Potential conflict of interest: Dr. Balas is an employee of F. Hoffmann-La Roche. Dr. Schenker is a consultant for and advises Sanofi-Aventis, Lilly, Roche, Schering-Plough, Novartis, and Pfizer.
Pioglitazone treatment improves insulin resistance (IR), glucose metabolism, hepatic steatosis, and necroinflammation in patients with nonalcoholic steatohepatitis (NASH). Because abnormal lipid metabolism/elevated plasma free fatty acids (FFAs) are important to the pathophysiology of NASH, we examined the impact of pioglitazone therapy on adipose tissue insulin resistance (Adipo-IR) during the treatment of patients with NASH. To this end, we assessed glucose/lipid metabolism in 47 patients with impaired glucose tolerance/type 2 diabetes mellitus and NASH and 20 nondiabetic controls. All individuals underwent a 75-g oral glucose tolerance test (OGTT) in which we measured glucose tolerance, IR, and suppression of plasma FFAs. We also measured Adipo-IR index (fasting, FFAs × insulin), hepatic fat by magnetic resonance spectroscopy, and liver histology (liver biopsy). Patients were randomized (double-blind) to diet plus pioglitazone (45 mg/day) or placebo for 6 months, and all measurements were repeated. We found that patients with NASH had severe Adipo-IR and low adiponectin levels. Fasting FFAs were increased and their suppression during the OGTT was impaired. Adipo-IR was strongly associated with hepatic fat (r= 0.54) and reduced glucose clearance both fasting (r=0.34) and during the OGTT (r=0.40, all P <0.002). Pioglitazone significantly improved glucose tolerance and glucose clearance, steatosis and necroinflammation (all P<0.01-0.001 versus placebo). Fasting/postprandial plasma FFAs decreased to levels of controls with pioglitazone (P<0.02 versus placebo). Adipo-IR decreased by 47% and correlated with the reduction of hepatic fat (r=0.46, P=0.009) and with the reduction in hepatic necroinflammation (r=0.47, P=0.0007). Conclusion: Patients with NASH have severe Adipo-IR independent of the degree of obesity. Amelioration of Adipo-IR by pioglitazone is closely related to histological improvement and plays an important role during treatment of patients with NASH. (HEPATOLOGY 2009)
Nonalcoholic fatty liver disease (NAFLD) and its more severe form associated with steatohepatitis (NASH) are common clinical problems now reaching epidemic proportions.1, 2 Hepatic and muscle insulin resistance (IR) are known to be a primary feature of patients with fatty liver3–8 and/or steatohepatitis.9 However, adipose tissue insulin resistance (Adipo-IR) has received considerably less attention, although fat is another important target of insulin action. Insulin inhibits triglyceride lipolysis and promotes lipid storage. Failure to do so leads to the oversupply of free fatty acids (FFAs) and “lipotoxicity” from ectopic accumulation of lipids in organs (i.e., liver) that do not normally store large amounts of fat.10 In this setting, lipid is shunted into harmful pathways of nonoxidative metabolism with accumulation of toxic metabolites that promote the activation of inflammatory pathways (i.e., IκB/nuclear factor-κB and c-Jun N-terminal kinase pathways).11In vitro and animal models of NASH confirm the close link between FFA overload and steatohepatitis.12–15 Under normal living conditions, FFA supply to the liver in humans is largely (66%) of endogenous origin (i.e., adipose tissue lipolysis) and to a lesser degree from exogenous (i.e., dietary) sources.16 In obese patients with NASH, Donnelly et al.17 clearly demonstrated that most of the FFAs used for triglyceride synthesis and very low-density lipoprotein secretion derives from adipose tissue. Taken together, there is an urgent need to better understand the relationship between Adipo-IR and steatohepatitis in humans.
