AJCN Tufts Nutrition Symposium, Boston & Online Sept 2009
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American Journal of Clinical Nutrition, Vol. 87, No. 2, 295-302, February 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Increased fat accumulation in liver may link insulin resistance with subcutaneous abdominal adipocyte enlargement, visceral adiposity, and hypoadiponectinemia in obese individuals1,2,3

Juraj Koska, Norbert Stefan, Paska A Permana, Christian Weyer, Mina Sonoda, Clifton Bogardus, Steven R Smith, Denis R Joanisse, Tohru Funahashi, Jonathan Krakoff and Joy C Bunt

1 From the Obesity and Diabetes Clinical Research Section, NIDDK/NIH/DHHS, Phoenix, AZ (JK, NS, CW, CB, JK, and JCB); Carl T Hayden Veterans Affairs Medical Center, Phoenix, AZ (PAP); the Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan (MS and TF); Pennington Biomedical Research Center, Baton Rouge, LA (SRS); and the Department of Kinesiology, Laval University, Quebec, Canada (DRJ)

2 Supported by an intramural research program of the NIDDK/NIH/DHHS (to JK, NS, PAP, CW, CB, JK, and JCB) and a new investigator award from the Fonds de la Recherche en Santé du Québec (to DRJ).

3 Reprints not available. Address correspondence to J Krakoff, ODCRS/NIDDK/NIH/DHHS; 4212 N 16th Street, Phoenix, AZ 85016. E-mail: jkrakoff{at}mail.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Enlargement of adipocytes from subcutaneous abdominal adipose tissue (SAT), increased intrahepatic lipid content (IHL), intramyocellular lipid content (IMCL), and low circulating adiponectin concentrations are associated with insulin resistance.

Objective: Because adiponectin increases fat oxidation in skeletal muscle and liver, and the expression of the adiponectin gene in SAT is inversely associated with adipocyte size, we hypothesized that hypoadiponectinemia links hypertrophic obesity with insulin resistance via increased IMCL and IHL.

Design: Fifty-three obese Pima Indians with a mean (±SD) age of 27 ± 8 y, body fat of 35 ± 5%, and normal glucose regulation (normal fasting and 2-h glucose concentration per WHO 1999 criteria) underwent euglycemic-hyperinsulinemic clamp, biopsies of SAT and vastus lateralis muscle, and magnetic resonance imaging of the abdomen.

Results: Adipocyte diameter (AD) correlated positively with body fat (P < 0.0001) and IHL (estimated from magnetic resonance imaging intensity of liver; P = 0.047). No association was found between AD and plasma adiponectin or IMCL. Plasma adiponectin negatively correlated with type II IMCL (IIA, P = 0.004; IIX, P = 0.009) or IHL (P = 0.02). In a multivariate analysis, plasma adiponectin, AD, and visceral adipose tissue (VAT) independently predicted IHL. Low insulin-mediated glucose disposal was associated with low plasma adiponectin (P = 0.02) and high IHL (P = 0.0003), SAT (P = 0.02), and VAT (P = 0.04). High IHL was the only predictor of reduced insulin-mediated suppression of hepatic glucose production (P = 0.02) and the only independent predictor of insulin-mediated glucose disposal in a multivariate analysis.

Conclusions: Increased lipid content in the liver may independently link hypoadiponectinemia, hypertrophic obesity, and increased visceral adiposity with peripheral and hepatic insulin resistance.

Key Words: Obesity • adipocyte size • adiponectin • intrahepatic fat • intramyocellular fat • insulin action


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Obesity is characterized by a growing adipose tissue mass associated with increases in adipocyte size and number (1). It has been known for a few decades that obese people with enlarged subcutaneous adipocytes (ie, with hypertrophic obesity) are, on average, more hyperinsulinemic than those with a similar degree of adiposity but relatively smaller adipocytes (2). More recent analyses have shown that the enlargement of subcutaneous abdominal adipocytes is associated with reduced insulin-mediated glucose disposal independent of overall adiposity (3, 4).

