Nonalcoholic fatty liver disease (NAFLD), characterized by excessive accumulation of fat in the liver, is commonly associated with obesity-related comorbidities such as type 2 diabetes, dyslipidemia, and metabolic syndrome. It is estimated that the incidence of NAFLD in the United States is approximately 25% of the adult population (1). The natural history of NAFLD includes a slow progression from simple steatosis (hepatocyte ballooning) to steatohepatitis (steatosis + inflammation) and then a faster progression toward fibrosis, cirrhosis, and, in a more limited number of cases, hepatocellular carcinoma (2). The severity of NAFLD and its progression toward cirrhosis and liver failure are associated with escalating risk toward atherogenesis and cardiovascular disease (3,4). Slowing down, or possibly reversing, the progression of NAFLD to advanced fibrosis has an immense potential to reduce the societal cardiovascular and general health risk burden.
There are no effective and safe pharmacological agents for treating NAFLD (5), as metformin and thiazolidinediones have been shown to improve liver histology only after inflammation sets in (commonly referred to as nonalcoholic steatohepatitis, or NASH). Previously, it was reported that bariatric surgery induced long-term improvements in liver histology in patients with grade 3 obesity and NASH (6,7). Interestingly, the long-term effectiveness of bariatric surgery for improving liver pathology was predicted by early improvements in whole-body insulin sensitivity. Weight loss (~5% to 10%) achieved by a combination of physical activity and hypocaloric diet is the mainstay of therapy for NAFLD and NASH (5). However, the amount weight loss achieved, even in a tightly regulated setting, is highly variable (8). In this study, we sought to determine whether short-term aerobic exercise training could manifest metabolic improvement in liver-related outcomes.
The liver plays a critical role in maintaining peripheral insulin concentrations relative to insulin secretion by regulating the first-pass metabolism of insulin, a phenomenon known as “hepatic insulin extraction” (HIE). In healthy adults, ~50% to 80% of secreted insulin (in the portal vein) is extracted and cleared by hepatocytes (9). Meier et al. (9) found that both the amount of insulin secreted in a pulsatile fashion and the amplitude of the pulsatile peak were positively correlated with hepatic insulin clearance and delivery of insulin to the circulation. Incretins, like GLP-1, can also theoretically influence HIE primarily by modulating the degree of insulin secretion. Although the presence of hepatic insulin resistance is established in NAFLD (10), the relationship with liver fat content is strongly debated (11,12). Understanding these intricate relationships is important to devise targeted therapies to manage NAFLD. In this study, we explored whether HIE is related to liver fat content and/or peripheral insulin resistance. Second, we evaluated whether HIE is modulated by aerobic exercise, independent of weight loss.
The recruitment and methods have been previously described (13) (Fig. 1). Briefly, 13 obese, sedentary adults (age, 58 ± 3.4 yr; body mass index, 34.3 ± 1.1 kg·m−2) with radiographically confirmed NAFLD (>5% intrahepatic lipid (IHL) content) were enrolled into a short-term, 7-d exercise intervention. After obtaining informed consent, individuals underwent a medical screening, physical examination, oral glucose tolerance test (OGTT), and blood analyses including a lipid panel and liver enzymes to estimate hepatic function. The exclusion criteria included individuals with any active disease condition, use of medications known to affect study outcomes, individual alcohol consumption >20 g·d−1 for men and >10 g·d−1 for women, ongoing exercise or weight loss programs, any contraindications to exercise (as detected during a 12-lead electrocardiogram exercise test), and postmenopausal status or hormonal replacement therapy in women. This study was approved by Cleveland Clinic Institutional Review Board. All participants signed informed consent forms before study participation.
Physical activity levels were estimated using the Minnesota Leisure Time Physical Activity Questionnaire (14). The subjects were deemed sedentary if they expended <300 kcal·d−1 of energy through their leisure time activity. An incremental graded treadmill exercise test was conducted to determine the participant’s maximal oxygen consumption (V˙O2max) as previously reported (15). Expired air was continuously sampled using an automated sampler (Jaeger Oxycon Pro; Viasys, Yorba Linda, CA). Heart rate was continuously captured during each exercise session. Maximal heart rate was used to prescribe exercise intensity during the exercise training sessions (Polar Electro Inc., Woodbury, NY). The test was deemed satisfactory if ≥3 of the following criteria were attained: 1) a respiratory quotient of >1.10, 2) self-determined fatigue, 3) heart rate at <10 bpm of age-predicted maximum, and (4) plateau in oxygen consumption with increasing workloads. Participants abstained from coffee and alcohol consumption for 12 and 48 h, respectively, before exercise testing.