Thiazolidinediones (TZDs) have a significant effect on insulin sensitivity in insulin-resistant states and in type 2 diabetes mellitus,18, 19 as well as in patients with NAFLD or NASH.1, 2 TZDs exert their metabolic effects through activation of the gamma isoform of the peroxisome proliferator-activated receptor (PPARγ), a nuclear receptor primarily involved in lipid metabolism.21–23 PPARγ is predominantly expressed in adipose tissue, but are also present in muscle, liver, pancreas, heart, and spleen.12, 24 Activation of PPARγ results in adipocyte differentiation25 and adipogenesis.26 At the level of the liver, TZDs stimulate fatty acid oxidation and inhibit hepatic fatty acid synthesis via activation of adenosine monophosphate–activated protein kinase (AMPK).27 In obesity and type 2 diabetes mellitus, TZDs increase plasma adiponectin levels, decrease excessive rates of lipolysis, and cause a redistribution of fat from liver and visceral depots to subcutaneous adipose tissue.18, 19, 28, 29 Using glucose turnover techniques, TZDs have also been shown to improve hepatic and peripheral (i.e., muscle) insulin sensitivity in patients with NASH.9 Thiazolidinediones have also been shown to have anti-inflammatory effects in patients with NASH.20, 30, 31 Despite the wealth of knowledge regarding the effects of TZDs, their impact on Adipo-IR in NASH has not been fully evaluated, in particular in relation to histological changes following treatment in patients with NASH.
To gain insights into the relationship between the degree of Adipo-IR, steatohepatitis, and the impact of TZD therapy in humans, we studied a group of lean and obese patients with NASH and compared them with a group of healthy controls matched for total body adiposity. We then evaluated the effect of pioglitazone treatment on Adipo-IR in the patients with NASH and assessed its relationship with changes in liver histology.
Participants included 47 patients with NASH and 20 nondiabetic subjects (controls) that were recruited from the University of Texas Health Science Center at San Antonio, the Audie L. Murphy Veteran Administration Medical Center, and Brooke Army Medical Center, San Antonio, TX. After excluding other etiologies for liver disease, the diagnosis of NASH was confirmed by a liver biopsy. Volunteers were excluded if they had aspartate aminotransferase and alanine aminotransferase elevated to ≥2.5 times the upper limit of normal, history of heavy alcohol use (>1 drink/day), fasting glucose ≥240 mg/dL, type 1 diabetes, heart, hepatic (other than NASH) or renal disease, or were taking metformin, thiazolidinediones, or insulin. The study was approved by Institutional Review Board of the UTHSCSA, and informed written consent was obtained from each patient before participation. All subjects gave written informed consent prior to participation.
The protocol has been previously described.9 In brief, during the 4-week run-in period, subjects were instructed by the research dietician not to change the calorie content of their diet or their physical activity level. Subjects were started on placebo and compliance was assessed by pill count on follow-up visits. Baseline metabolic measurements were performed between weeks 3 and 4, and were repeated at the end of the study. They included: (1) fasting plasma glucose, hemoglobin A1c, lipid profile, insulin, free fatty acid (FFA), and adiponectin concentrations; (2) whole-body fat by dual energy X-ray absorptiometry (DXA); (3) hepatic fat content by magnetic resonance spectroscopy (MRS); and (4) a 4-hour glucose tolerance test (OGTT) with the use of the double-tracer technique to measure glucose turnover and glucose clearance as previously described.9 Plasma glucose, insulin, and FFA concentration were determined every 15–30 minutes during the study.
After baseline metabolic measurements, participants were randomized to either placebo or pioglitazone (ACTOS, Takeda Pharmaceuticals) 30 mg/day, titrated after 2 months to 45 mg/day until the end of the study (6 months). Randomization was computer-generated by the research pharmacy and blinded to investigators. All patients were instructed at baseline by a research dietician to reduce their intake by 500 kcal/day. This instruction continued during follow-up visits in all participants. Every 2 weeks, subjects were followed by the investigators, who recorded vital signs, performed a physical examination, reviewed home glucose monitoring results if individuals had diabetes, and assessed study drug compliance (confirmed by pill count) and adverse events. Blood was drawn for liver function tests and metabolic measurements. After 6 months of treatment, subjects repeated all baseline measurements and the study was considered completed.