It has been hypothesized that enlargement of subcutaneous adipocytes is a sign of reduced adipogenic capacity of subcutaneous adipose tissue (SAT), which in turn leads to increased accumulation of fat in visceral adipose tissue (VAT), skeletal muscle, and liver and a subsequent worsening of insulin action and glucose tolerance (5). In support of this hypothesis, implantation of adipose tissue in lipodystrophic mice (mice with no adipogenic potential) is followed by reversal of hepatic steatosis, insulin resistance, and hyperglycemia (6, 7). Moreover, administration of thiazolidinediones (TZDs), a class of antihyperglycemic medications that increase proliferation of adipose tissue (8), leads to a reduction of fat content in the liver and improvement of insulin action despite increased body weight (9).

The mechanism underlying abnormal partitioning of lipids in hypertrophic obesity, and metabolic derangements reminiscent of those seen in lipodystrophic syndromes, may involve other factors in addition to simple overflow of fat from large dysfunctional adipocytes. One such factor may be the adipocyte-derived, insulin-sensitizing hormone adiponectin. It has been suggested that large adipocytes are characterized by reduced production and secretion of adiponectin compared with small newly differentiated adipocytes (10). Hypoadiponectinemia is a common characteristic of both lipodystrophy and obesity (11, 12). Administration of adiponectin to mice with lipodystrophic diabetes reduces the fat content in liver and skeletal muscle, improves insulin action, and reverses hyperglycemia (13). In nondiabetic humans, low plasma adiponectin concentrations are associated with increased intramyocellular and intrahepatic fat content (14, 15). Finally, increases in circulating adiponectin concentrations in patients with type 2 diabetes mellitus treated with TZDs are associated with improvements in insulin sensitivity and reduction of intrahepatic fat (9).

In the present study we tested 1) whether larger subcutaneous abdominal adipocyte size is associated with increased visceral, hepatic, and intramyocellular fat accumulation in obese individuals with normal glucose tolerance; 2) whether those relations might be mediated by low plasma adiponectin concentrations; and 3) whether increased accumulation of fat in VAT, liver, and skeletal muscle might explain the association of increased adipocyte size and low adiponectin concentrations with impairment of insulin action.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Subjects were at least 3 quarters Pima Indian, between 18 and 45 y of age, nonsmokers at the time of the study, and healthy on the basis of medical history, physical examination, and routine laboratory tests. The protocol was approved by the Tribal Council of the Gila River Indian Community and by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases, and all subjects provided written informed consent before participation. The female subjects were studied during the follicular phase of the menstrual cycle.

Methods
All subjects were admitted to a clinical research unit and were placed on a weight-maintaining diet (containing 50% of energy as carbohydrates, 30% as fat, and 20% as protein). Body composition was measured by dual-energy X-ray absorptiometry (DPX-L; Lunar Radiation, Madison, WI) (16) to confirm obesity status [percentage body fat (BF) ≥25 in men and ≥30 in women]. At least 3 d after admission and after a 12-h overnight fast, the subjects underwent a 2-h 75-g oral-glucose-tolerance test to exclude impaired glucose regulation or diabetes according to the World Health Organization 1999 criteria. Plasma glucose concentrations were determined by the glucose oxidase method (Beckman Instruments, Fullerton, CA). Plasma insulin concentrations were measured with an automated immunoassay (Access; Beckman Instruments). Blood samples for the measurement of fasting plasma adiponectin concentrations were drawn at 0700 with prechilled syringes, transferred into prechilled EDTA-coated tubes, and immediately placed on ice. All tubes were centrifuged at 4 °C within several minutes of collection and were stored at –70 °C until assayed. Total plasma adiponectin concentrations were determined with the use of a validated sandwich enzyme-linked immunosorbent assay that uses an adiponectin-specific antibody (intraassay and interassay CVs of 3.3% and 7.4%, respectively).

Insulin action was assessed at physiologic insulin concentrations during a hyperinsulinemic-euglycemic glucose clamp (17). Briefly, after the subjects fasted overnight, a primed (30 µCi) continuous (0.3 µCi/min) 3-[3H]glucose infusion was started to determine endogenous glucose production (EGP). Two hours after the isotope infusion was started, a primed continuous intravenous insulin infusion was administered for 100 min at a constant rate of 40 mU·m–2 body surface area·min–1. Blood samples for measurement of 3-[3H] glucose specific activity were collected from the end of the basal period to and every 10 min during the final 40 min of insulin infusion. Under basal conditions, EGP was calculated as the 3-[3H] glucose infusion rate divided by the steady state plasma 3-[3H] glucose specific activity. During the insulin clamp, EGP was calculated from Steele's equation (18). The degree of hepatic insulin sensitivity was calculated as the ratio of absolute differences (clamp minus fasting) in EGP and plasma insulin concentrations. The rate of total insulin-stimulated glucose disposal was calculated for the last 40 min of the insulin infusion and was corrected for the rate of EGP. Individual variation in plasma glucose and insulin concentrations during the clamp was taken into account in the calculation of the insulin-mediated glucose disposal (17, 19). All measurements derived from the clamp were normalized to estimated metabolic body size (or fat-free mass + 17.7 kg) (20).

