Relationship between disease severity, hyperinsulinemia, and impaired insulin clearance in patients with nonalcoholic steatohepatitis


  • Fernando Bril,

    1. Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL
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  • Romina Lomonaco,

    1. Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL
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  • Beverly Orsak,

    1. Diabetes, Diabetes and Metabolism, University of Florida, Gainesville, FL
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  • Carolina Ortiz-Lopez,

    1. Diabetes, Diabetes and Metabolism, University of Florida, Gainesville, FL
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  • Amy Webb,

    1. Hepatology, Diabetes and Metabolism, University of Florida, Gainesville, FL
    2. Audie L. Murphy Veterans Administration Medical Center (VAMC), San Antonio, TX
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  • Fermin Tio,

    1. Pathology Divisions, The University of Texas Health Science Center at San Antonio, San Antonio, TX
    2. Audie L. Murphy Veterans Administration Medical Center (VAMC), San Antonio, TX
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  • Joan Hecht,

    1. Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL
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  • Kenneth Cusi

    Corresponding author
    1. Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL
    2. Diabetes, Diabetes and Metabolism, University of Florida, Gainesville, FL
    3. Audie L. Murphy Veterans Administration Medical Center (VAMC), San Antonio, TX
    4. Malcom Randall VAMC, Gainesville, FL
    • Address reprints to: Kenneth Cusi, M.D., F.A.C.P., F.A.C.E., Division of Endocrinology, Diabetes and Metabolism, University of Florida, 1600 SW Archer Road, Room H-2, Gainesville, FL 32610. E-mail:; fax: 352-846-2231.

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  • Potential conflict of interest: Nothing to report.

  • This work was supported by the Burroughs Wellcome Fund (to K.C.), the American Diabetes Association (1-08-CR-08; to K. C.), and a VA Merit Award (1 I01 CX000167-01; to K. C.).


Hyperinsulinemia is believed to play a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH) and associated cardiovascular risk. However, the relative contribution of insulin clearance to hyperinsulinemia and its relationship to liver histology have not been carefully evaluated before. To examine this, we enrolled 190 patients (32 without nonalcoholic fatty liver disease [NAFLD], 36 with simple steatosis [SS], and 122 with biopsy-proven NASH). Insulin secretion and hepatic insulin clearance were estimated by means of an oral glucose tolerance test, whereas peripheral insulin sensitivity and whole-body insulin clearance were measured during a euglycemic insulin clamp. A liver biopsy was performed to assess histology (grade/stage). Patients with NASH had similar hepatic insulin sensitivity, compared to patients with SS, but more severe adipose tissue insulin resistance and worse hyperinsulinemia. Patients with SS and NASH had a similar ∼30% reduction (P < 0.01) in hepatic insulin clearance, when compared to patients without NAFLD. Reduced hepatic insulin clearance was not associated with severity of inflammation, ballooning, and fibrosis. In contrast, worse histological inflammation and ballooning (but not steatosis or fibrosis) were associated with a progressive reduction in whole-body insulin clearance (P < 0.001 for trend). There was no significant difference in insulin secretion between patients with SS versus NASH. Conclusion: Decreased hepatic insulin clearance develops with a mild increase in liver fat (LFAT) accumulation. It appears to be largely driven by hepatic steatosis, whereas steatohepatitis is more closely associated with reduced whole-body insulin clearance. Hyperinsulinemia in NAFLD correlated strongly with impaired insulin clearance, but not with insulin secretion. Strategies that reduce LFAT and improve insulin clearance hold the potential to revert the unfavorable effects of hyperinsulinemia in these patients. (Hepatology 2014;59:2178–2187)


analysis of variance


area under the curve


body mass index


cardiovascular disease


dipeptidyl peptidase 4


dual energy X-ray absorptiometry


endogenous glucose production


free fatty acids


fasting plasma insulin


hemoglobin A1c




insulin resistance


lean body mass


liver fat


metabolic syndrome


magnetic resonance imaging


magnetic resonance spectroscopy


nonalcoholic fatty liver disease


nonalcoholic steatohepatitis


not significant


oral glucose tolerance test


whole-body insulin-stimulated glucose disposal


simple steatosis


type 2 diabetes mellitus


total body fat




University of Texas Health Science Center at San Antonio, Texas

The liver is the main site where insulin is cleared from the plasma.[1] It is estimated that it metabolizes approximately 50% of endogenously secreted insulin during its presystemic first pass. Advanced liver disease (cirrhosis) has been associated with low insulin clearance and hyperinsulinemia.[2] This implies that hepatic histological damage may affect the liver's ability to clear insulin.