Measurements of Total Body and Liver Fat Content.
Whole-body fat content (FM) and fat-free mass (FFM) were measured by dual-energy absorptiometry (DXA) (Hologic Inc., Waltham, MA). For the measurement of hepatic fat content, localized proton nuclear magnetic resonance spectra of the liver were acquired on a 1.9-T magnetic resonance imaging scanner (Elscint Prestige Ltd., Haifa, Israel) as described.9
Analytical Methods and Calculations.
Plasma glucose was determined by the glucose oxidase method (Beckman Instruments Inc., Fullerton, CA). Plasma insulin concentration was measured by radioimmunoassay (Diagnostics Products, Los Angeles, CA) and plasma FFAs with standard colorimetric methods. Plasma adiponectin was measured by radioimmunoassay (Linco Research, St. Charles, MO). Plasma glucose radioactivity was measured from plasma extracts precipitated from barium oxide and zinc sulfate.
There is a linear relationship between the rise in the fasting plasma insulin level and the decline in the rate of basal (fasting) endogenous glucose production (EGP) in healthy subjects.32 The higher the rate of EGP and the level of fasting plasma insulin (FPI), the greater the severity of hepatic IR. Therefore, an hepatic IR index was calculated as the product of fasting EGP and FPI concentration (hepatic IR index = EGP × FPI [μmol/minute−1/kg FFM−1/pmol/L]). Insulin is also a strong inhibitor of lipolysis, and a similar relationship exists in healthy subjects between the FPI concentration and fasting plasma FFA levels.32 Thus, an index of Adipo-IR was calculated as the product of the fasting plasma FFA and insulin concentration (Adipo-IR index = FFA × FPI [mmol/L/pmol/L]).7, 32 Experimental validation for both indexes has been published previously.7, 9, 18, 19, 29, 33 Mean values during the OGTT were calculated as the area under the curve divided by the duration of the test (240 minutes).
Data are given as the mean ± standard error of the mean if normally distributed. Variables with skewed distribution are expressed as median and interquartile range (in parentheses). Group differences were analyzed by Student t test, Mann-Whitney U test, and χ2 test, for normally distributed, non-normally distributed, and noncontinuous variables, respectively. Univariate and multivariate analysis were used to estimate associations among continuous variables in the whole dataset. A two-tailed P < 0.05 was considered statistically significant.
To study the impact of pioglitazone changes in adiponectin versus variables of interest (Figs. 2–4), we performed a regression analysis using both pretreatment and posttreatment values in the subgroup of subjects treated with pioglitazone. Correlation coefficient and P values were reported in the graphs as well as in the text.
Lipid Metabolism in Patients with NASH.
In Table 1, we summarize the patient characteristics of the 47 patients with NASH and 20 controls. Both groups were well-matched for adiposity (Table 1). As expected, patients with NASH at baseline had increased liver fat content by MRS and plasma glucose–hemoglobin A1c and liver aminotransferases compared to control subjects (all P < 0.001). Plasma adiponectin was significantly lower in patients with NASH (13.5 ± 1.4 μg/mL versus 6.5 ± 0.5 μg/mL; P < 0.001), indicative of dysfunctional and insulin-resistant adipose tissue. No statistical difference was found between adiponectin concentrations in male and female patients with NASH, although female patients tended to have higher concentrations (female: 6.9 ± 0.7 versus male: 5.7 ± 0.6, P = 0.20). The fasting and postprandial plasma insulin concentration was two-fold to three-fold higher in patients compared to controls, which indicates systemic IR. Despite the elevated plasma insulin concentration in patients with NASH, FFA concentration was significantly higher in patients with NASH (739 ± 34 μmol/L versus controls 533 ± 42 μmol/L; P < 0.001). During the OGTT, insulin-mediated suppression of plasma FFAs was impaired (mean value 0–240 minutes: 359 ± 19 μmol/L versus 232 ± 19 μmol/L; P < 0.001), and the plasma FFA profile of subjects with NASH was ∼30%–35% higher and always above that in controls. Taken together, the fasting and postprandial plasma FFA results in the group of subjects with NASH suggested marked adipose tissue resistance to the antilipolytic effect of insulin (Fig. 1).
