Altered extracellular matrix dynamics is associated with insulin resistance in adolescent children with obesity.

OBJECTIVE: The objective of this study was to examine the hypothesis that abdominal and gluteal adipocyte turnover, lipid dynamics, and fibrogenesis are dysregulated among insulin-resistant (IR) compared with insulin-sensitive (IS) adolescents with obesity. METHODS: Seven IS and seven IR adolescents with obesity participated in a 3-h oral glucose tolerance test and a multi-section magnetic resonance imaging scan of the abdominal region to examine body fat distribution patterns and liver fat content. An 8-week 70% deuterated water (2 H2 O) labeling protocol examined adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from biopsied abdominal and gluteal fat. RESULTS: Abdominal and gluteal subcutaneous adipose tissue (SAT) turnover rates of lipid components were similar among IS and IR adolescents with obesity. However, the insoluble collagen (type I, subunit α2) isoform measured from abdominal, but not gluteal, SAT was elevated in IR compared with IS individuals. In addition, abdominal insoluble collagen Iα2 was associated with ratios of visceral-to-total (visceral adipose tissue + SAT) abdominal fat and whole-body and adipose tissue insulin signaling, and it trended toward a positive association with liver fat content. CONCLUSIONS: Altered extracellular matrix dynamics, but not expandability, potentially decreases abdominal SAT lipid storage capacity, contributing to the pathophysiological pathways linking adipose tissue and whole-body IR with altered ectopic storage of lipids within the liver among IR adolescents with obesity.

Estimating the contribution of CD4 T cell subset proliferation and differentiation to HIV persistence.

Persistence of HIV in people living with HIV (PWH) on suppressive antiretroviral therapy (ART) has been linked to physiological mechanisms of CD4+ T cells. Here, in the same 37 male PWH on ART we measure longitudinal kinetics of HIV DNA and cell turnover rates in five CD4 cell subsets: naïve (TN), stem-cell- (TSCM), central- (TCM), transitional- (TTM), and effector-memory (TEM). HIV decreases in TTM and TEM but not in less-differentiated subsets. Cell turnover is ~10 times faster than HIV clearance in memory subsets, implying that cellular proliferation consistently creates HIV DNA. The optimal mathematical model for these integrated data sets posits HIV DNA also passages between CD4 cell subsets via cellular differentiation. Estimates are heterogeneous, but in an average participant's year ~10 (in TN and TSCM) and ~104 (in TCM, TTM, TEM) proviruses are generated by proliferation while ~103 proviruses passage via cell differentiation (per million CD4). In simulations, therapies blocking proliferation and/or enhancing differentiation could reduce HIV DNA by 1-2 logs over 3 years. In summary, HIV exploits cellular proliferation and differentiation to persist during ART but clears faster in more proliferative/differentiated CD4 cell subsets and the same physiological mechanisms sustaining HIV might be temporarily modified to reduce it.

Characterization of the Intraclonal Complexity of Chronic Lymphocytic Leukemia B Cells: Potential Influences of B-Cell Receptor Crosstalk with Other Stimuli.

Chronic lymphocytic leukemia (CLL) clones contain subpopulations differing in time since the last cell division ("age"): recently born, proliferative (PF; CXCR4DimCD5Bright), intermediate (IF; CXCR4IntCD5Int), and resting (RF; CXCR4BrightCD5Dim) fractions. Herein, we used deuterium (2H) incorporation into newly synthesized DNA in patients to refine the kinetics of CLL subpopulations by characterizing two additional CXCR4/CD5 fractions, i.e., double dim (DDF; CXCR4DimCD5Dim) and double bright (DBF; CXCR4BrightCD5Bright); and intraclonal fractions differing in surface membrane (sm) IgM and IgD densities. Although DDF was enriched in recently divided cells and DBF in older cells, PF and RF remained the most enriched in youngest and oldest cells, respectively. Similarly, smIgMHigh and smIgDHigh cells were the youngest, and smIgMLow and smIgDLow were the oldest, when using smIG levels as discriminator. Surprisingly, the cells closest to the last stimulatory event bore high levels of smIG, and stimulating via TLR9 and smIG yielded a phenotype more consistent with the in vivo setting. Finally, older cells were less sensitive to in vivo inhibition by ibrutinib. Collectively, these data define additional intraclonal subpopulations with divergent ages and phenotypes and suggest that BCR engagement alone is not responsible for the smIG levels found in vivo, and the differential sensitivity of distinct fractions to ibrutinib might account, in part, for therapeutic relapse.