Nonalcoholic fatty liver disease (NAFLD) usually develops within an insulin-resistant environment and is therefore strongly related to metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM).[3-6] Some studies have shown that the amount of liver fat (LFAT) is associated with a reduction in insulin clearance in both people with[7] and without T2DM.[2] However, although liver triglyceride (TG) accumulation and impaired insulin clearance may appear to be causally associated, they could just represent the common final pathway of liver insulin resistance (IR). Because insulin clearance was shown to be decreased in people with obesity,[8] high visceral adiposity,[9] and T2DM,[7] it has been suggested that it could represent a compensatory response to induce peripheral hyperinsulinemia in patients with IR.[10] Yet, when insulin clearance was measured in patients with and without T2DM, results have been rather inconsistent, with some studies showing lower insulin clearance in patients with T2DM[7, 11] and others showing no difference in insulin clearance.[9, 12] In addition, studies do not usually distinguish whole-body (largely muscle and kidney) versus hepatic insulin clearance, making data interpretation difficult. The degree of LFAT accumulation may link reduced insulin clearance with obesity and T2DM and help explain the inconsistencies of previous works that have not quantified LFAT in the setting of IR.[13, 14] However, whether LFAT per se is responsible for reduction of insulin clearance in insulin-resistant states remains to be elucidated. No previous study has carefully assessed the role of insulin clearance in patients with biopsy-proven nonalcoholic steatohepatitis (NASH), and decreases in insulin clearance could have simply mirrored more advanced steatohepatitis.[15]

The aim of this study was to assess the role of histological damage in patients with biopsy-proven NASH in whole-body and hepatic insulin clearance and their final influence on hyperinsulinemia.

Patients and Methods


A total of 190 subjects were recruited from the general population of San Antonio, Texas, as previously reported, between late 2007 and 2011.16 Briefly, patients between ages 18 and 70 were identified either from responses to local newspaper advertisements or from referrals from hepatology clinics at the University of Texas Health Science Center at San Antonio, Texas (UTHSCSA). Participants were excluded if there was evidence of any significant chronic disease (other than NASH, T2DM, or components of MetS), as determined by history, physical exam, routine blood and urine chemistries, and electrocardiography. Subjects with T2DM had to be on stable doses of their antidiabetic medications and were excluded if they were on thiazolidinediones, incretin-mimetics, or dipeptidyl peptidase 4 (DPP-4) inhibitors. Volunteers were also excluded if they had a history of alcohol abuse (≥20 g/day), liver disease other than NASH (i.e., hepatitis B or C, autoimmune hepatitis, hemochromatosis, Wilson's disease, drug-induced, or others), type 1 diabetes, or a history of clinically significant renal, pulmonary, or heart disease (New York Heart Classification greater than grade II). The study was approved by the UTHSCSA Institutional Review Board, and an informed written consent was obtained from each patient before participation.

Study Design

All studies were performed at the Frederic C. Bartter Clinical Research Unit, Audie L. Murphy Veterans Administration Medical Center (San Antonio, TX), except for the liver magnetic resonance imaging and spectroscopy (MRS) performed at the UTHSCSA Research Imaging Center and the liver biopsy at the Veterans Administration Radiology Department. Metabolic and imaging measurements included the following: (1) fasting plasma glucose, hemoglobin A1c (HbA1c), lipid profile, liver function tests, fasting plasma insulin (FPI), and fasting free fatty acids (FFAs); (2) total body fat (TBF) by dual energy X-ray absorptiometry (DXA); (3) LFAT content by the gold-standard technique of MRS; (4) 75-g oral glucose tolerance test (OGTT) to establish the diagnosis of normal glucose tolerance or T2DM according to current criteria[17] and to measure insulin and C-peptide areas under the curve (AUCs) as previously described18; (5) euglycemic hyperinsulinemic clamp with 3-[3H] glucose to measure endogenous glucose production and tissue-specific and total body insulin sensitivity and clearance; and 6) a liver biopsy to establish the diagnosis of NASH and the stage and grade of the disease.

