👤 Masanori Aikawa

Apolipoprotein A4's (APOA4's) functions on HDL in humans are not well understood. A unique feature of APOA4 is that it is an intestinal apolipoprotein secreted on HDL and chylomicrons. The goal of this study was to gain a better understanding of the origin and function of APOA4 on HDL by studying its metabolism across 6 HDL sizes. Twelve participants completed a metabolic tracer study. HDL was isolated by APOA1 immunopurification and separated by size. Tracer enrichments for APOA4 and APOA1 were determined by targeted mass spectrometry, and metabolic rates were derived by compartmental modeling. APOA4 metabolism on small HDL (alpha3, prebeta, and very small prebeta) was distinct from that of APOA4 on large HDL (alpha0, 1, 2). APOA4 on small HDL appeared in circulation by 30 minutes and was relatively rapidly catabolized. In contrast, APOA4 on large HDL appeared in circulation later (1-2 hours) and had a much slower catabolism. The unique metabolic profiles of APOA4 on small and large HDL likely indicate that each has a distinct origin and function in humans. This evidence supports the notion that APOA4 on small HDL originates directly from the small intestine while APOA4 on large HDL originates from chylomicron transfer. Show less

Recent in vivo tracer studies demonstrated that targeted mass spectrometry (MS) on the Q Exactive Orbitrap could determine the metabolism of HDL proteins 100s-fold less abundant than apolipoprotein A1 (APOA1). In this study, we demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than APOA1, specifically the lipid transfer proteins phospholipid transfer protein (PLTP), cholesterol ester transfer protein (CETP), and lecithin-cholesterol acyl transferase (LCAT). Relative to the Q Exactive, the Lumos improved tracer detection by reducing tracer enrichment compression, thereby providing consistent enrichment data across multiple HDL sizes from 6 participants. We determined by compartmental modeling that PLTP is secreted in medium and large HDL (alpha2, alpha1, and alpha0) and is transferred from medium to larger sizes during circulation from where it is catabolized. CETP is secreted mainly in alpha1 and alpha2 and remains in these sizes during circulation. LCAT is secreted mainly in medium and small HDL (alpha2, alpha3, prebeta). Unlike PLTP and CETP, LCAT's appearance on HDL is markedly delayed, indicating that LCAT may reside for a time outside of systemic circulation before attaching to HDL in plasma. The determination of these lipid transfer proteins' unique metabolic structures was possible due to advances in MS technologies. Show less

Monogenic diabetes is clinically heterogeneous and differs from common forms of diabetes (type 1 and 2). We aimed to investigate the clinical usefulness of a comprehensive genetic testing system, comprised of targeted next-generation sequencing (NGS) with phenotype-driven bioinformatics analysis in patients with monogenic diabetes, which uses patient genotypic and phenotypic data to prioritize potentially causal variants. We performed targeted NGS of 383 genes associated with monogenic diabetes or common forms of diabetes in 13 Japanese patients with suspected (n = 10) or previously diagnosed (n = 3) monogenic diabetes or severe insulin resistance. We performed in silico structural analysis and phenotype-driven bioinformatics analysis of candidate variants from NGS data. Among the patients suspected having monogenic diabetes or insulin resistance, we diagnosed 3 patients as subtypes of monogenic diabetes due to disease-associated variants of INSR, LMNA, and HNF1B. Additionally, in 3 other patients, we detected rare variants with potential phenotypic effects. Notably, we identified a novel missense variant in TBC1D4 and an MC4R variant, which together may cause a mixed phenotype of severe insulin resistance. This comprehensive approach could assist in the early diagnosis of patients with monogenic diabetes and facilitate the provision of tailored therapy. Show less

Retinoic acid (RA) is a ligand for nuclear receptors that modulate gene transcription and cell differentiation. Whether RA controls ectopic calcification in humans is unknown. We tested the hypothesis that RA regulates osteogenic differentiation of human arterial smooth muscle cells and aortic valvular interstitial cells that participate in atherosclerosis and heart valve disease, respectively. Approach and Results: Human cardiovascular tissue contains immunoreactive RAR (RA receptor)-a retinoid-activated nuclear receptor directing multiple transcriptional programs. RA stimulation suppressed primary human cardiovascular cell calcification while treatment with the RAR inhibitor AGN 193109 or These results establish retinoid regulation of human cardiovascular calcification, provide new insight into mechanisms involved in these responses, and suggest selective retinoid modulators, like acyclic retinoids may allow for treating cardiovascular calcification without the adverse effects associated with cyclic retinoids. Show less

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Show less

Clinical evidence has linked low HDL (high-density lipoprotein) cholesterol levels with high cardiovascular disease risk; however, its significance as a therapeutic target remains unestablished. We hypothesize that HDLs functional heterogeneity is comprised of metabolically distinct proteins, each on distinct HDL sizes and that are affected by diet. Approach and Results: Twelve participants were placed on 2 healthful diets high in monounsaturated fat or carbohydrate. After 4 weeks on each diet, participants completed a metabolic tracer study. HDL was isolated by Apo (apolipoprotein) A1 immunopurification and separated into 5 sizes. Tracer enrichment and metabolic rates for 8 HDL proteins-ApoA1, ApoA2, ApoC3, ApoE, ApoJ, ApoL1, ApoM, and LCAT (lecithin-cholesterol acyltransferase)-were determined by parallel reaction monitoring and compartmental modeling, respectively. Each protein had a unique, size-specific distribution that was not altered by diet. However, carbohydrate, when replacing fat, increased the fractional catabolic rate of ApoA1 and ApoA2 on alpha3 HDL; ApoE on alpha3 and alpha1 HDL; and ApoM on alpha2 HDL. Additionally, carbohydrate increased the production of ApoC3 on alpha3 HDL and ApoJ and ApoL1 on the largest alpha0 HDL. LCAT was the only protein studied that diet did not affect. Finally, global proteomics showed that diet did not alter the distribution of the HDL proteome across HDL sizes. This study demonstrates that HDL in humans is composed of a complex system of proteins, each with its own unique size distribution, metabolism, and diet regulation. The carbohydrate-induced hypercatabolic state of HDL proteins may represent mechanisms by which carbohydrate alters the cardioprotective properties of HDL. Show less

Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor liver X receptor (LXR) is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knock down Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knock-out of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation, and ER stress from the negative effects on lipogenesis. Show less

The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm. Show less