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach.

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (2H)-labeled water. The incorporation of 2H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Selective decrease of donor-reactive Tregs after liver transplantation limits Treg therapy for promoting allograft tolerance in humans.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

HIV-1 Genomes Are Enriched in Memory CD4+ T-Cells with Short Half-Lives.

Future HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4+ T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1. IMPORTANCE The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically-intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Our investigations into the possible cellular mechanisms maintaining the HIV-1 reservoir in different T-cell subsets have revealed a link between the half-lives of T-cells and the level of proviruses they contain. Taken together, we believe our study shows that more differentiated and proliferative cells, such as transitional and effector memory T-cells, contain the highest levels of genetically-intact proviruses, and the rapid turnover rate of these cells contributes to the expansion of genetically-intact proviruses within them. Therefore, our study delivers an in-depth assessment of the cellular mechanisms, such as cellular proliferation and half-life, that contribute to and maintain the latent HIV-1 reservoir.

Exercise reduced the formation of new adipocytes in the adipose tissue of mice in vivo.

Exercise has beneficial effects on metabolism and health. Although the skeletal muscle has been a primary focus, exercise also mediates robust adaptations in white adipose tissue. To determine if exercise affects in vivo adipocyte formation, fifty-two, sixteen-week-old C57BL/6J mice were allowed access to unlocked running wheels [Exercise (EX) group; n = 13 males, n = 13 females] or to locked wheels [Sedentary (SED) group; n = 13 males, n = 13 females] for 4-weeks. In vivo adipocyte formation was assessed by the incorporation of deuterium (2H) into the DNA of newly formed adipocytes in the inguinal and gonadal adipose depots. A two-way ANOVA revealed that exercise significantly decreased new adipocyte formation in the adipose tissue of mice in the EX group relative to the SED group (activity effect; P = 0.02). This reduction was observed in male and female mice (activity effect; P = 0.03). Independent analysis of the depots showed a significant reduction in adipocyte formation in the inguinal (P = 0.05) but not in the gonadal (P = 0.18) of the EX group. We report for the first time that exercise significantly reduced in vivo adipocyte formation in the adipose tissue of EX mice using a physiologic metabolic 2H2O-labeling protocol.

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

Adipose depot-specific effects of 16 weeks of pioglitazone on in vivo adipogenesis in women with obesity: a randomised controlled trial.