Total Body and LFAT Content and Analytical Measurements

Total body fat content was measured by DXA (Hologic Inc, Waltham, MA). For measurement of hepatic fat content, localized proton nuclear magnetic resonance spectra of the liver were acquired on a Siemens TIM-Trio 3.0T MRI whole-body scanner and using methodology previously described.[16] An LFAT content of >5.5% was considered diagnostic of NAFLD.[19] Plasma insulin and C-peptide levels were measured by radioimmunoassay (Siemens, Los Angeles, CA).

Euglycemic Hyperinsulinemic Clamp

Patients were admitted to the research unit at 6:30 a.m. after a 12-hour overnight fast, and the study was performed as reported by our group.[20] In brief, upon arrival at the unit, a polyethylene catheter was inserted into an antecubital vein for infusion of all test substances. A second catheter was inserted retrogradely into an ipsilateral wrist vein on the dorsum of the hand for collection of arterialized blood sampling, and the hand was kept in a heated box at 65°C. A primed (25 µCi × [fasting glucose/100])–continuous (0.25 µCi/minute) infusion of 3-[3H] glucose (DuPont-NEN, Boston, MA) was initiated and continued until the end of the study. During the last 30 minutes of the basal equilibration period (150-180 minutes), plasma samples were taken at 5- to 10-minute intervals for determination of plasma glucose, insulin concentrations, and 3-[3H] glucose-specific activity. After the basal equilibration period, insulin was administered as a primed-continuous infusion at 10 mIU/m2 · min for 120 minutes to assess suppression of endogenous (mainly hepatic) glucose production (EGP), followed by another 2 hours at an infusion rate of 80 mIU/m2 · min for 120 minutes to assess whole-body insulin-stimulated glucose disposal (Rd). Plasma glucose level was measured every 5 minutes after start of insulin, and a variable infusion of 20% glucose was adjusted based on the negative feedback principle to maintain plasma glucose concentration at approximately 90-100 mg/dL, with a coefficient of variation <5%. Plasma samples were collected every 5-10 minutes for determination of plasma glucose, insulin, and FFA concentrations and 3-[3H] glucose-specific activity.

Liver Biopsy

An ultrasound-guided liver biopsy was performed in patients with elevated liver transaminases when all other causes of liver disease were ruled out, or in patients with normal liver transaminases if they were diagnosed with NAFLD by MRS and had significant risk factors for development of NASH, such as T2DM, MetS, and/or high IR. Biopsies were evaluated by a pathologist who was unaware of the subjects' identity or clinical information. Histologic characteristics for the diagnosis of NASH were determined using standard criteria.[21]


For hepatic and adipose tissue IR, we used the percentage of suppression of EGP and the percentage of suppression of plasma FFA during the low-dose insulin infusion (2 hours) of the euglycemic hyperinsulinemic clamp. Skeletal muscle insulin sensitivity was estimated as the insulin-stimulated muscle glucose uptake (Rd) during the second 2-hour insulin clamp step (high-dose insulin infusion), as previously reported by our group.[16] Whole-body insulin clearance (mainly kidney and muscle) was calculated dividing the insulin infusion rate by the steady-state plasma insulin concentration during the last 30 minutes of the hyperinsulinemic euglycemic clamp and expressed per kilogram of lean body mass (LBM). For estimating hepatic insulin clearance, we used the C-peptide/insulin AUC ratio calculated by the trapezoidal rule during the OGTT, as previously described.[18, 20] Similarly, the C-peptide/glucose AUC ratio was used as a measure of insulin secretion during the OGTT (with and without adjustment for IR). Of note, these two measurements are only indirect estimates of hepatic insulin clearance and insulin secretion, respectively, and not direct calculations of these parameters.

Statistical Analysis

Data were summarized in percentages for categorical variables and as mean ± standard error for numeric variables. Categorical variables were compared performing the chi-squared or Fisher's exact test. For comparisons between two groups, we performed Kruskal-Wallis' or Student t test for numeric variables, depending on variables' distribution. Comparisons among three or more groups were performed with analysis of variance (ANOVA; Bonferroni's method for post-hoc testing) or Kruskal-Wallis' test. Pearson's or Spearman's correlations were used for numerical variables according to their characteristics. A two-tailed P value of less than 0.05 was considered to indicate statistical significance. Analyses were performed with Stata 11.1 statistical software (StataCorp LP, College Station, TX).


