Fernando Bril

Limited value of plasma cytokeratin-18 as a biomarker for NASH and fibrosis in patients with non-alcoholic fatty liver disease

BACKGROUND & AIMS Liver biopsy is the only reliable way of diagnosing and staging NASH but its invasive nature limits its use. Plasma caspase-generated cytokeratin-18 fragments (CK-18) have been proposed as a non-invasive alternative. We studied its clinical value in a large multiethnic NAFLD population and examined its relationship to clinical/metabolic/histological parameters. METHODS 424 middle-aged subjects in whom we measured adipose tissue, liver and muscle insulin resistance (IR), liver fat by MRS (n=275) and histology (n=318). RESULTS Median CK-18 were elevated in patients with vs. without NAFLD by MRS (209 [IQR: 137-329] vs. 122 [IQR: 98-155]U/L) or with vs. without NASH (232 [IQR: 151-387] vs. 170 [IQR: 135-234]U/L, both p<0.001). Plasma CK-18 raised significantly with any increase in steatosis, inflammation and fibrosis, but there was a significant overlap across disease severity. The CK-18 AUROC to predict NAFLD, NASH or fibrosis were 0.77 (95% CI=0.71-0.84), 0.65 (95% CI=0.59-0.71) and 0.68 (95% CI=0.61-0.75), respectively. The overall sensitivity/specificity for NAFLD, NASH and fibrosis were 63% (57-70%)/83% (69-92%), 58% (51-65%)/68% (59-76%) and 54% (44-63%)/85% (75-92%), respectively. CK-18 correlated most strongly with ALT (r=0.57, p<0.0001) and adipose tissue IR (insulin-suppression of FFA: r=-0.43; p<0.001), less with steatosis, lobular inflammation and fibrosis (r=0.28-0.34, all p<0.001), but not with ballooning, BMI, metabolic syndrome or T2DM. CONCLUSIONS Plasma CK-18 has a high specificity for NAFLD and fibrosis, but its limited sensitivity makes it inadequate as a screening test for staging NASH. Whether combined as a diagnostic panel with other biomarkers or clinical/laboratory tests may prove useful requires further study.

High Prevalence of Nonalcoholic Fatty Liver Disease in Patients With Type 2 Diabetes Mellitus and Normal Plasma Aminotransferase Levels.

CONTEXT AND OBJECTIVE Nonalcoholic fatty liver disease (NAFLD) and its more severe form with steatohepatitis (NASH) are common in patients with type 2 diabetes mellitus (T2DM). However, they are usually believed to largely affect those with elevated aminotransferases. The aim of this study was to determine the prevalence of NAFLD by the gold standard, liver magnetic resonance spectroscopy ((1)H-MRS) in patients with T2DM and normal aminotransferases, and to characterize their metabolic profile. PARTICIPANTS AND METHODS We recruited 103 patients with T2DM and normal plasma aminotransferases (age, 60 ± 8 y; body mass index [BMI], 33 ± 5 kg/m(2); glycated hemoglobin [A1c], 7.6 ± 1.3%). We measured the following: 1) liver triglyceride content by (1)H-MRS; 2) systemic insulin sensitivity (homeostasis model assessment-insulin resistance); and 3) adipose tissue insulin resistance, both fasting (as the adipose tissue insulin resistance index: fasting plasma free fatty acids [FFA] × insulin) and during an oral glucose tolerance test (as the suppression of FFA). RESULTS The prevalence of NAFLD and NASH were much higher than expected (50% and 56% of NAFLD patients, respectively). The prevalence of NAFLD was higher in obese compared with nonobese patients as well as with increasing BMI (P = .001 for trend). Higher plasma A1c was associated with a greater prevalence of NAFLD and worse liver triglyceride accumulation (P = .01). Compared with nonobese patients without NAFLD, patients with NAFLD had severe systemic (liver/muscle) and, particularly, adipose tissue (fasting/postprandial) insulin resistance (all P < .01). CONCLUSIONS The prevalence of NAFLD is much higher than previously believed in overweight/obese patients with T2DM and normal aminotransferases. Moreover, many are at increased risk of NASH. Physicians should have a lower threshold for screening patients with T2DM for NAFLD/NASH.