AIMS/HYPOTHESIS: In vitro and rodent studies suggest that pioglitazone, a thiazolidinedione, can promote adipogenesis in adipose tissue (AT); however, there is a lack of in vivo studies in humans to support these findings. The objectives of this randomised, placebo-controlled, parallel-arm trial were to test if pioglitazone stimulates in vivo adipogenesis in the subcutaneous adipose tissue depots and if these measures were related to metabolic health outcomes in women with obesity. METHODS: Forty-one healthy women with obesity (20 black; 21 white; 29 ± 6 years; BMI 32.0 ± 1.7 kg/m2; 44.0 ± 3.6% body fat) were randomised to consume 30 mg/day of pioglitazone (n = 21) or placebo (n = 20) for 16 weeks. SAS v9.4 was used to generate the block randomisation code sequence (stored in password-protected files) with a 1:1 allocation ratio. The participants and study staff involved in assessing and analysing data outcomes were blinded to the group assignments. The trial was conducted at Pennington Biomedical Research Center and ended in 2016. At baseline and post-intervention, subcutaneous abdominal (scABD) and femoral (scFEM) AT biopsies were collected, and in vivo cellular kinetics (primary endpoint of the trial) were assessed by an 8 week labelling protocol of deuterium (2H) into the DNA of adipose cells. Body composition was measured by dual-energy x-ray absorptiometry (DXA), scABD and visceral AT (VAT) by MRI, ectopic fat by 1H-MRS, and insulin sensitivity by an OGTT. RESULTS: After the 16 week intervention, there was a significant decrease in visceral fat (VAT:total abdominal AT [as a %]; p = 0.002) and an increase in the Matsuda index (i.e. improved insulin sensitivity; p = 0.04) in the pioglitazone group relative to the placebo group. A significant increase in the formation of new adipocytes was observed in the scFEM (Δ = 3.3 ± 1.6%; p = 0.04) but not the scABD depot (Δ = 2.0 ± 2.1%; p = 0.32) in the pioglitazone group relative to the placebo group. No serious adverse events were reported. CONCLUSIONS/INTERPRETATION: Pioglitazone may elicit distinct differences in in vivo adipogenesis in subcutaneous adipose depots in women with obesity, with increased rates in the protective scFEM. Trial registration ClinicalTrials.gov NCT01748994 Funding This study was funded by R01DK090607, P30DK072476, and R03DK112006 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health. The Robert C. and Veronica Atkins Foundation. Graphical abstract.

Human sebum requires de novo lipogenesis, which is increased in acne vulgaris and suppressed by acetyl-CoA carboxylase inhibition.

Sebum plays important physiological roles in human skin. Excess sebum production contributes to the pathogenesis of acne vulgaris, and suppression of sebum production reduces acne incidence and severity. We demonstrate that sebum production in humans depends on local flux through the de novo lipogenesis (DNL) pathway within the sebocyte. About 80 to 85% of sebum palmitate (16:0) and sapienate (16:1n10) were derived from DNL, based on stable isotope labeling, much higher than the contribution of DNL to triglyceride palmitate in circulation (~20%), indicating a minor contribution by nonskin sources to sebum lipids. This dependence on local sebocyte DNL was not recapitulated in two widely used animal models of sebum production, Syrian hamsters and Göttingen minipigs. Confirming the importance of DNL for human sebum production, an acetyl-CoA carboxylase inhibitor, ACCi-1, dose-dependently suppressed DNL and blocked synthesis of fatty acids, triglycerides, and wax esters but not free sterols in human sebocytes in vitro. ACCi-1 dose-dependently suppressed facial sebum excretion by ~50% (placebo adjusted) in human individuals dosed orally for 2 weeks. Sebum triglycerides, wax esters, and free fatty acids were suppressed by ~66%, whereas non-DNL-dependent lipid species, cholesterol, and squalene were not reduced, confirming selective modulation of DNL-dependent lipids. Last, individuals with acne vulgaris exhibited increased sebum production rates relative to individuals with normal skin, with >80% of palmitate and sapienate derived from DNL. These findings highlight the importance of local sebocyte DNL for human skin sebaceous gland biology and illuminate a potentially exploitable therapeutic target for the treatment of acne vulgaris.

An ANGPTL4-ceramide-protein kinase Cζ axis mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia in mice.

Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders.

Altered In Vivo Lipid Fluxes and Cell Dynamics in Subcutaneous Adipose Tissues Are Associated With the Unfavorable Pattern of Fat Distribution in Obese Adolescent Girls.