Baseline Characteristics

A total of 190 patients were enrolled in this study. Thirty-two patients without NAFLD by MRS were used as controls. A liver biopsy was performed in the rest of the patients (n = 158), and NASH was diagnosed in 122 of these patients.[22] Table 1 summarizes the demographic and metabolic characteristics of patients divided into three groups: (1) those without NAFLD by MRS; (2) those with a diagnosis of NAFLD by MRS, but that did not fulfill criteria for NASH on liver histology (simple steatosis; SS); and (3) patients with histological criteria for definite NASH.[21] Eighteen patients diagnosed with borderline NASH by biopsy were excluded from analyses. Patients without NAFLD had a better metabolic profile, with less obesity, T2DM, and lower IR. However, as can be appreciated, SS and NASH groups were well matched for all relevant clinical variables, such as age, gender, body mass index (BMI), TBF, prevalence of T2DM, and amount of LFAT accumulation. No patients were treated with pioglitazone, incretin mimetics, or DPP-4 inhibitors. Most patients with T2DM were well controlled with diet only because many were newly diagnosed during the screening OGTT. In those pharmacologically treated, the majority were on metformin (∼80%) and/or a sulfonylurea (∼50%), with a similar proportion of these agents in both groups (SS vs. NASH). This was consistent with the near-normal HbA1c of 6.9% among patients with T2DM.

Table 1. Clinical and Laboratory Characteristics of Patients
CharacteristicsNo NAFLD by MRS (n = 32)SS (n = 36)NASH (n = 122)P ValueaP Valueb SS vs. NASH
  1. Abbreviations: LDL-C, low-density lipoprotein; HDL-C, high-density lipoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LBM, lean body mass.

  2. a

    P values for comparisons among the three groups using ANOVA/Kruskall-Wallis.

  3. b

    P values for post-hoc analysis comparing SS versus NASH.

Age, years48 ± 250 ± 252 ± 10.181.00
Gender (male), %5967750.211.00
Body mass index, kg/m228.9 ± 1.032.6 ± 0.634.2 ± 0.4<0.0010.18
TBF, %29.2 ± 1.632.3 ± 1.333.3 ±
LFAT, %2.6 ± 0.324.3 ± 2.326.3 ± 1.3<0.0011.00
T2DM, %3147580.020.73
Total cholesterol, mg/dL175 ± 5168 ± 7183 ± 40.170.22
LDL-C, mg/dL104 ± 5101 ± 7109 ± 40.530.89
TGs, mg/dL99 ± 9149 ± 12196 ± 12<0.0010.11
HDL-C, mg/dL52 ± 239 ± 137 ± 1<0.0011.00
ALT, IU/mL27 ± 445 ± 366 ± 4<0.0010.005
AST, IU/mL27 ± 330 ± 247 ± 2<0.001<0.001
Fasting plasma glucose, mg/dL107 ± 4114 ± 3123 ± 20.0040.18
HbA1c, %5.7 ± 0.26.1 ± 0.26.5 ± 0.1<0.0010.10
FPI, μIU/ml4 ± 110 ± 115 ± 1<0.0010.01
Fasting plasma C-peptide, ng/mL2.5 ± 0.34.0 ± 0.44.7 ± 0.2<0.0010.17
Fasting FFA, mmol/L0.47 ± 0.040.51 ± 0.030.56 ±
Suppression of FFA, %76 ± 356 ± 445 ± 2<0.0010.03
Suppression of EGP, %56 ± 542 ± 440 ± 20.021.00
Muscle insulin sensitivity (Rd), mg/kgLBM · min9.1 ± 0.64.9 ± 0.43.9 ± 0.2<0.0010.09

Patients with NASH had a significantly higher FPI level (P ≤ 0.01) and had a significantly worse adipose tissue IR (insulin suppression of plasma FFA; P = 0.03). However, there was no difference in insulin suppression of EGP during the low-dose (or high-dose; data not shown) insulin infusion during the euglycemic insulin clamp. Table 2 summarizes liver histology grade and stage of both groups. As expected, patients with NASH had more-severe hepatic steatosis, inflammation, necrosis, and fibrosis.

Table 2. Histological Characteristics of Patients With SS or NASH
Histological ParameterSSa n (%)NASH n (%)
  1. a

    SS defined as NAFLD by MRS (and without a diagnosis of NASH by biopsy).