Nonalcoholic Fatty Liver Disease: Current Issues and Novel Treatment Approaches

Nonalcoholic fatty liver disease (NAFLD) is considered the most common liver disorder in the Western world. It is commonly associated with insulin resistance, obesity, dyslipidaemia, type 2 diabetes mellitus (T2DM) and cardiovascular disease. Nonalcoholic steatohepatitis (NASH) is characterized by steatosis with necroinflammation and eventual fibrosis, which can lead to end-stage liver disease and hepatocellular carcinoma. Its pathogenesis is complex, and involves a state of ‘lipotoxicity’ in which insulin resistance, with increased free fatty acid release from adipose tissue to the liver, play a key role in the onset of a ‘lipotoxic liver disease’ and its progression to NASH. The diagnosis of NASH is challenging, as most affected patients are symptom free and the role of routine screening is not clearly established. A complete medical history is important to rule out other causes of fatty liver disease (alcohol abuse, medications, other). Plasma aminotransferase levels and liver ultrasound are helpful in the diagnosis of NAFLD/NASH, but a liver biopsy is often required for a definitive diagnosis. However, there is an active search for plasma biomarkers and imaging techniques that may non-invasively aid in the diagnosis. The treatment of NASH requires a multifaceted approach. The goal is to reverse obesity-associated lipotoxicity and insulin resistance via lifestyle intervention. Although there is no pharmacological agent approved for the treatment of NAFLD, vitamin E (in patients without T2DM) and the thiazolidinedione pioglitazone (in patients with and without T2DM) have shown the most consistent results in randomized controlled trials. This review concentrates on our current understanding of the disease, with a focus on the existing therapeutic approaches and potential future pharmacological developments for NAFLD and NASH.

The role of liver fat and insulin resistance as determinants of plasma aminotransferase elevation in nonalcoholic fatty liver disease

Plasma aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) are usually increased in patients with nonalcoholic fatty liver disease (NAFLD). However, the factors behind their elevation remain unclear. The aim of this study was to assess the role of insulin resistance (IR) and liver triglyceride content in relation to histology in patients with NAFLD/nonalcoholic steatohepatitis (NASH) with normal or elevated ALT levels. To this end, we enrolled 440 patients, divided into three groups: no NAFLD (n = 60); NAFLD with normal ALT (n = 165); and NAFLD with elevated ALT (n = 215). We measured: (1) liver fat by proton magnetic resonance spectroscopy (1H‐MRS); (2) severity of liver disease by biopsy (n = 293); and (3) insulin sensitivity in liver, muscle, and adipose tissue by a euglycemic hyperinsulinemic clamp with 3‐3H‐glucose. Patients with NAFLD and elevated ALT, even when well matched for body mass index to those with normal ALT, had worse adipose tissue insulin resistance (ATIR; P < 0.0001), higher liver triglyceride content (P < 0.0001), and lower plasma adiponectin (P < 0.05), but no differences in hepatic insulin resistance. Similar results were found when only patients with NASH were compared: both ATIR (P < 0.0001) and liver triglyceride content by 1H‐MRS (P < 0.0001) were worse in NASH with elevated ALT. Consistent with the 1H‐MRS data, steatosis on liver biopsy was also significantly increased in patients with NASH and elevated ALT levels (P < 0.0001). However, and most important, there were no differences in inflammation (P = 0.62), ballooning (P = 0.13), or fibrosis (P = 0.12). Conclusion: In patients with NAFLD or NASH, ATIR (but not HIR) and liver triglyceride content are major factors in the elevation of plasma aminotransferase levels. Patients with normal versus elevated ALT had similar severity of NASH, suggesting that plasma aminotransferase levels are misleading parameters for guiding clinical management. (Hepatology 2015;61:153–160)

Relationship of vitamin D with insulin resistance and disease severity in non-alcoholic steatohepatitis.

