Srilaxmi Kalavalapalli

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.

Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets.

Response to Pioglitazone in Patients With Nonalcoholic Steatohepatitis With vs Without Type 2 Diabetes

Background & Aims Pioglitazone is effective for long‐term treatment of patients with nonalcoholic steatohepatitis (NASH) with prediabetes or type 2 diabetes. However, it is not clear how the presence of type 2 diabetes affects the drug’s efficacy. We compared metabolic and histologic responses to pioglitazone in patients with NASH and prediabetes vs type 2 diabetes. Methods We performed a prospective study of adults with biopsy‐proven NASH (52 with type 2 diabetes and 49 with prediabetes), enrolled from the general population of San Antonio, Texas, from 2008 through 2014. After a run‐in period of approximately 4 weeks, when all baseline measurements were made (liver magnetic resonance proton spectroscopy, euglycemic insulin clamp with glucose turnover measurements, dual‐energy absorptiometry, and liver biopsy), subjects were randomly assigned to groups given pioglitazone or placebo (45 mg/d) for 18 months; all procedures performed at baseline were then repeated. The primary outcome was a reduction in nonalcoholic fatty liver disease activity score of 2 points or more (for at least 2 components) without worsening of fibrosis (and expressed as difference vs placebo). Secondary outcomes included NASH resolution, individual histologic components, intrahepatic triglyceride content (measured by 1H magnetic resonance spectroscopy), and insulin sensitivity (measured by euglycemic insulin clamp). Results The primary outcome was met by 48% of patients with type 2 diabetes vs 46% without diabetes. Resolution of NASH was achieved in 44% of patients with type 2 diabetes vs 26% without diabetes. A significant reduction in fibrosis, from baseline, was observed only in patients with type 2 diabetes (P = .035). Intrahepatic triglyceride content was reduced by 11% ± 2% in patients with diabetes vs a reduction of 9% ± 2% in patients without diabetes (P = .62); the plasma level of alanine aminotransferase was reduced by 50 ± 10 U/L in patients with diabetes vs a reduction of 36 ± 5 U/L in patients without diabetes (P = .22). Pioglitazone was associated with a significantly greater insulin sensitivity in adipose tissue of patients with diabetes vs without diabetes (P < .001), but nonsignificant differences in responses in hepatic (P = .49) and skeletal muscle (P = .32) insulin sensitivity. Conclusions In a prospective study, we found pioglitazone to be effective in patients with and without type 2 diabetes. However, pioglitazone reduced liver fibrosis and increased adipose tissue insulin sensitivity at significantly greater levels in patients with type 2 diabetes than in patients with prediabetes. Further studies are needed to determine the mechanisms by which pioglitazone reduces liver disease in patients with type 2 diabetes. ClinicalTrials.gov: NCT00994682.

Clinical and Histologic Characterization of Nonalcoholic Steatohepatitis in African American Patients

OBJECTIVE There has been a widespread misconception among physicians that African Americans are protected from developing nonalcoholic steatohepatitis (NASH). However, a formal histologic and metabolic comparison against well-matched Caucasians has never been performed. RESEARCH DESIGN AND METHODS Sixty-seven African American patients were matched 2:1 to Caucasians (n = 134) for age, sex, BMI, hemoglobin A1c, and prevalence of type 2 diabetes mellitus (T2DM). Screening for NASH included measurement of intrahepatic triglyceride content by proton MRS (1H-MRS), followed by a liver biopsy if patients had hepatic steatosis. Insulin resistance was estimated during an oral glucose tolerance test using the Matsuda Index. RESULTS Compared with Caucasians, African American patients had a lower intrahepatic triglyceride content (mean ± SD 6.1 ± 6.8% vs. 9.4 ± 7.5%, P = 0.007) and the presence of nonalcoholic fatty liver disease (NAFLD) was less common (25.0% vs. 51.9%, P = 0.003). However, prevalence of NASH was not different between ethnicities in patients with NAFLD (57.1% vs. 73.3%, P = 0.12). Moreover, they showed similar severity in each of the individual histologic parameters (inflammation, ballooning, and fibrosis). Among patients with NAFLD, insulin resistance was similar between both ethnic groups (Matsuda Index: 3.3 ± 1.8 vs. 3.1 ± 1.9, P = 0.61; adipose tissue insulin resistance [Adipo-IR] index: 5.7 ± 4.6 vs. 6.4 ± 4.7 mmol/L ⋅ µU/mL, P = 0.53) but appeared to be worse in African American versus Caucasian patients without NAFLD (Matsuda Index: 4.9 ± 3.6 vs. 7.0 ± 4.9, P = 0.11; Adipo-IR: 3.9 ± 2.8 vs. 2.7 ± 2.3 mmol/L ⋅ µU/mL, P = 0.06). African American patients also had lower plasma triglycerides and higher HDL cholesterol, independent of the severity of intrahepatic triglyceride. CONCLUSIONS Although African Americans have lower intrahepatic triglyceride accumulation, once NAFLD develops, NASH occurs as frequently, and as severe, as in Caucasian patients. Therefore, African Americans with NAFLD should be screened for NASH with the same degree of clinical resolve as in Caucasian patients.

