Insulin activates sterol regulatory element-binding protein-1c in liver, thereby increasing fatty acid and triglyceride synthesis. We created a line of transgenic rats that produce epitopetagged human SREBP-1c in liver under control of the constitutive apolipoprotein E promoter/enhancer. This system allows us to dissect the pathway by which insulin stimulates SREBP-1c processing without interference by the insulin-mediated increase in SREBP-1c mRNA. Rats are used because freshly isolated rat hepatocytes respond much more robustly to insulin than do mouse hepatocytes. The data reveal that insulin-mediated stimulation of SREBP-1c processing requires the mechanistic target of rapamycin complex 1 (mTORC1), which also is required for insulin-mediated SREBP-1c mRNA induction. However, in contrast to mRNA induction, insulin stimulation of SREBP-1c processing is blocked by an inhibitor of p70 S6-kinase. The data indicate that the pathways for insulin enhancement of SREBP-1c mRNA and proteolytic processing diverge after mTORC1. Stimulation of processing requires the mTORC1 target p70 S6-kinase, whereas induction of mRNA bypasses this enzyme. Insulin stimulation of both processes is blocked by glucagon. The transgenic rat system will be useful in further defining the molecular mechanism for insulin stimulation of lipid synthesis in liver in normal and diabetic states. T he liver plays a unique role in lipid metabolism because it is the only organ that synthesizes fatty acids (FAs) and triglycerides (TGs) for export to other tissues. These synthetic processes are controlled reciprocally by insulin and glucagon, which are secreted by the pancreas and delivered directly to the liver via the portal vein. Precise control is important because excess FA synthesis leads to elevated FAs in muscle, thereby contributing to the peripheral insulin resistance and lipotoxicity seen in type 2 diabetes. Excess FA synthesis also causes fatty liver, which sometimes leads to cirrhosis and liver failure (1, 2).Insulin stimulates FA synthesis in liver by increasing the mRNA and the processed nuclear form of sterol regulatory elementbinding protein-1c (SREBP-1c), a transcription factor that activates all the genes needed to produce FAs and TGs in liver (3). Of the three SREBP isoforms, SREBP-1c is the one whose expression is highest in liver, and it is the only one that is controlled primarily by insulin. For this reason, definitive studies of insulin-mediated activation of SREBP-1c must be performed with liver cells.Studies of insulin action on liver cells are difficult because none of the established hepatocyte cell lines responds to insulin with the robustness observed in the livers of living animals. Moreover, freshly isolated hepatocytes lose their insulin responsiveness within 48-72 h after isolation. Therefore, studies must be performed in living animals or with freshly isolated hepatocytes that are less than 72 h old. Even more perplexing are species differences. Although mouse and rat livers manifest robust elevations in SREBP-1c mRNA...
We probed an epigenetic regulatory path from small molecule to neuronal gene activation. Isoxazole small molecules triggered robust neuronal differentiation in adult neural stem cells, rapidly signaling to the neuronal genome via Ca(2+) influx. Ca(2+)-activated CaMK phosphorylated and mediated nuclear export of the MEF2 regulator HDAC5, thereby de-repressing neuronal genes. These results provide new tools to explore the epigenetic signaling circuitry specifying neuronal cell fate and new leads for neuro-regenerative drugs.
Insig-1 and Insig-2, two closely related proteins, are essential for feedback inhibition of cholesterol biosynthesis. Here, we characterized a line of epidermal-specific, Insig-double knockout (Epi-Insig-DKO) mice lacking both Insigs in epidermis. At birth, Epi-Insig-DKO mice were indistinguishable from control littermates, but thereafter they failed to thrive and died before 6 weeks of age. By 14 days of age, 100% of Epi-Insig-DKO mice exhibited defects in hair growth along with other skin abnormalities, including hyperkeratosis. Hair follicles in Epi-Insig-DKO mice developed normally through postnatal day 7, but they failed to progress to later stages and thus exhibited defects in postnatal hair cycling. Insig deficiency caused a marked buildup of cholesterol precursors in skin associated with a marked increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase protein. Topical treatment of Epi-Insig-DKO mice with simvastatin, an inhibitor of reductase, reduced sterol precursors in skin and corrected the hair and skin defects. We conclude that Insig deficiency in skin causes accumulation of cholesterol precursors, and this impairs normal hair development. These findings have implications for several human genetic diseases in which mutations in cholesterol biosynthetic enzymes lead to accumulation of sterol precursors and multiple cutaneous abnormalities.