Height and body weight were measured using standard techniques. Whole-body fat distribution was measured by dual-energy x-ray absorptiometry (Lunar model iDXA, Madison, WI).
Aerobic exercise intervention
Study participants completed 60 min of supervised aerobic exercise on a treadmill at ~85% of HRmax daily for seven consecutive days. They were instructed to maintain current diet during this intervention period.
After an overnight fast, participants were admitted to the Clinical Research Unit, rested for ~30 min, and indirect calorimetry was conducted to estimate energy expenditure and substrate metabolism. Exhaled air was continuously sampled for ~15 min using an automated system under a ventilated hood until steady state was achieved. Energy expenditure, and rates of carbohydrate and fat oxidation were calculated as previously described (16).
After indirect calorimetry, a standard 75-g OGTT was performed at ~8:00 am before and after the 7-d exercise intervention. An intravenous catheter was placed in an antecubital vein and secured with a saline lock. After the baseline draws, the glucose drink was consumed and whole-blood samples were drawn in plastic tubes containing EDTA at 30, 60, 90, 120, and 180 min after ingestion. Incremental area under the curve (iAUC) was calculated using the trapezoidal equation after baseline correction. Insulin sensitivity index (ISI) was estimated using the Soonthorpun model (17):
Here, BW is the body weight in kilograms; AUCglu, area under the glucose curve after glucose load for 3 h; Uglu, urinary loss of glucose; and AUCins, area under the insulin curve for 3 h during the OGTT. HIE was calculated from molar areas under the insulin and C-peptide curves and expressed as fold change relative to baseline (18). Hepatic insulin resistance index (HIRI) was estimated as the product of AUCgluc and AUCins during the first 30 min of the test (19). The C-peptide-genic index, which is the increase in the peripheral C-peptide concentration relative to increase in glucose concentration within 30 min of oral glucose loading, was calculated from [ΔC-peptide(30–0)/ ΔGlucose(30–0)] (19).
Plasma glucose was determined at the time of testing using a YSI 2300 STAT Plus (Yellow Springs, OH). The remaining plasma was immediately stored at −80°C. Plasma insulin concentrations were determined using a radioimmunoassay (Millipore, Billerica, MA). Plasma C-peptide was measured using enzyme-linked immunosorbent assay (ELISA; Linco Research, St. Charles, MO). Plasma free fatty acid concentrations were determined using a colorimetric assay (Wako Pure Chemical Industries, Richmond, VA). GLP-1 and HMW adiponectin were measured through the 120-min time point of the OGTT using commercially available ELISA kits (Millipore, St. Charles, MO). MNC-derived TNF-α was determined via high-sensitivity ELISA (R&D Systems, Minneapolis, MN).
IHL content was measured by 1H-magnetic resonance spectroscopy on a 3-T magnetic resonance system (Siemens Sonata, Erlangen, Germany), before and after the exercise intervention. After an overnight fast, the subjects arrived at the laboratory at ~6:00 am. A body-array magnetic resonance imaging coil with center aligning to the participants’ spine and shoulders was affixed using Velcro straps. Participants were placed in a prone position and head first into the scanner on a memory foam mattress to further minimize respiratory artifact. To accurately scan the liver, an 8-cm3 voxel was placed within the right posterior lobe of the participants’ liver, and magnetic resonance spectra with and without water suppression were acquired with a single-voxel PRESS acquisition (repetition time, 5000 ms; echo time, 30 ms) (20). Final data were Fourier-transformed, filtered, baseline-corrected, and phased. The diagnosis of NAFLD was confirmed if IHL was >5%.
Normality of the data was determined using the Shapiro–Wilk test. Continuous variables are expressed as mean and SEM for parametric measures, and median and interquartile ranges for nonparametric measures. Categorical variables are expressed as percentages. Differences between the preintervention and postintervention groups were analyzed for statistical significance using paired Student t-tests (parametric), and Wilcoxon signed rank test (nonparametric) for continuous variables and χ2 test for categorical variables. α was assumed at 0.05. Effect size was calculated using Cohen’s D formula as the difference in the means divided by the SD of the difference. With the current paired sample size of 13, effect size of 0.84, SD of 0.075, and α of 5%, the calculated power for our primary outcome of HIE was >95%. Correlation analyses were performed using Pearson’s and Spearman methods, and the association between biochemical and anthropometric parameters was evaluated using multivariate linear regression, with and without subgroup analysis, after power transformation. All graphical and descriptive statistical analyses were performed using R Studio, version 1.1.453.
