin sensitivity and leptin administration in the following experiments. We demonstrate the centrality of leptin action in hepatic TG storage by leptin infusion in young lean and old obese rats. Leptin improved hepatic TG content beyond its anorectic effect in young leptin-sensitive rats. However, the reduction in hepatic TG content was accompanied by an improvement in hepatic insulin action as well (Fig. 1A ). The effects of leptin on hepatic TG and insulin action were completely abolished in old leptin-resistant rats (Fig. 1B ). Improving hepatic insulin sensitivity in older rats (by VF removal) resulted in both improved leptin sensitivity and hepatic TG content. Of note, the reduction in TG content was greater than expected for the improvement in hepatic insulin action (Fig. 2A ), suggesting an additive effect when the improvement is attained in both leptin and insulin sensitivity with VF removal. The use of genetically leptin-resistant model (ZDF) allowed us to create an experimental design that would selectively improve one and not the other and thus assist in delineating the individual roles. An improvement in hepatic insulin sensitivity by visceral fat removal in these genetically leptin-resistant rats did not cause a reduction in hepatic TG amount (Fig. 2B ), which highlights the central role of leptin in hepatic TG accumulation.

Regardless of the strong association, evidence is accumulating against the causative role of hepatic insulin resistance in TG accumulation. For instance, it was previously shown that deletion of PTEN, a negative regulator of insulin signaling in murine liver, resulted in enhancement of hepatic insulin action but also in fatty liver (18) . In another transgenic mice model, it was demonstrated that increased expression of transcription factor Foxa2, which physiologically is regulated by insulin, increased the expression of lipogenic enzymes and caused transient steatosis while euglycemia was maintained (37) . Finally, studies have shown that hepatic fat accumulation preceded the development of hepatic insulin resistance (12) .

Growing evidence indicates that leptin, in addition to its anorectic action, is involved in certain metabolic processes such as body fat distribution and hepatic TG accumulation (10) . It was shown in ob/ob mice and rat models that leptin treatment causes a specific loss of fat mass, whereas pair-feeding depletes both lean and fat mass (10) . Furthermore, it was shown that in the absence of leptin, ob/ob mice developed enlarged livers engorged with TG, and leptin replacement reversed this pathology (38) . In our studies, a week-long infusion of leptin in young rats caused preferential visceral fat and hepatic TG depletion despite constant FFA, supporting the role of leptin in FFA oxidation (21) . In addition to these effects, leptin also improved hepatic insulin sensitivity (Fig. 1A ). The ability of leptin to exert "insulin-like" effects on both peripheral and hepatic insulin sensitivity has been previously demonstrated. Leptin has been shown to reduce total hepatic Glc production by redistributing hepatic Glc fluxes (20) . Improvement of hepatic insulin sensitivity in leptin-resistant rats (ZDF) did not change hepatic TG content (Fig. 2B ), demonstrating that hepatic TG depletion is attributable to leptin beyond its insulinergic actions.

A few models frequently employed in steatosis studies include genetic models, nutritionally manipulated models, and pharmacological models (39) . However, alternations in basic physiological processes in these models emanate from a genetic defect or nutritional deficiency, limiting their usefulness in studying the natural history of hepatic fat accumulation associated with IR and leptin resistance. Since the hallmarks of the metabolic decline of aging are the development of obesity and changes in fat distribution and insulin resistance (29 30 31 32) , we found an aging insulin-resistant model perfectly suited for studying the natural course of hepatic fat accumulation associated with the development of the metabolic syndrome. Aging FBN rats have been adopted by the NIA as a model that exhibits many of the phenotype of human aging, and the leptin resistance of aging is suggested in human studies as well. The use of ZDF rats, which bear a mutant leptin receptor, helped to clearly distinguish the individual roles of leptin and insulin resistance. However, in contrast, a marked genetic model such as the ZDF rats may have secondary alterations that may not necessarily be applicable to other steatosis models. In conclusion, we have shown that leptin has important functions in improving hepatic insulin sensitivity and modulating hepatic TG content. The beneficial effect of leptin on hepatic TG storage is exerted by mechanisms beyond its insulinergic properties.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health (RO1-AG 18381 and P01-AG 21654), the American Diabetes Association, and by the Core Laboratories of the Albert Einstein Diabetes Research and Training Center (DK 20541). S. F. is supported by a grant from American Physicians Fellowship for Medicine in Israel.

Received for publication May 19, 2006. Accepted for publication July 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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