Upon infection, our immune cells produce a small protein called interferon, which in turn signals a protective response through a series of biochemical reactions that involves lowering the cells' ability to make cholesterol by targeting a gene essential for controlling the pathway for cholesterol metabolism.
Sterol regulatory element binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic micro-RNA locus that is activated directly by SREBP-2. Two of the encoded miRs, miR-182 and miR-96, negatively regulate expression of Fbxw7 and Insig-2 respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miR pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a new mechanism for regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRs that are expressed from the same SREBP-2 regulated miR locus and each targets a different protein of the multi-step pathway that regulates SREBP function. These studies reveal a miR “operon” analogous to the classic model for genetic control in bacterial regulatory systems.
The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10 6 50% tissue culture infective doses (TCID 50 ) of SHRV/ ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10 5 TCID 50 of SHRV/ ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.
ObjectiveNicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding.MethodsInsulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal).ResultsNAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice.ConclusionsOur studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance.
SignificanceThere is a growing appreciation for a fundamental connection between lipid metabolism and the innate immune response. Phagocytosis is a key macrophage innate immune response to pathogen exposure, and cytoplasmic membrane expansion is required to surround and capture the target pathogen prior to internalization. Sterol regulatory element binding proteins (SREBPs) are gene regulatory factors that sense the intracellular lipid environment and modulate key genes that drive fatty acid and cholesterol synthesis to maintain lipid homeostasis. In this study, we show that, in mutant cells that lack a key SREBP isoform, phagocytosis is impaired, and we track the defect to altered lipid composition of membrane phospholipids that results in decreased interaction between membrane lipid rafts and the actin cytoskeletal network.
SUMMARY
Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2-dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice; both systems of elevated SREBP-1c driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition on active SREBPs. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions.
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