Table 1. Subject Characteristics
Controls (n = 20)
Patients with NASH (n = 47)
P < 0.001 NASH versus controls. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; DEXA, dual energy X-ray absorptiometry; FFAs, free fatty acids; OGTT, oral glucose tolerance test.
To further examine this, we calculated the Adipo-IR index and found that it was clearly abnormal with a 4.5-fold increase in patients with NASH (72.6 ± 7.9 mmol/L × pmol/L versus 16.1 ± 2.5 mmol/L × pmol/L; P < 0.0001). A significant finding was that this was independent on the degree of obesity (Fig. 2). In the entire group, the almost three-fold higher hepatic fat content of patients with NASH was associated with increased Adipo-IR (r = 0.54, P < 0.0001) and with decreased plasma adiponectin (r = −0.31, P < 0.02). Patients with NASH showed increased hepatic IR that was associated with the elevated fasting and postprandial (OGTT) plasma FFA concentration (r = 0.37, P = 0.003 and r = 0.30, P = 0.02, respectively). Both hepatic IR and Adipo-IR were strongly associated with reduced glucose clearance both during fasting (r = 0.43 and r = 0.34) and during the OGTT (r = 0.46 and r = 0.40, all P < 0.002). The Adipo-IR was higher in subjects with increased steatosis (score >1, P = 0.04), or MRS-measured hepatic fat greater than 20% (P = 0.02). However, no direct correlation was found between Adipo-IR and scores, probably because the score is a discrete number.
Effect of Pioglitazone Treatment on Lipid Metabolism in NASH.
After the baseline evaluation, patients with NASH were randomized to pioglitazone or placebo for 6 months. Compared to placebo, plasma FFA concentration decreased by ∼20% with pioglitazone (P = 0.01), and the plasma FFA profile during the OGTT was reduced to levels that were superimposable to controls (Fig. 3). In contrast, subjects treated with placebo the change in the plasma FFA concentration was modest and non-significant. Adipo-IR decreased by ∼47% (from 73.6 ± 12.8 mmol/L × pmol/L to 38.7 ± 8.4 mmol/L × pmol/L; P = 0.03), and this was strongly correlated to the reduction of hepatic fat accumulation (r = 0.46, P = 0.009).
As previously reported, pioglitazone treatment significantly improved the histological score.9 We found that the improvement in FFA metabolism observed after pioglitazone treatment was a strong determinant of the improvement in liver histology (Fig. 4). The combined mean necroinflammation score did not change significantly in patients receiving diet and placebo (∼12%), while it improved by ∼44% in patients receiving diet and pioglitazone (P < 0.001 versus placebo). Improvement in the necroinflammation score was strongly correlated to the reduction in Adipo-IR (r = 0.47, P = 0.0007). Decrease in the steatosis score (∼50%) was also related to the reduction in Adipo-IR (r = 0.29, P = 0.049), whereas the relationship with the changes in fibrosis did not reach statistical significance (r = 0.25, P = 0.09) (Fig. 4). There was a correlation also between the improvement in histology and hepatic IR, but the correlation with improvement in Adipo-IR was stronger.
It is recognized that chronically elevated plasma FFA concentrations can impair insulin sensitivity and insulin secretion.33–36 Subjects with NASH have an increased release of FFAs,4 even when they are not obese.5, 6 This study provides a novel role for Adipo-IR, because it establishes its role in relation to histological changes and pioglitazone therapy in patients with NASH. In addition, it offers insights into postprandial FFA metabolism in patients with NASH. Taken together, this study highlights the importance of dysfunctional fat on the severity of NASH and its potential reversal through the removal of lipotoxicity.