Abdominal SAT was removed from the periumbilical region by percutaneous needle biopsy under local anesthesia (1% lidocaine). The specimen was placed on a sterile nylon mesh, rinsed with sterile 0.9% NaCl solution, and cleaned of visible connective tissue and blood vessels in Hank's Buffered Saline Solution (HBBS) supplemented with 5.5 mmol/L glucose. Adipose tissue was then digested in HBBS buffer containing 5.5 mmol/L glucose, 5% fatty acid–free bovine serum albumin (Introgen/Serologicals, Norcross, GA) and 3.3 mg/mL type I collagenase (Worthington Biochemical Corp, Lakewood, NJ) for 30 min in a 37 °C water bath. The digestion mixture was passed through a sterile 230-µm stainless steel tissue sieve (Thermo EC, Holbrook, NY). Adipocytes were allowed to float by gravity, and an aliquot of the supernatant containing adipocytes was collected for adipocyte size measurement (21). Briefly, packed adipocytes were mixed at a ratio of 1:2 with Medium 199 (Life Technologies, Grand Island, NY) containing 1% heat-inactivated fetal bovine serum (Life Technologies), 1% bovine serum albumin (Introgen/Serologicals), and 50 nmol/L adenosine (Sigma-Aldrich, St Louis, MO) on a chambered slide covered with a cover slip. Representative pictures of the adipocytes were taken with a Polaroid Microcam (Polaroid, Waltham, MA) on an inverted microscope (Eclipse TE200), and the adipocyte cell perimeters on scanned images were measured by using Scion Image (Scion, Frederick, MD) by 3 independent readers (interreader variability: 4.7%). Only samples with ≥100 sized cells (n = 48) were included in further analyses (22).

Muscle samples were obtained under local anesthesia (1% lidocaine) from the vastus lateralis muscle via needle biopsy. Muscle was rapidly trimmed of visible connective tissue and blotted free of blood, and small pieces were mounted on corkboard, frozen by immersion in isopentane cooled on liquid nitrogen (–160 °C), and stored at –70 °C until analyzed.

The amount and quality of the sample allowed histochemical analyses in 41 subjects. Muscle cross sections (10 µm) obtained with a microtome at –20 °C were stained for myofibrillar ATPase by established techniques (23) for the determination of fiber types (I, IIA, and IIX). The intramyocellular lipid content (IMCL) of muscle fibers was measured by using the oil-red-O Sudan-type dye staining technique, which mainly reveals triacylglycerols (23). Briefly, a stock solution was prepared by solubilizing 300 mg oil-red-O dye (Sigma, St Louis, MO) in 100 mL concentrated isopropanol (99%). Muscle sections were incubated at room temperature for 10 min in 12 mL freshly prepared stock solution (filtered through a Whatmann paper no. 42) diluted in 8 mL distilled water. Thereafter, stained samples were immersed 4 times in distilled water and rinsed with running water for another 10 min. Glass slides were overlaid with a glycerol drop (to preserve lipids) and sealed with a cover slip and an acetone-based nail polish. All sections were examined under a light microscope (Leitz Dialux 20, Wetzlar, Germany) connected to a CCD camera (Sony C-350) with an analog-to-digital conversion system for image capture. Image analysis was performed by using the public domain NIH Image program (Internet: http://rsb.info.nih.gov/nih-image/). Myosin ATPase and oil-red-O–stained fibers were matched, and the oil-red-O staining intensity of type I, IIA, and IIX muscle fibers was quantified as the average intensity signal over the entire surface of the fiber.