Patterns of abdominal fat distribution (for example, a high vs. low visceral adipose tissue [VAT]/[VAT + subcutaneous adipose tissue (SAT)] ratio), independent of obesity, during adolescence carry a high risk for insulin resistance and type 2 diabetes. Longitudinal follow-up of a cohort of obese adolescents has recently revealed that a high ratio (high VAT/[VAT + SAT]) is a major determinant of fatty liver and metabolic impairment over time, with these effects being more pronounced in girls than in boys. To unravel the underlying metabolic alterations associated with the unfavorable VAT/(VAT + SAT) phenotype, we used the 2H2O labeling method to measure the turnover of adipose lipids and cells in the subcutaneous abdominal and gluteal/femoral adipose tissue (SAT) of weight-stable obese adolescent girls with a similar level of obesity but discordant VAT/(VAT + SAT) ratios. Girls with the unfavorable (high VAT/[VAT + SAT]) phenotype exhibited higher in vivo rates of triglyceride (TG) turnover (representing both lipolysis and synthesis at steady state), without significant differences in de novo lipogenesis in both abdominal and gluteal depots, compared with obese girls with the favorable phenotype. Moreover, mature adipocytes had higher turnover, with no difference in stromal vascular cell proliferation in both depots in the metabolically unfavorable phenotype. The higher TG turnover rates were significantly correlated with higher intrahepatic fat stores. These findings are contrary to the hypothesis that impaired capacity to deposit TGs or proliferation of new mature adipocytes are potential mechanisms for ectopic fat distribution in this setting. In summary, these results suggest that increased turnover of TGs (lipolysis) and of mature adipocytes in both abdominal and gluteal SAT may contribute to metabolic impairment and the development of fatty liver, even at this very early stage of disease.

ERß activation in obesity improves whole body metabolism via adipose tissue function and enhanced mitochondria biogenesis.

OBJECTIVE: Estrogens play a key role in the distribution of adipose tissue and have their action by binding to both estrogen receptors (ER), α and ß. Although ERß has a role in the energy metabolism, limited data of the physiological mechanism and metabolic response involved in the pharmacological activation of ERß is available. METHODS: For clinical relevance, non-ovariectomized female mice were subjected to high fat diet together with pharmacological (DIP - 4-(2-(3,5-dimethylisoxazol-4-yl)-1H-indol-3-yl)phenol) interventions to ERß selective activation. The physiological mechanism was assessed in vivo by magnetic resonance imaging and spectroscopy, and oral glucose and intraperitoneal insulin tolerance test before and after DIP treatment. Liver and adipose tissue metabolic response was measured in HFD + vehicle and HFD + DIP by stable isotope, RNA sequencing and protein content. RESULTS: HFD-fed females treated with DIP had a tissue-specific response towards ERß selective activation. The metabolic profile showed an improved fasting glucose level, insulin sensitivity and reduced liver steatosis. CONCLUSIONS: Our data demonstrate that selective activation of ERß exerts a tissue-specific activity which promotes a beneficial effect on whole body metabolic response to obesity.

Measurement of Leukemic B-Cell Growth Kinetics in Patients with Chronic Lymphocytic Leukemia.

Cell proliferation plays a central role in the pathogenesis of every neoplastic disease as well as many other types of illness. Labeling of newly replicated DNA with deuterium (2H), a nonradioactive isotope of hydrogen, administered to the patients in drinking water (2H2O) is a safe and reliable method to measure the in vivo birth rates of cells. Here, we describe a protocol to measure chronic lymphocytic leukemia B-cell birth/proliferation and death rates over time using this approach.

Racial differences in in vivo adipose lipid kinetics in humans.

The storage of lipids in the form of triglycerides (TGs) and the de novo synthesis (lipogenesis) of fatty acids from nonlipid precursors [de novo lipogenesis (DNL)] are important functions of adipose tissue (AT) that influence whole-body metabolism. Yet, few studies have reported in vivo estimates of adipose lipid kinetics in humans. Fifty-two women with obesity (27 African-American and 25 Caucasian; 29.7 ± 5.5 years; BMI 32.2 ± 2.8 kg/m2; 44.3 ± 4.0% body fat) were enrolled in the study. In vivo synthesis (or replacement) of TGs (fTG) as well as the synthesis of the fatty acid, palmitate [a measure of adipose DNL (fDNL)], were assessed using an 8 week incorporation of deuterium into lipids (glycerol and palmitate moieties of TGs) in subcutaneous abdominal (scABD) and subcutaneous femoral (scFEM) AT. We report, for the first time, significant race differences in both TG synthesis and absolute DNL, with Caucasians having higher fTG and fDNL as compared with African-Americans. The DNL contribution to newly synthesized TG (corrected fDNL) was not different between races. Interestingly, our findings also show that the scFEM adipose depot had higher TG replacement rates relative to the scABD. Finally, the replacement rate of TG (fTG) was negatively correlated with changes in body weight over the 8 week labeling period. Our results provide the first evidence that in vivo TG replacement (synthesis and breakdown) rates differ by ethnicity. In addition, TG turnover varies by depot location in humans, implying an increased capacity for TG storage and higher lipolytic activity in the scFEM AT.