Data from a subset of the individuals included herein have been previously reported (13,21); this secondary analysis is focused on HIE and hepatic insulin resistance. Participant characteristics are shown in Table 1. Blood and metabolic screening data revealed that subjects were prediabetic with a fasting blood glucose concentration of 5.9 ± 0.3 mM and met the diagnostic criteria for hepatic steatosis based on IHL measured with magnetic resonance spectroscopy (average, 14% ± 9%).
The 7-d aerobic exercise training prescription induced a 9.8% ± 3.1% increase in V˙O2max (P < 0.01), and 20.7% ± 6.5% and 21.5% ± 10.4% decrease in the iAUC for glucose and insulin after the glucose load (P < 0.05), respectively (Fig. 2). There was no change in body weight or body fat percentage after the exercise program. Although fasting glucose concentration and C-peptide did not change with exercise training, peripheral insulin concentrations decreased significantly (11.9% ± 4.3%, P < 0.05). There was also an 11.5% ± 4.2% and 13.5% ± 3.9% (P < 0.05) decrease in fasting and iAUC for GLP-1 after the exercise intervention, respectively (Fig. 2). Other plasma metabolic and biochemical parameters did not significantly change.
HIE and ISIOGTT increased significantly (9.8% ± 3.2% and 49.2% ± 14.7%, respectively; P < 0.05). HIRI decreased by 4.0% ± 18% from baseline (P < 0.05) (Fig. 3). The C-peptide-genic index trended toward a decrease; however, this was not statistically significant (pre: 721.6 ± 222.5 pM·mM−1 vs post: 489.6 ± 90.5 pM·mM−1, P = 0.17). There was a 63.5% ± 32.9% increase in hepatic polyunsaturated lipid content (P < 0.05), although the saturated fat percentage and the triglyceride content did not differ for these participants. Whole-body fasting carbohydrate oxidation rate decreased by 28.2% ± 8.1% (P < 0.01).
At baseline, a higher HIE percentage correlated with a lower HIRI, higher peripheral insulin sensitivity, lower hepatic saturated fat percentage, and higher plasma HDL concentration (r = −0.5, 0.67, −0.48, and 0.6, respectively; all, P < 0.05; Fig. 4). The change in HIE correlated significantly with the change in peripheral insulin sensitivity and the change in peak glucose concentration after OGTT (r = −0.69 and 0.61, respectively; P < 0.05; Fig. 5). After the intervention, HIE correlated positively with adiponectin (r = 0.56, P < 0.05) and negatively with plasma TNF-α concentration (r = −0.78, P < 0.001).
NAFLD is a complex disease that arises from a combination of reduced insulin sensitivity (both hepatic and peripheral), hyperinsulinemia, and their related downstream metabolic effects. There is conflicting evidence that HIE is associated with liver fat content, owing to differences in methodologies used and study populations (11,18,22). In this study, we found that lower HIE was significantly associated with a higher saturated fraction of liver fat. The positive association of plasma HDL with HIE was further increased by its interaction with body weight (adjusted R2 = 0.3 vs 0.58, P < 0.05), potentially through ApoA-1 (23), which suggests a central role for insulin dynamics in the development and the progression of NAFLD. ApoA-1 is the major protein component of HDL and is found to have an inverse relationship with hepatic fat content. Decreased HIE, and thus prevailing hyperinsulinemia, promotes insulin resistance (24,25) (Fig. 4). More recently, Viskochil et al. (26) described elevations in plasma insulin concentrations after a 7-d increase in sedentary time owing to dynamic reductions in total HIE (AUC). Hence, exploiting a therapeutic option, which can independently affect each of these components in a dose-dependent and time-dependent manner, should seemingly result in a robust treatment response.
The American Association for the Study of Liver Diseases treatment guidelines recommend exercise as the most important component of treatment for NAFLD (5). The physiological adaptations to exercise are multifactorial; however, to date, the independent effects of exercise on NAFLD have not been isolated from concomitant weight loss. Our study provides evidence that aerobic exercise training quickly reverses some of the underlying pathophysiological derangements of NAFLD, even before the weight-loss phase sets in. These effects may be due to discrete and interdependent increments in HIE, and peripheral and hepatic insulin sensitivity (27). The associations between changes in HIE and peripheral and hepatic insulin resistance support our working hypothesis. Given that skeletal muscle is the largest insulin responsive organ in the body, it stands to reason that the change in skeletal muscle insulin sensitivity may be contributing to the change in HIE. Although the short-term exercise intervention used in this study did not significantly reduce liver triglyceride content or change percentage of saturated fat, small but likely clinically important changes were predicted by the interaction between changes in HIE and V˙O2max. Our findings are in agreement with Utzschneider et al. (11), who compared HIE in NAFLD and non-NAFLD subjects and determined that HIE was not related to liver fat but instead was related to peripheral insulin resistance.