We observed a markedly increased fasting and postprandial (i.e., OGTT) plasma FFA level in lean and obese patients with NASH. Moreover, insulin-mediated suppression of lipolysis and FFA release was markedly impaired. This is important because for most of the day humans live in a postprandial state. In addition, the postprandial state is characterized by hyperinsulinemia, and frequently by hyperglycemia, in patients with NASH; both of these conditions are major drivers of hepatic lipogenesis.17, 37 Thus, elevated plasma FFA levels can cause or aggravate the major pathogenic disturbances that are responsible for impaired glucose homeostasis. This creates the conditions for ectopic fat accumulation not only in the liver but also in cardiac38 and skeletal muscle,39 as well as in beta cells.10, 11 Therefore, understanding adipose tissue metabolism is extremely important for the prevention and treatment of NASH.
In this study, we observed that patients with NASH had a 30% increase in fasting plasma FFA concentration despite high fasting insulin levels. Therefore, it was not surprising that patients with NASH had an Adipo-IR index that was several-fold higher (worse) than controls matched for total body adiposity (Fig. 2). This is consistent with recent work in rodents from our laboratory in which the combination of elevated plasma insulin and FFAs were much more steatogenic than elevated glucose and FFAs alone.40 An unexpected finding was that both obese and nonobese patients with NASH had similar Adipo-IR, a finding that deserves confirmation in a larger cohort of patients. This can be interpreted as evidence that dysfunctional fat, and not total body fat per se, is the driving force for hepatic lipotoxicity in NASH.
Uncontrolled studies41–43 and short-term randomized controlled trials9, 44, 45 with TZDs have shown them to be effective for the treatment of NASH. There is strong evidence in rodents that TZDs ameliorate lipotoxicity, although the evidence in humans is scarce.9–11, 20, 31, 46 This analysis offers new insights on how TZDs might improve steatohepatitis by ameliorating lipotoxicity. As shown in Fig. 3, 6 months of pioglitazone treatment was sufficient to restore normal insulin action on adipocytes, so that postprandial FFA concentrations were matched with control levels. This is consistent with the reduction observed in Adipo-IR and is in line with previous reports that pioglitazone up-regulates genes involved in adipogenesis as well as fatty acid oxidation.47, 48
A very important finding of this work is that restoration of adipose tissue insulin sensitivity by pioglitazone translated into a frank improvement in the severity of steatohepatitis. There may be multiple reasons for such an effect, including a reduction of plasma FFA supply combined with a reduction of hyperglycemia and hyperinsulinemia, as well as an increase in hepatic FFA oxidation as a function of an improvement in hepatic AMPK activity (either directly or from the increase in plasma adiponectin levels49), both having indirect and direct effects on hepatic glucose and lipid signaling. Improvement in mitochondrial function has also been reported as a direct effect of pioglitazone treatment.48 Amelioration of mitochondrial stress would be expected to reduce the production of reactive oxygen species, lipid peroxidation, and activation of inflammatory pathways12, 50 and could explain the close relationship between improvement of Adipo-IR, lipotoxicity, and necroinflammation observed in this study.
In summary, patients with NASH showed severe Adipo-IR. Pioglitazone treatment improved it, and this was closely related to histological improvements (i.e., reduction in liver fat and associated necroinflammation). Thus, Adipo-IR plays a crucial role in the pathogenesis of NASH, and it should be a specific target of treatment. Future studies should investigate the role of early intervention (such as lifestyle and/or pharmacological therapy) for the prevention and treatment of dysfunctional fat in NASH.
We thank our study volunteers, the nursing staff, and the nutrition and laboratory staff for their assistance in performing the described studies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources of the National Institutes of Health.