Acetyl-CoA Carboxylase Inhibitor GS-0976 for 12 Weeks Reduces Hepatic De Novo Lipogenesis and Steatosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Increased de novo lipogenesis (DNL) contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase catalyzes the rate-limiting step in DNL. We evaluated the safety and efficacy of GS-0976, a small molecule inhibitor of acetyl-CoA carboxylase, in patients with NASH. METHODS: In an open-label prospective study, patients with NASH (n = 10) received GS-0976 20 mg orally once daily for 12 weeks. NASH was diagnosed based on a proton density fat fraction estimated by magnetic resonance imaging (MRI-PDFF) ≥10% and liver stiffness by magnetic resonance elastography (MRE) ≥2.88 kPa. The contribution from hepatic DNL to plasma palmitate was measured by 14 days of heavy water labeling before and at the end of treatment. We performed the same labelling protocol in an analysis of healthy volunteers who were not given DNL (controls, n = 10). MRI-PDFF and MRE at baseline, and at weeks 4 and 12 of GS-0976 administration, were measured. We analyzed markers of liver injury and serum markers of fibrosis. RESULTS: The contribution of hepatic DNL to plasma palmitate was significantly greater in patients with NASH compared with controls (43% vs 18%) (P = .003). After 12 weeks administration of GS-0976, the median hepatic DNL was reduced 22% from baseline in patients with NASH (P = .004). Compared with baseline, reductions in MRI-PDFF at week 12 (15.7% vs 9.1% at baseline; P = .006), liver stiffness by MRE (3.4 kPa vs 3.1 kPa at baseline; P = .049), TIMP metallopeptidase inhibitor 1 (275 ng/mL vs 244 ng/mL at baseline; P = .049), and serum level of alanine aminotransferase (101 U/L vs 57 U/L at baseline; P = .23) were consistent with decreased hepatic lipid content and liver injury. At week 12, 7 patients (70%) had a ≥30% decrease in MRI-PDFF. CONCLUSION: In an open-label study, patients with NASH given GS-0976 for 12 weeks had reduced hepatic DNL, steatosis, and markers of liver injury. ClinicalTrials.gov no: NCT02856555.

LRH-1 regulates hepatic lipid homeostasis and maintains arachidonoyl phospholipid pools critical for phospholipid diversity.

Excess lipid accumulation is an early signature of nonalcoholic fatty liver disease (NAFLD). Although liver receptor homolog 1 (LRH-1) (encoded by NR5A2) is suppressed in human NAFLD, evidence linking this phospholipid-bound nuclear receptor to hepatic lipid metabolism is lacking. Here, we report an essential role for LRH-1 in hepatic lipid storage and phospholipid composition based on an acute hepatic KO of LRH-1 in adult mice (LRH-1AAV8-Cre mice). Indeed, LRH-1-deficient hepatocytes exhibited large cytosolic lipid droplets and increased triglycerides (TGs). LRH-1-deficient mice fed high-fat diet displayed macrovesicular steatosis, liver injury, and glucose intolerance, all of which were reversed or improved by expressing wild-type human LRH-1. While hepatic lipid synthesis decreased and lipid export remained unchanged in mutants, elevated circulating free fatty acid helped explain the lipid imbalance in LRH-1AAV8-Cre mice. Lipidomic and genomic analyses revealed that loss of LRH-1 disrupts hepatic phospholipid composition, leading to lowered arachidonoyl (AA) phospholipids due to repression of Elovl5 and Fads2, two critical genes in AA biosynthesis. Our findings reveal a role for the phospholipid sensor LRH-1 in maintaining adequate pools of hepatic AA phospholipids, further supporting the idea that phospholipid diversity is an important contributor to healthy hepatic lipid storage.