Magnetic resonance imaging (MRI) of the abdomen was performed by using a 1.5-T Sigma scanner (General Electric, Milwaukee, WI), which acquired a series of cross-sectional T1-weighted scans centered on L4–L5 for measurement of fat distribution and around portal hilus for estimation of the relative intensity of liver. MRI images were not acquired in 2 subjects because of scanner unavailability at the time of their admission. VAT and abdominal SAT areas were measured by using ImageJ software (Internet: http://rsb.info.nih.gov/ij/) as the sum of pixels above the intensity corresponding to the nadir between the lean and fat peaks within and outside the abdominal wall (24). Liver fat content was estimated from the relative intensity of liver to that of SAT and spleen (assumed to be fat free). Regions of interest were drawn by using the auto-trace tool, which enables threshold-based segmentation by connecting all pixels within a specified threshold range around a selected seed pixel using Analyze Direct software (Mayo Clinic, Rochester, MN). Intrahepatic lipid content (IHL) was estimated by using the formula

Formula 1(1)
where AI is average intensity.

Statistical analysis
Statistical analyses were performed by using SAS (version 8.02; SAS Institute, Cary, NC). The values of nonnormally distributed variables were logarithmically transformed to approximate normal distribution. Student t test was used for comparisons between men and women, and Pearson correlations were used to test for simple or partial correlations between the variables. General linear regression analysis was used to test for independent predictors of the outcomes after adjustment for confounders. P values less than 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical characteristics of the study population are presented in Table 1Go. On average, women had a higher percentage of body fat (BF), larger adipocytes, less VAT, a higher IMCL in type IIA and type IIX fibers, and a lower fasting plasma adiponectin concentration than did men. Both mean subcutaneous abdominal adipocyte diameter (AD) and fasting plasma adiponectin concentration correlated with BF but not with SAT or VAT (Figure 1Go). After adjustment for sex in the partial correlation, the association between BF and AD was weaker but still significant, whereas the association between BF and plasma adiponectin concentration disappeared (Figure 1Go). IHL was positively associated with AD, negatively associated with fasting plasma adiponectin concentration, and not related to BF (Figure 2Go). IMCL in type I fibers was related neither to BF nor to AD or plasma adiponectin concentration. IMCL in type II fibers was related only to plasma adiponectin concentration (Figure 2Go). These associations were slightly attenuated after adjustment for sex (Figure 2Go). After adjustment for age and sex, both AD and fasting plasma adiponectin concentration were independent predictors of IHL before as well as after additional adjustment for VAT, which was a borderline independent predictor of IHL (Table 2Go).


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  		TABLE 1. Anthropometric and metabolic characteristics of the study population1

 

Figure 1
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  		FIGURE 1.. Correlation of mean subcutaneous abdominal adipocyte diameter and fasting plasma adiponectin concentration with measures of total and abdominal adiposity in men (•) and women ({circ}). Data are Pearson correlation coefficients. Solid lines represent the regression, and the dotted lines represent 95% CIs for significant correlations only.

 

Figure 2
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  		FIGURE 2.. Correlation of intramyocellular lipid content (IMCL) and intrahepatic lipid content (IHL) with adiposity, mean subcutaneous abdominal adipocyte diameter, and fasting plasma adiponectin concentration in men (•) and women ({circ}). Data are Pearson correlation coefficients. Solid lines represent the regression, and the dotted lines represent 95% CIs for significant correlations only.

 

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  		TABLE 2. Multiple linear regression analysis of intrahepatic fat content

 
Fasting plasma insulin concentrations correlated positively with BF, SAT, VAT, and IHL and correlated negatively with plasma adiponectin concentration (Table 3Go). Endogenous glucose production (EGP) during insulin infusion was fully suppressed in 22 subjects. Insulin action on glucose uptake and production ({Delta}EGP/{Delta}insulin) was not related to AD, BF, or IMCL (Table 3Go). Insulin-mediated glucose disposal was negatively associated with SAT (Table 3Go), VAT, and IHL (Table 3Go and Figure 3Go) and positively correlated with fasting plasma adiponectin concentration (Table 3Go and Figure 3Go). {Delta}EGP/{Delta}insulin was related only to IHL (Table 3Go). In the multivariate analysis, IHL but not VAT or plasma adiponectin concentration was an independent predictor of insulin-mediated glucose disposal (Table 4Go).