Exercise is known to independently change both glucose and insulin kinetics (28). Fasting plasma glucose and C-peptide concentrations did not change after the intervention. However, the insulin concentrations required to maintain blood glucose levels decreased significantly, indicating the important contribution of increased HIE to maintaining overall glucose homeostasis. In support, we observed that the overall change in HIE correlated positively with change in the peak glucose concentration (90 min after OGTT; Fig. 5, change in HIE and glucose), a measure that also contributes significantly to reduced cardiovascular disease risk (29).
As previously published (30), there was a decrease in fasting and total circulating GLP-1 with short-term exercise training in these patients. However, we did not observe any significant statistical associations between changes in HIE and GLP-1 and any significant change in insulin secretion based on the C-peptide-genic index. This suggests that exercise-induced improvements in glucose homeostasis in people with NAFLD are potentially due to an early increase in HIE and are independent of modulations of the GLP-1/insulin-secretion pathway. The distinct changes in GLP-1 may eventually contribute to sustained improvements in the capacity to secrete insulin. Furthermore, the significant relationship between HIE and adiponectin (r = 0.6) and TNF-α (r = −0.78) after exercise intervention highlights the contribution of HIE to a metabolically healthy and lower systemic inflammatory milieu. These effects may be mediated through circulating free fatty acid concentrations, which could directly reduce HIE independent of prevailing glucose concentrations (31).
First-pass extraction of insulin is ~50% to 80%, and is predominately driven by the liver (32), followed by the kidney at ~20% (33). In the liver, HIE involves binding and internalization via receptor-mediated endocytosis and later degradation by insulin-degrading enzyme (IDE) via lysosome proteolysis (34). Hyperinsulinemia has been implicated to reduce HIE by insulin-receptor saturation (35), although it was recently counterargued that acute changes in HIE are essential for dampening systemic insulin oscillations relative to pulsatile insulin secreted from β-cells (9). Kirwan et al. (36) have previously shown that acute exercise increases HIE in untrained adults, which may be attributed to increased expression of hepatic IDE (37). Kurauti et al. (37) found that exercise can increase the expression and activity of IDE at least via IL-6 (myokine)–mediated transduction and activation of STAT3 in the liver. Long-term aerobic exercise training increases mitochondrial biogenesis, activates hepatic PGC1-α expression, and alters hepatic metabolism by altering the IRS1/IRS2 ratio (38), eventually leading to decreased steatosis. Thus, chronic exercise training could potentially lead to systemic PPAR-α activation and reduced CEACAM1-mediated HIE in the context of improved disposition index (39). We speculate that the increase in HIE in our study may be due to early changes in PPAR-α and mitochondrial biogenesis. We also found that whole-body carbohydrate oxidation rate was decreased in our participants. It is still unclear if this finding is due to change in HIE or peripheral insulin sensitivity, or both. The exploration regarding the potential mechanistic links between substrate oxidation and HIE is still underway.
One of the potential limitations of our study is the use of OGTT modeling for estimating HIE, instead of the gold standard hepatic portal vein sampling. We minimized the error of model estimation during the non–steady state by using AUC calculations of C-peptide, and insulin after the concentration curves returned to baseline levels (40). Other potential limitations could be that our study population is not gender-matched, limited sample size, nonexercising controls, or absence of isotope tracers.
In conclusion, we found that the pathophysiology contributing to NAFLD is quickly reversed by 7 d of aerobic exercise training. Even in the absence of weight loss, exercise reduced hyperinsulinemia through an increase in HIE and, concomitantly, muscle and liver insulin sensitivity. These effects were independent of changes in fasting and aggregate plasma incretin concentrations. Changes in total GLP-1 and adiponectin sensitivity may add more potency to the effectiveness of longer-term exercise in these patients. Our findings further strengthen the rationale for the use of exercise in managing NAFLD.
The authors wish to thank the research volunteers for their outstanding dedication and effort, and the staff of the Clinical Research Unit who helped with the implementation of the study and assisted with data collection.
This research was supported by National Institutes of Health Grant R01-AG-12834 (J. P. K) and was supported in part by the National Institutes of Health National Center for Research Resources, CTSA-1UL1-RR-024989, and the Case Center for Imaging Research, Case Western Reserve University, Cleveland, OH.
The authors declare that they have no conflict of interest. The results of this study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The authors report that results of the present study do not constitute endorsement by the American College of Sports Medicine.
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