SUMMARY Nonalcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular and liver-related mortality. NAFLD is characterized by both triglyceride and free cholesterol (FC) accumulation without a corresponding increment in cholesterol esters. The aim of this study was to evaluate the expression of cholesterol metabolic genes in NAFLD and relate these to disease phenotype. NAFLD was associated with increased SREBP-2 maturation, HMG CoA reductase (HMGCR) expression and decreased phosphorylation of HMGCR. Cholesterol synthesis was increased as measured by the circulating desmosterol:cholesterol ratio. miR-34a, a microRNA increased in NAFLD, inhibited sirtuin-1 with downstream dephosphorylation of AMP kinase and HMGCR. Cholesterol ester hydrolase was increased while ACAT-2 remained unchanged. LDL receptor expression was significantly decreased and similar in NAFLD subjects on or off statins. HMGCR expression was correlated with FC, histologic severity of NAFLD and LDL-cholesterol. These data demonstrate dysregulated cholesterol metabolism in NAFLD which may contribute to disease severity and cardiovascular risks.

No abstract

IMPORTANCE Low-density lipoprotein cholesterol (LDL-C), a key cardiovascular disease marker, is often estimated by the Friedewald or Martin equation, but calculating LDL-C is less accurate in patients with a low LDL-C level or hypertriglyceridemia (triglyceride [TG] levels Ն400 mg/dL). OBJECTIVE To design a more accurate LDL-C equation for patients with a low LDL-C level and/or hypertriglyceridemia. DESIGN, SETTING, AND PARTICIPANTS Data on LDL-C levels and other lipid measures from 8656 patients seen at the National Institutes of Health Clinical Center between January 1, 1976, and June 2, 1999, were analyzed by the β-quantification reference method (18 715 LDL-C test results) and were randomly divided into equally sized training and validation data sets. Using TG and non-high-density lipoprotein cholesterol as independent variables, multiple least squares regression was used to develop an equation for very low-density lipoprotein cholesterol, which was then used in a second equation for LDL-C. Equations were tested against the internal validation data set and multiple external data sets of either β-quantification LDL-C results (n = 28 891) or direct LDL-C test results (n = 252 888). Statistical analysis was performed from August 7, 2018, to July 18, 2019. MAIN OUTCOMES AND MEASURES Concordance between calculated and measured LDL-C levels by β-quantification, as assessed by various measures of test accuracy (correlation coefficient [R 2 ], root mean square error [RMSE], mean absolute difference [MAD]), and percentage of patients misclassified at LDL-C treatment thresholds of 70, 100, and 190 mg/dL. RESULTSCompared with β-quantification, the new equation was more accurate than other LDL-C equations (slope, 0.964; RMSE = 15.2 mg/dL; R 2 = 0.9648; vs Friedewald equation: slope, 1.056; RMSE = 32 mg/dL; R 2 = 0.8808; vs Martin equation: slope, 0.945; RMSE = 25.7 mg/dL; R 2 = 0.9022), particularly for patients with hypertriglyceridemia (MAD = 24.9 mg/dL; vs Friedewald equation: MAD = 56.4 mg/dL; vs Martin equation: MAD = 44.8 mg/dL). The new equation calculates the LDL-C level in patients with TG levels up to 800 mg/dL as accurately as the Friedewald equation does for TG levels less than 400 mg/dL and was associated with 35% fewer misclassifications when patients with hypertriglyceridemia (TG levels, 400-800 mg/dL) were categorized into different LDL-C treatment groups. CONCLUSIONS AND RELEVANCEThe new equation can be readily implemented by clinical laboratories with no additional costs compared with the standard lipid panel. It will allow for more accurate calculation of LDL-C level in patients with low LDL-C levels and/or hypertriglyceridemia (TG levels, Յ800 mg/dL) and thus should improve the use of LDL-C level in cardiovascular disease risk management.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesistwo crucial arms of cancer growth-AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.metabolism | glioma A MP-activated protein kinase (AMPK) is a molecular hub for cellular metabolic control (1-4). It is a heterotrimer of catalytic α, regulatory β, and γ subunits. The rising AMP:ATP ratio during energy stress leads to AMP-dependent phosphorylation of the catalytic α subunits. This activates AMPK which then phosphorylates numerous substrates to restore energy homeostasis. It phosphorylates acetyl CoA carboxylase (ACCα) to inhibit fatty acid (FA) synthesis (5) and TSC2 and RAPTOR (6, 7) to inhibit mammalian target of rapamycin (mTOR)C1. Because fatty acid synthesis and mTORC1 activity are essential for cell proliferation and growth (8), AMPK activation with two indirect AMPK agonists AICAR and metformin have been correlated with suppression of cell proliferation and growth (9-11).AICAR is metabolized to an AMP mimetic, ZMP that activates AMPK (12). Although AICAR does inhibit proliferation (11-15), it also causes AMPK-independent cellular and metabolic effects (12, 16) including inhibition of glucokinase, glycogen phosphorylase, and nucleotide biosynthesis (17, 18). Whether AICAR requires AMPK to suppress proliferation is questionable because although both AICAR and 2-deoxyglucose activated AMPK, only AICAR inhibited proliferation of trisomic mouse fibroblasts (11). Moreover, although AICAR strongly increases glucose uptake through AMPK activation in muscle cells, it reduced fluorodeoxyglucose-PET signals and inhibited glioma gro...