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  		TABLE 3. Pearson correlation of fasting plasma insulin concentration and peripheral (M) and hepatic ({Delta}EGP/{Delta}insulin) insulin action with measures of adiposity and fasting plasma adiponectin concentration1

 

Figure 3
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  		FIGURE 3.. Pearson correlation of insulin-mediated glucose disposal (M) with visceral adipose tissue, intrahepatic fat content, and fasting plasma adiponectin concentration in men (•) and women ({circ}). Dotted lines represent 95% CIs.

 

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  		TABLE 4. Multiple linear regression analysis of insulin-mediated glucose disposal (M)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study of obese individuals with normal glucose regulation, mean size of adipocytes from abdominal SAT was positively associated with increased lipid content in liver but not in skeletal muscle. Contrary to our hypothesis, mean adipocyte size was not associated with fasting plasma adiponectin concentration. However, both increased mean adipocyte size and low adiponectin concentration were significant predictors of increased fat content in liver, which in multivariate analysis was the only significant predictor of decreased peripheral and hepatic insulin action.

Adipose tissue expands by recruiting new adipocytes and accumulating lipids in existing ones (1). However, even adipocytes ultimately have limited capacity to store lipids (2). Individuals with larger adipocytes in SAT may therefore have a lower capacity for further lipid storage, so the subsequent excesses of fat may be stored in VAT, liver, and skeletal muscle (5). As further increase in adipocyte size becomes limited, increased accumulation of lipid in the visceral organs and in skeletal muscle was suggested to link obesity characterized by large adipocytes with insulin resistance and increased risk of diabetes (5). To test this hypothesis, we studied obese Pima Indians with normal glucose tolerance. We found that mean adipocyte size positively correlated with intrahepatic fat but not with IMLC. A similar finding was recently reported in a cohort of overweight individuals of diverse ethnic origins (4). It is well known that the negative association of insulin action with total body adiposity is attenuated in individuals at the upper spectra of obesity (25-27). In addition, our data indicate that in obese individuals even enlargement of subcutaneous abdominal adipocytes is not a significant predictor of additional worsening of insulin sensitivity.

Although a negative association between insulin-mediated glucose disposal and IMCL was reported by several authors (28, 29), other studies, as well as our present study, have failed to find such an association (4, 30). It has been postulated that muscle triacylglycerols are only a surrogate for other lipid species, having a more direct effect on insulin action, such as diacylglycerol or fatty acid acyl-CoA (30). There is also evidence that muscle oxidative capacity is the major predictor of peripheral insulin action, not the levels of these lipid metabolites in skeletal muscle (31). It is plausible that insulin resistance and increased IMCL are 2 independent characteristics related to mitochondrial dysfunction.

In the liver, insulin suppresses the endogenous production of glucose. Previous studies in healthy individuals have shown that the suppressibility of EGP is negatively related to intrahepatic fat content (32). In our study, both the hepatic and peripheral action of insulin were inversely associated with IHL. This agrees with data showing that both hepatic and peripheral insulin action are lower in individuals with nonalcoholic fatty liver disease (NAFLD) than in healthy controls (33). Whether increased accumulation of fat in the liver is the cause or the consequence of peripheral insulin resistance is not clear. Rats with a genetically increased accumulation of fat in the liver are characterized by reduced insulin-mediated glucose disposal in the skeletal muscle (34). In addition, fatty liver in animals and humans is associated with the increased production of fetuin-A (35), which impairs insulin signaling in the liver and skeletal muscle (36). The prevailing opinion, however, is that fat increasingly accumulates in the liver secondary to peripheral insulin resistance via insulin-mediated activation of lipogenesis and inhibition of lipid oxidation (37). An increase in IHL may further worsen the inhibitory effect of insulin on hepatic glucose production. In fact, previous studies in Pima Indians showed that serum alanine aminotransferase activity, considered to be a marker of liver fat content, predicted the prospective decline in hepatic but not in peripheral insulin action (38), and that hyperinsulinemia predicted the development of type 2 diabetes mellitus independently of low insulin-mediated glucose disposal (39).