A B S T R A C T Germline mutations in SAMD9 and SAMD9L genes cause MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) (OMIM: *610456) and ataxia-pancytopenia (OMIM: *611170) syndromes, respectively, and are associated with chromosome 7 deletions, myelodysplastic syndrome (MDS), and bone marrow failure. In this retrospective series, we report outcomes of allogeneic hematopoietic cell transplantation (HCT) in patients with hematologic disorders associated with SAMD9/SAMD9L mutations. Twelve patients underwent allogeneic HCT for MDS (n = 10), congenital amegakaryocytic thrombocytopenia (n = 1), and dyskeratosis congenita (n = 1). Exome sequencing revealed heterozygous mutations in SAMD9 (n = 6) or SAMD9L (n = 6) genes. Four SAMD9 patients had features of MIRAGE syndrome. Median age at HCT was 2.8 years (range, 1.2 to 12.8 years). Conditioning was myeloablative in 9 cases and reduced intensity in 3 cases. Syndrome-related comorbidities (diarrhea, infections, adrenal insufficiency, malnutrition, and electrolyte imbalance) were present in MIRAGE syndrome cases. One patient with a familial SAMD9L mutation, MDS, and morbid obesity failed to engraft and died of refractory acute myeloid leukemia. The other 11 patients achieved neutrophil engraftment. Acute post-transplant course was complicated by syndrome-related comorbidities in MIRAGE cases. A patient with SAMD9L-associated MDS died of diffuse alveolar hemorrhage. The other 10 patients had resolution of hematologic disorder and sustained peripheral blood donor chimerism. Ten of 12 patients were alive with a median follow-up of 3.1 years (range, 0.1 to 14.7 years). More data are needed to refine transplant approaches in SAMD9/ SAMD9L patients with significant comorbidities and to develop guidelines for their long-term follow-up.
Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.
Purpose: Treatment failure from drug resistance is the primary reason for relapse in acute lymphoblastic leukemia (ALL). Improving outcomes by targeting mechanisms of drug resistance is a potential solution.Patients and Methods: We report results investigating the epigenetic modulators decitabine and vorinostat with vincristine, dexamethasone, mitoxantrone, and PEG-asparaginase for pediatric patients with relapsed or refractory B-cell ALL (B-ALL). Twentythree patients, median age 12 years (range, 1-21) were treated in this trial.Results: The most common grade 3-4 toxicities included hypokalemia (65%), anemia (78%), febrile neutropenia (57%), hypophosphatemia (43%), leukopenia (61%), hyperbilirubinemia (39%), thrombocytopenia (87%), neutropenia (91%), and hypocalcemia (39%). Three subjects experienced dose-limiting toxicities, which included cholestasis, steatosis, and hyperbilirubinemia (n ¼ 1); seizure, somnolence, and delirium (n ¼ 1); and pneumonitis, hypoxia, and hyperbilirubinemia (n ¼ 1). Infectious complications were common with 17 of 23 (74%) subjects experiencing grade ≥3 infections including invasive fungal infections in 35% (8/23). Nine subjects (39%) achieved a complete response (CR þ CR without platelet recovery þ CR without neutrophil recovery) and five had stable disease (22%). Nine (39%) subjects were not evaluable for response, primarily due to treatment-related toxicities. Correlative pharmacodynamics demonstrated potent in vivo modulation of epigenetic marks, and modulation of biologic pathways associated with functional antileukemic effects.Conclusions: Despite encouraging response rates and pharmacodynamics, the combination of decitabine and vorinostat on this intensive chemotherapy backbone was determined not feasible in B-ALL due to the high incidence of significant infectious toxicities. This study is registered at http://www. clinicaltrials.gov as NCT01483690.
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