Objective Coronary heart disease (CHD) is the leading cause of death in the United States, yet assessing risk of its development remains challenging. The present study evaluates a new automated assay of small dense low-density lipoprotein cholesterol content (sdLDL-C) and whether sdLDL-C is a risk factor for CHD compared with LDL-C or small LDL particle concentrations derived from nuclear magnetic resonance spectroscopy. Approach and Results sdLDL-C was measured using a new automated enzymatic method, and small LDL concentrations were obtained by nuclear magnetic resonance in 4387 Multi-Ethnic Study of Atherosclerosis participants. Cox regression analysis estimated hazard ratios for developing CHD for 8.5 years after adjustments for age, race, sex, systolic blood pressure, hypertension medication use, high-density lipoprotein cholesterol, and triglycerides. Elevated sdLDL-C was a risk factor for CHD in normoglycemic individuals. Those in the top sdLDL-C quartile showed higher risk of incident CHD (hazard ratio, 2.41; P=0.0037) compared with those in the bottom quartile and indicated greater CHD risk than the corresponding quartile of LDL-C (hazard ratio, 1.75; P=0.019). The association of sdLDL-C with CHD risk remained significant when LDL-C (<2.57 mmol/L) was included in a multivariate model (hazard ratio, 2.37; P=0.012). Nuclear magnetic resonance–derived small LDL concentrations did not convey a significant risk of CHD. Those with impaired fasting glucose or diabetes mellitus showed higher sdLDL-C and small LDL concentrations but neither was associated with higher CHD risk in these individuals. Conclusions This new automated method for sdLDL-C identifies risk for CHD that would remain undetected using standard lipid measures, but only in normoglycemic, nondiabetic individuals.

Objective We aimed to examine associations of Lp(a) concentrations with coronary heart disease (CHD) and determine whether current Lp(a) clinical laboratory cut points identify risk of disease incidence in four races/ethnicities of the Multi-Ethnic Study of Atherosclerosis (MESA). Approach and Results A subcohort of 1,323 Black, 1,677 Caucasian, 548 Chinese-American, and 1,044 Hispanic MESA participants were followed over a mean 8.5 year period in which 235 incident CHD events were recorded. Lp(a) mass concentrations were measured using a turbidimetric immunoassay. Cox regression analysis determined associations of Lp(a) with CHD risk with adjustments for lipid and non-lipid variables. Lp(a) concentrations were continuously associated with risk of CHD incidence in Black [hazard ratio (HR)=1.49; 95% CI: 1.09 – 2.04] and Caucasian participants (HR=1.22; 95% CI: 1.02 – 1.45). Examining Lp(a) risk by the 50 mg/dL cut point revealed higher risks of incident CHD in all races except Chinese Americans: Blacks (HR=1.69; 95% CI: 1.03 – 2.76); Caucasians (HR=1.82; 95% CI: 1.15 – 2.88); Hispanics (HR=2.37; 95% CI: 1.17 – 4.78). The lower Lp(a) cut point of 30 mg/dL identified higher risk of CHD in Black participants alone (HR: 1.87; 95% CI: 1.08 – 3.21). Conclusions Our findings suggest that the 30 mg/dL cutoff for Lp(a) is not appropriate in Caucasian and Hispanic individuals, and the higher 50 mg/dL cutoff should be considered. In contrast, the 30 mg/dL cutoff remains suitable in Black individuals. Further research is necessary to develop the most clinically useful Lp(a) cutoff values in individual races/ethnicities.

No abstract

The expression of connexin43, the primary gap-junction constituent of glial cells, was evaluated at the messenger RNA and protein levels in different grades of astrocytoma to investigate the relevance of gap junctions in herpes simplex virus-thymidine kinase (HSV-tk)-mediated gene therapy of brain tumors. Transduction of the retroviral-mediated HSV-tk gene into tumor cells with subsequent administration of ganciclovir has recently been used as an experimental therapeutic strategy for treatment of brain tumors. One aspect of this approach is the bystander effect, which augments the efficacy of this therapeutic approach. Glioblastoma cells with minimum levels of connexin43 protein were transfected with a connexin43 complementary DNA. These cells manifested a marked increase in the in vitro bystander effect, supporting the contention that the in vitro bystander effect is a consequence of metabolic cooperation between cells mediated by gap junctions. To assess relative levels of gap-junction protein expression in the relevant tumor type, we examined primary astrocytomas, primary astrocytoma cell cultures, and glioblastoma cell lines. Although most astrocytoma tumor samples expressed connexin43, they differed in the level of expression, with the greatest variation exhibited in high-grade astrocytomas. Primary glioblastoma cell cultures and established glioblastoma cell lines also displayed some variability in connexin43 levels. In aggregate, our results anticipate that glioblastomas will have a varied bystander effect during HSV-tk gene therapy depending on the level of connexin43 expression.