Impact of Moderate Alcohol Discontinuation on Insulin Action and Secretion in Latinos With and Without Hepatitis C.

BACKGROUND: Insulin resistance (IR) is associated with hepatitis C virus (HCV), and Latinos are both at risk of IR and are disproportionately affected by HCV. Moderate alcohol consumption improves insulin sensitivity and may modify HCV-associated IR. We investigated the impact of moderate alcohol discontinuation on insulin sensitivity and secretion in Latinos using direct measurements. METHODS: Twenty-five nondiabetic, noncirrhotic Latino adults without (n = 17) or with (n = 8) HCV underwent 3-day metabolic assessment before and after prescription of 6 weeks of moderate alcohol discontinuation. Peripheral IR was measured via steady-state plasma glucose (SSPG) and hepatic IR using endogenous glucose production during a 2-step 240-minute insulin suppression test. Insulin secretion was measured using graded glucose infusion test. RESULTS: Baseline mean age was 46 ± 11 years, 63% male, 29% had HCV, and mean body mass index was 27 ± 4 kg/m2 . Compared to non-HCV, HCV patients had a higher median SSPG (132 vs. 98.8 mg/dl, p = 1.0), hepatic IR (13.5 vs. 11.3, p = 0.24), and insulin secretion rate (ISR-AUC, 1,290 vs. 1,250 pmol/min, p = 0.98). After confirmed alcohol discontinuation, hepatic IR was the only parameter that changed significantly (increased, mean change 2.6 ± 4.8, p = 0.02). Higher baseline alanine aminotransferase (ALT) was also associated with a greater change in hepatic IR (average 4.0 points/ALT doubling, p = 0.004), and HCV was associated with a lesser change (average -7.3 points, p = 0.002), independent of ALT. CONCLUSIONS: Short-term moderate alcohol discontinuation adversely impacted hepatic IR in Latinos which was influenced by level of ALT at baseline independent of etiology. Although reduction in ALT through weight loss and HCV eradication remains a priority in improving IR, the observed nonharmful effect of moderate alcohol use represents a potentially confounding variable that warrants further study.

Origin and differentiation of human memory CD8 T cells after vaccination.

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Effect of Weight Gain and Weight Loss on In Vivo Colonocyte Proliferation Rate in People with Obesity.

OBJECTIVE: To evaluate the effects of diet-induced changes in energy balance and body weight on in vivo colonocyte fractional proliferation rates (FPR) in people with obesity. METHODS: In vivo colonocyte FPR was assessed in 31 men and women with obesity (BMI: 35.4 ± 4.0 kg/m2 , age: 52.6 ± 8.9 years) before and after diet-induced weight loss, weight gain, or weight maintenance. Subjects ingested aliquots of 2 H2 O (heavy water) daily for 4 to 7 days, followed by flexible sigmoidoscopy with colon biopsies to assess the incorporation of 2 H into the DNA of dividing colonocytes. RESULTS: Colonocyte FPR averaged 12.7% ± 3.8% per day and correlated directly with intra-abdominal adipose tissue (IAAT) volume (r = 0.364, P = 0.044). Colonocyte FPR decreased in the weight loss group, did not change in the weight maintenance group, and increased in the weight gain group. The change in colonocyte FPR correlated directly with the percent change in body weight (r = 0.409, P = 0.028) and IAAT volume (r = 0.598, P = 0.001). CONCLUSIONS: A high-calorie diet and weight gain increase, whereas a low-calorie diet and weight loss decrease, in vivo colonocyte proliferation rate in people with obesity. These results suggest that changes in energy balance influence the risk of developing colon cancer in people with obesity by regulating colonic mucosal growth rates.