Plasma thyroid hormone concentration is associated with hepatic triglyceride content in patients with type 2 diabetes

The underlying mechanisms responsible for the development and progression of non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM) are unclear. Since the thyroid hormone regulates mitochondrial function in the liver, we designed this study in order to establish the association between plasma free T4 levels and hepatic triglyceride accumulation and histological severity of liver disease in patients with T2DM and NAFLD. This is a cross-sectional study including a total of 232 patients with T2DM. All patients underwent a liver MR spectroscopy (1H-MRS) to quantify hepatic triglyceride content, and an oral glucose tolerance test to estimate insulin resistance. A liver biopsy was performed in patients with a diagnosis of NAFLD. Patients were divided into 5 groups according to plasma free T4 quintiles. We observed that decreasing free T4 levels were associated with an increasing prevalence of NAFLD (from 55% if free T4≥1.18 ng/dL to 80% if free T4<0.80 ng/dL, p=0.016), and higher hepatic triglyceride accumulation by 1H-MRS (p<0.001). However, lower plasma free T4 levels were not significantly associated with more insulin resistance or more severe liver histology (ie, inflammation, ballooning, or fibrosis). Decreasing levels of plasma free T4 are associated with a higher prevalence of NAFLD and increasing levels of hepatic triglyceride content in patients with T2DM. These results suggest that thyroid hormone may play a role in the regulation of hepatic steatosis and support the notion that hypothyroidism may be associated with NAFLD. No NCT number required.

Mitochondrial ATP transporter depletion protects mice against liver steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.

Concentration-dependent response to pioglitazone in nonalcoholic steatohepatitis

Pioglitazone is a safe and effective option to manage patients with type 2 diabetes and nonalcoholic steatohepatitis (NASH). However, there is marked variability in treatment response.

Pioglitazone improves hepatic mitochondrial function in a mouse model of nonalcoholic steatohepatitis

Pioglitazone is effective in improving insulin resistance and liver histology in patients with nonalcoholic steatohepatitis (NASH). Because dysfunctional mitochondrial metabolism is a central feature of NASH, we hypothesized that an important target of pioglitazone would be alleviating mitochondrial oxidative dysfunction. To this end, we studied hepatic mitochondrial metabolism in mice fed high-fructose high-transfat diet (TFD) supplemented with pioglitazone for 20 wk, using nuclear magnetic resonance-based 13C isotopomer analysis. Pioglitazone improved whole body and adipose insulin sensitivity in TFD-fed mice. Furthermore, pioglitazone reduced intrahepatic triglyceride content and fed plasma ketones and hepatic TCA cycle flux, anaplerosis, and pyruvate cycling in mice with NASH. This was associated with a marked reduction in most intrahepatic diacylglycerol classes and, to a lesser extent, some ceramide species (C22:1, C23:0). Considering the cross-talk between mitochondrial function and branched-chain amino acid (BCAA) metabolism, pioglitazones impact on plasma BCAA profile was determined in a cohort of human subjects. Pioglitazone improved the plasma BCAA concentration profile in patients with NASH. This appeared to be related to an improvement in BCAA degradation in multiple tissues. These results provide evidence that pioglitazone-induced changes in NASH are related to improvements in hepatic mitochondrial oxidative dysfunction and changes in whole body BCAA metabolism.

Improved experimental data processing for UHPLC–HRMS/MS lipidomics applied to nonalcoholic fatty liver disease

IntroductionUntargeted metabolomics workflows include numerous points where variance and systematic errors can be introduced. Due to the diversity of the lipidome, manual peak picking and quantitation using molecule specific internal standards is unrealistic, and therefore quality peak picking algorithms and further feature processing and normalization algorithms are important. Subsequent normalization, data filtering, statistical analysis, and biological interpretation are simplified when quality data acquisition and feature processing are employed.ObjectivesMetrics for QC are important throughout the workflow. The robust workflow presented here provides techniques to ensure that QC checks are implemented throughout sample preparation, data acquisition, pre-processing, and analysis.MethodsThe untargeted lipidomics workflow includes sample standardization prior to acquisition, blocks of QC standards and blanks run at systematic intervals between randomized blocks of experimental data, blank feature filtering (BFF) to remove features not originating from the sample, and QC analysis of data acquisition and processing.ResultsThe workflow was successfully applied to mouse liver samples, which were investigated to discern lipidomic changes throughout the development of nonalcoholic fatty liver disease (NAFLD). The workflow, including a novel filtering method, BFF, allows improved confidence in results and conclusions for lipidomic applications.ConclusionUsing a mouse model developed for the study of the transition of NAFLD from an early stage known as simple steatosis, to the later stage, nonalcoholic steatohepatitis, in combination with our novel workflow, we have identified phosphatidylcholines, phosphatidylethanolamines, and triacylglycerols that may contribute to disease onset and/or progression.

Use of a metabolomic approach to non-invasively diagnose non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus

To assess the utility of existing metabolomics scores to classify liver disease in patients with type 2 diabetes mellitus (T2DM).