Similar to previous reports (14, 15, 40), a lower fasting plasma adiponectin concentration was associated with reduced peripheral insulin action and with higher IMCL and IHL. These data are supported by experimental evidence that adiponectin increases glucose uptake in skeletal muscle and increases fatty acid oxidation and reduces fatty acid synthesis in both skeletal muscle and liver (41). The activation of the AMP-kinase pathway is thought to provide the mechanistic link for these effects of adiponectin (41). In rat skeletal muscle, AMP-kinase stimulation by adiponectin or synthetic agonist is followed by increased glucose uptake and fatty acid oxidation in fast-twitch but not slow-twitch muscle (42, 43). In lean humans, although a low plasma adiponectin concentration is associated with high IMCL in slow-twitch muscle, it predicts high-fat-diet–induced fat accumulation in fast-twitch muscle (14). In our study, the plasma adiponectin concentration was inversely associated with IMCL in fast-twitch (type II) but not in slow-twitch (type I) muscle fibers. It is conceivable that, with overfeeding or with the development of obesity, type I fibers become easily saturated with lipids because they have a high lipid transport capacity, whereas lipid metabolism in type II fibers depends more on the adiponectin-induced activation of AMP-kinase pathway.

We cannot exclude that the average cell size might have been underestimated in subjects with the largest fat cells due to their increased propensity for disruption or lysis when treated by collagenase (44). It must also be noted that hepatic insulin sensitivity reflects primarily fasting endogenous glucose output in subjects with complete suppression of EGP (43% of the group) warranting some caution when interpreting the results on hepatic insulin sensitivity. Another possible limitation of our study is that a standard MRI has a low sensitivity to detect small amounts of fat and low specificity to distinguish steatosis from other types of liver pathology (45). However, we attempted to minimize those limitations by inclusion of subjects without clinical and laboratory signs of liver dysfunction, which is typical for more advanced liver pathologies and by inclusion of obese subjects who are likely to have a higher liver fat content. In addition, because we have replicated several relations shown by others using gold standard techniques (4, 15, 32, 33), we believe that it provided a valid estimate of relative IHL. Finally, although we used the same biopsy sites as in previous studies reporting the relation of insulin resistance to adipocyte size (3) and skeletal muscle triacylglycerol content (46), our conclusions refer to a small part of a much larger and heterogeneous adipose tissue and skeletal muscle compartment.

In conclusion, our data indicate that the enlargement of subcutaneous abdominal adipocytes is not directly related to insulin action but predicts an increased liver fat content in obese individuals with normal glucose tolerance. This association was independent of fasting plasma adiponectin concentration. Furthermore, liver fat content was the only independent predictor of reduced peripheral and hepatic insulin action in the multivariate analysis, which indicates a pivotal role of increased liver fat accumulation in predicting metabolic risk attributed to subtypes of obesity with enlarged subcutaneous abdominal adipocytes, hypoadiponectinemia, or excess visceral adiposity (Figure 4Go).


Figure 4
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  		FIGURE 4.. Central role of increased liver fat accumulation for impaired insulin action in obesity. Enlargement of adipocytes in the abdominal subcutaneous adipose tissue (AT), decreased production of adiponectin in the adipocytes from subcutaneous AT, and increased intraabdominal adipose tissue mass are independent risk factors for the increased accumulation of fat in the liver, which in turn has a negative effect on both hepatic and peripheral insulin action. Fat accumulation in liver may be further aggravated by the prolipogenic action of excess insulin, which compensates for insulin resistance.

 


   ACKNOWLEDGMENTS
 
We acknowledge Thomas Brookshire, Kathy Trinidad, and the nursing and dietary staff for the care of the volunteers; Josée St-Onge, Emma Rousseau, and Shannon Parrington for technical assistance; and Nicola Pannacciulli, Duc Son Le, and Angelo DelParigi for help with the data evaluation. We are grateful to the members and leaders of the Gila River Indian Community for their continuing cooperation in our studies.

The authors' responsibilities were as follows—JK: data collection and analysis and manuscript writing; NS: study design and data collection; PAP: study design and analyses of adipocytes; CW: study design; MS: analysis of plasma samples (adiponectin); CB: study design; SRS: advice and consultation on magnetic resonance imaging; DRJ: analyses of skeletal muscle samples; TF: design and supervision of the assay (adiponectin); JK: manuscript writing; JCB: study design and manuscript writing. None of the authors had a conflict of interest to disclose.


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 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication June 8, 2007. Accepted for publication September 13, 2007.




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