Grade 07 (19.4)0
Grade 119 (52.8)41 (33.6)
Grade 26 (16.7)53 (43.4)
Grade 34 (11.1)28 (23.0)
Grade 05 (13.9)0
Grade 126 (72.2)60 (49.2)
Grade 25 (13.9)61 (50.0)
Grade 301 (0.8)
Grade 036 (100.0)0
Grade 10114 (93.4)
Grade 208 (6.6)
Stage 036 (100.0)31 (25.4)
Stage 1072 (59.0)
Stage 209 (7.4)
Stages 3-4010 (8.2)

Hepatic and Whole-Body Insulin Clearance in SS and NASH

Hepatic insulin clearance was significantly lower in patients with SS or NASH, when compared to patients without NAFLD (∼30%; P < 0.01; Fig. 1A), but was similar between both groups with NAFLD (9.2 ± 1.0 vs. 8.8 ± 0.4; P = 0.71). This remained true even after excluding patients with T2DM from the analysis. Both patients with SS or NASH (without T2DM) had a similarly ∼40% (P ≤ 0.001) reduction in hepatic insulin clearance, when compared to nondiabetic patients without NAFLD.

Figure 1.

Hepatic and whole-body insulin clearance in patients without NAFLD, with SS or with NASH on liver histology. (A) Hepatic insulin clearance measured as the C-peptide to insulin area under the curve (AUC) ratio and (B) whole-body insulin clearance (mL/min) per kg of lean body mass (LBM). ^P < 0.01, when compared to patients without NAFLD; #P < 0.05, when compared to patients with SS.

Patients with SS had a ∼12% reduction in whole-body insulin clearance, when compared to controls without NAFLD (26.3 ± 1.5 vs. 29.5 ± 1.8 ml/min · KgLBM, respectively; P = 0.16; Fig.1B). However, there was a significant further 16% decrease in whole-body insulin clearance in patients with NASH, when compared to those with SS (22.2 ± 0.7 vs. 26.3 ± 1.5 ml/min · KgLBM; P = 0.03) and a 25% decrease, when compared to controls (P < 0.001). Overall results for both hepatic insulin clearance (controls: 12.8 ± 1.1 vs. SS: 7.2 ± 1.3 vs. NASH: 7.2 ± 0.5; P < 0.0001) and whole-body insulin clearance (controls: 30.4 ± 2.1 vs. SS: 26.0 ± 1.8 vs. NASH: 22.6 ± 1.1 ml/min · KgLBM; P < 0.01) were similar when only patients without T2DM were included in the analysis.

Hepatic and Whole-Body Insulin Clearance—Role of Histology

To further assess the role of NASH on insulin clearance, patients with a liver biopsy were divided according to disease grade and stage. Figure 2 summarizes the effect of steatosis, inflammation, ballooning, and fibrosis on hepatic insulin clearance in patients with NAFLD (both SS and NASH combined). Hepatic insulin clearance was almost maximally impaired by mild steatosis (grade 1) with minimal change, because LFAT increased up to 65% (grade 2). However, at the extreme of hepatic fat accumulation (>66%; grade 3), hepatic steatosis further impaired hepatic insulin clearance (P for trend <0.001). This was associated with progressive hyperinsulinemia that increased from 4 in controls to 12, 15, and 17 µIU/mL in grades 1, 2, and 3, respectively (P for trend <0.0001). However, whereas hepatic insulin clearance was impaired at any given grade of inflammation or ballooning in patients with versus without NAFLD (all P ≤ 0.01, except for grade 0 of inflammation: P < 0.05), higher grades of inflammation or ballooning did not translate into worse hepatic insulin clearance. This was also true for patients with NAFLD with different fibrosis stages of the disease (all P < 0.05 vs. controls, but not significant [NS] between groups).

Figure 2.

Changes in hepatic insulin clearance according to severity of liver histology. (A) Steatosis, (B) inflammation, (C) ballooning, and (D) fibrosis. *P < 0.05, when compared to patients without NAFLD.

Figure 3 summarizes the effect of steatosis, inflammation, ballooning, and fibrosis on whole-body (primarily muscle and kidney) insulin clearance. As can be observed, whole-body insulin clearance was similarly reduced at any given grade of steatosis (i.e., once patients developed liver steatosis, whole-body insulin clearance was fully impaired, independently of the amount of LFAT accumulation). For liver inflammation and necrosis (ballooning), we observed a stepwise trend for a reduction in whole-body insulin clearance with higher grade of the disease (P for trend <0.001 for both variables). In contrast, the presence of fibrosis was not associated with an overall lower whole-body insulin clearance.