BACKGROUND & AIMS The role of plasma vitamin D deficiency in the development of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) remains poorly understood. Previous studies have suggested a role for vitamin D deficiency in the pathogenesis of NAFLD/NASH, but they have been rather small, and/or NAFLD was diagnosed using only aminotransferases or liver ultrasound. This study aimed to assess the role of vitamin D deficiency in relationship to liver fat accumulation and severity of NASH. METHODS A total of 239 patients were recruited and state-of-the-art techniques were used to measure insulin resistance (euglycemic insulin clamp with 3-(3)H-glucose), liver fat accumulation (magnetic resonance spectroscopy or (1)H-MRS), total body fat (dual energy X-ray absorptiometry), and severity of liver disease (liver biopsy). RESULTS Patients were divided into 3 groups according to plasma 25-hydroxyvitamin D levels (normal: >30 ng/ml; insufficiency: 20-30 ng/ml; deficiency: <20 ng/ml). When well-matched for clinical parameters (BMI, total adiposity, or prevalence of prediabetes/type 2 diabetes), no significant differences were observed among groups in terms of skeletal muscle, hepatic, or adipose tissue insulin sensitivity, the amount of liver fat by (1)H-MRS, or the severity of histological inflammation, ballooning, or fibrosis. Patients were then divided according to liver histology into those with definite NASH and those without NASH. Although patients with NASH had higher insulin resistance, plasma vitamin D concentrations were similar between both groups. CONCLUSIONS Our results suggest that plasma vitamin D levels are not associated with insulin resistance, the amount of liver fat accumulation, or the severity of NASH.

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

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)

Management of Nonalcoholic Fatty Liver Disease in Patients With Type 2 Diabetes: A Call to Action

Traditionally a disease of hepatologists, nonalcoholic fatty liver disease (NAFLD) has recently become a major concern for a broad spectrum of health care providers. Endocrinologists and those caring for patients with type 2 diabetes mellitus (T2DM) are at center stage, as T2DM appears to worsen the course of NAFLD and the liver disease makes diabetes management more challenging. However, the nature of this relationship remains incompletely understood. Although the increasing prevalence of NAFLD is frequently attributed to the epidemic of obesity and is often oversimplified as the “hepatic manifestation of the metabolic syndrome,” it is a much more complex disease process that may also be observed in nonobese individuals and in patients without clinical manifestations of the metabolic syndrome. It carries both metabolic and liver-specific complications that make its approach unique among medical conditions. Diabetes appears to promote the development of nonalcoholic steatohepatitis (NASH), the more severe form of the disease, and increases the risk of cirrhosis and hepatocellular carcinoma. Patients and physicians face many uncertainties, including fragmented information on the natural history of the disease, challenges in the diagnosis of NASH, and few pharmacological agents with proven efficacy. However, recent advances in diagnosis and treatment, combined with the risk of serious consequences from inaction, call for health care providers to be more proactive in the management of patients with T2DM and NASH.

Clinical value of liver ultrasound for the diagnosis of nonalcoholic fatty liver disease in overweight and obese patients.

Liver ultrasound (US) is usually used in the clinical setting for the diagnosis and follow‐up of patients with nonalcoholic fatty liver disease (NAFLD). However, no large study has carefully assessed its performance using a semiquantitative ultrasonographic scoring system in overweight/obese patients, in comparison to magnetic resonance spectroscopy (1H‐MRS) and histology.

Mitochondrial Adaptation in Nonalcoholic Fatty Liver Disease: Novel Mechanisms and Treatment Strategies

Nonalcoholic fatty liver disease (NAFLD) is prevalent in patients with obesity or type 2 diabetes. Nonalcoholic steatohepatitis (NASH), encompassing steatosis with inflammation, hepatocyte injury, and fibrosis, predisposes to cirrhosis, hepatocellular carcinoma, and even cardiovascular disease. In rodent models and humans with NAFLD/NASH, maladaptation of mitochondrial oxidative flux is a central feature of simple steatosis to NASH transition. Induction of hepatic tricarboxylic acid cycle closely mirrors the severity of oxidative stress and inflammation in NASH. Reactive oxygen species generation and inflammation are driven by upregulated, but inefficient oxidative flux and accumulating lipotoxic intermediates. Successful therapies for NASH (weight loss alone or with incretin therapy, or pioglitazone) likely attenuate mitochondrial oxidative flux and halt hepatocellular injury. Agents targeting mitochondrial dysfunction may provide a novel treatment strategy for NAFLD.

Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.