Figure 3.

Changes in whole-body insulin clearance according to severity of liver histology. (A) Steatosis, (B) inflammation, (C) ballooning, and (D) fibrosis. *P < 0.05, when compared to patients without NAFLD.

Insulin Secretion

Insulin secretion, expressed as C-peptide/glucose AUC ratio, was similar between patients with SS and those with NASH (0.059 ± 0.005 vs. 0.057 ± 0.003; P = 0.81). Even when patients were divided according to disease grade and stage, there was no significant difference in insulin secretion (Fig. 4). When these measurements were adjusted for IR (1/Rd), patients without NAFLD had a higher insulin secretion. However, there were no differences among the different disease grades and stages in patients with NAFLD (data not shown).

Figure 4.

Insulin secretion (C-peptide/glucose AUC ratio) according to severity of liver histology. (A) steatosis, (B) inflammation, (C) ballooning, and (D) fibrosis. All P values NS, except for inflammation grade 1 versus grade 2-3: P = 0.01.


Hyperinsulinemia correlated strongly with insulin clearance (hepatic: r = −0.41, P < 0.001 and whole-body: r = −0.49, P < 0.001), but not with insulin secretion (r = 0.11; NS). LFAT measured by MRS correlated closely with whole-body insulin clearance when expressed as a logarithmic function (r = −0.36; P < 0.001) and with hepatic insulin clearance in a linear way (r = −0.27; P < 0.001). This is consistent with the previously reported abrupt (Fig. 3A) and stepwise (Fig. 2A) reductions of whole-body and hepatic insulin clearance associated with hepatic steatosis (see Discussion below).

Insulin clearance correlated best with metabolic parameters related to IR. Muscle insulin sensitivity (Rd; r = 0.52; P < 0.001) and suppression of plasma FFA by low-dose insulin (r = 0.39; P < 0.001) were significantly associated with whole-body insulin clearance in patients with NASH. BMI (r = −0.19; P < 0.01) and muscle insulin sensitivity (Rd; r = 0.33; P < 0.01) were weakly correlated to hepatic insulin clearance.

In order to assess a potential link between liver inflammation and ballooning (NASH) and impaired whole-body insulin clearance, we performed Pearson's correlations between different inflammatory biomarkers (interleukin [IL]-6, IL-8, soluble intercellular adhesion molecule, vascular cell adhesion molecule, adiponectin, tumor necrosis factor alpha, transforming growth factor beta, high-sensitivity C-reactive protein, and others) and whole-body insulin clearance. Of these inflammatory biomarkers, only adiponectin had a statistically significant correlation with whole-body insulin clearance (r = 0.32; P < 0.001). None of these biomarkers correlated with hepatic insulin clearance (data not shown).


Plasma insulin concentration depends on a tightly regulated balance between insulin secretion and its clearance.[10] Although much consideration has been given to the importance of insulin secretion and IR to the development of hyperinsulinemia and eventually T2DM, insulin clearance has attracted much less attention as an important determinant of plasma insulin concentration. This is an important topic because hyperinsulinemia has been linked, in epidemiological studies, to cardiovascular disease[23, 24] and hepatocellular carcinoma[25] and both are the leading causes of death in NASH. Previous studies on the role of hepatic steatosis on insulin clearance have been small, usually without simultaneous measurements of LFAT and insulin clearance or relied exclusively on imaging techniques that did not explore the role of steatohepatitis severity.[2, 7] Without a liver biopsy, it is not possible to separate the relative contribution of LFAT per se versus steatohepatitis on insulin clearance. In addition, LFAT imaging may be a poor surrogate of the severity of NAFLD because it is well established that advanced liver disease in NASH is associated with a reduction in LFAT.[26] In our study, we have tried to overcome the above-described limitations by being the first to study a large cohort of patients with NAFLD and biopsy-proven NASH. Finally, another unique aspect of this study is differentiating whole-body from hepatic insulin clearance when assessing the effect of liver histology in insulin clearance. Previous studies assessing insulin clearance in patients with NAFLD relied only on a single measurement of insulin clearance (mainly whole-body) and did not distinguish between hepatic and extrahepatic (mainly renal and skeletal muscle) insulin clearance. This is important to examine because it offers a more comprehensive view of insulin and glucose metabolism with implications for treatment.

Our findings suggest that even a mild increase in liver steatosis (i.e., grade 1) significantly impairs hepatic and whole-body insulin clearance, without further worsening as LFAT increases to 34%-65% (grade 2) and only minimally the hepatic insulin clearance when LFAT is ≥66% (grade 3). This implies that metabolic alterations in patients with NAFLD occur at a low threshold for LFAT. The clinical implication is that an early diagnosis of NAFLD, and eventual treatment, may be important to avoid severe chronic hyperinsulinemia. Chronically elevated plasma insulin levels may drive hepatic lipogenesis and very-low-density lipoprotein oversecretion in patients with NAFLD.[15, 27] Whereas the role of hyperinsulinemia in the pathogenesis of CVD remains controversial, the association between NAFLD and CVD cannot be ignored.[23, 24, 28] Thus, whereas a causal relationship between chronic hyperinsulinemia and CVD needs further validation, amelioration of LFAT and hyperinsulinemia, at the current time, appear desirable. Consistent with this view, hepatic insulin clearance and LFAT were significantly correlated (P < 0.001). However, the presence of inflammation and ballooning was not associated with worse hepatic insulin clearance, compared to simple steatosis (Fig. 2A-C), indicating that LFAT accumulation affects hepatic insulin clearance independently of inflammation, ballooning, or fibrosis.

When the role of NASH on whole-body insulin clearance was assessed, we found that patients with NASH had a lower whole-body insulin clearance than patients with simple steatosis (Fig. 1A). Of note, this difference occurred regardless of patients being well matched for frequent confounders, such as age, BMI, TBF, and prevalence of diabetes. When we divided these patients according to their grades and stages of NAFLD, severity of inflammation and ballooning was associated with a reduction in whole-body insulin clearance (Fig. 3B and 3C). One can only speculate whether liver histology (inflammation and ballooning) may be the driver for the impairment in whole-body insulin clearance or, on the contrary, adipose tissue or muscle IR, both tightly correlated with whole-body insulin clearance (both P < 0.001), are indeed affecting the liver. For the former, several inflammatory biomarkers have been reported to be elevated in NASH[29] and believed to contribute to subclinical inflammation and IR.[30] For the latter, IR in adipose tissue promotes an overflow of plasma FFA to the liver, triggering inflammation and lipotoxicity.[15] Compensatory hyperinsulinemia from muscle IR can also promote hepatocyte lipogenesis, TG accumulation, inflammation, and apoptosis in patients with NASH.[27]

Of note, fibrosis played no role in hepatic or whole-body insulin clearance impairment. This implies that hyperinsulinemia and impaired insulin clearance observed in cirrhosis may not be related to the presence of fibrosis itself, but rather to a different mechanism related to severe IR. Activation of inflammatory cells in the liver has been shown to induce IR, and this can partly explain hyperinsulinemia observed in patients with cirrhosis.[31] Moreover, the relationship between hyperglycemia, hyperinsulinemia, and/or advanced glycation endproducts with hepatic stellate cells and their promotion of fibrosis is complex and not well understood.[32]

One potential limitation of this study is that the prevalence of T2DM was slightly higher in patients with liver disease (no NAFLD, 31%; SS, 47%; NASH, 58%). Because the presence of T2DM could be associated with a decrease in insulin clearance, it could have been acting as a confounding factor. However, in order to overcome this issue, we repeated the analyses excluding patients with T2DM. When only patients without T2DM were considered, results were similar to those of the entire population, with a similarly reduced hepatic insulin clearance in patients with SS and NASH and a further reduction on whole-body insulin clearance in patients with NASH, when compared to patients with SS. These results suggest that differences in insulin clearance among the three groups are independent of the presence of T2DM.

In summary, we have shown that patients with NASH have impaired insulin clearance, both at the hepatic and whole-body levels. Decreased hepatic insulin clearance develops even after a relatively mild increase in liver TG accumulation and is independent of the severity of liver ballooning, inflammation, or fibrosis. However, steatohepatitis is associated with a further reduction in whole-body insulin clearance. Whether severity of NASH has a direct role in impairing whole-body insulin clearance, or they are both the common final pathway of severe IR, remains to be elucidated. Impaired insulin clearance, rather than changes in insulin secretion, lead to hyperinsulinemia in NAFLD. Strategies that ameliorate hepatic steatosis hold the potential to revert the unfavorable effects of hyperinsulinemia in these patients, such as dyslipidemia and CVD.