The role of PACT in the RNA silencing pathway

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.     

Adipose Tissue-specific Inhibition of Hypoxia-inducible Factor 1α Induces Obesity and Glucose Intolerance by Impeding Energy Expenditure in Mice

Hypoxia in adipose tissue has been postulated as a possible contributor to obesity-related chronic inflammation, insulin resistance, and metabolic dysfunction. HIF1α (hypoxia-inducible factor 1α), a master signal mediator of hypoxia response, is elevated in obese adipose tissue. However, the role of HIF1α in obesity-related pathologies remains to be determined. Here we show that transgenic mice with adipose tissue-selective expression of a dominant negative version of HIF1α developed more severe obesity and were more susceptible to high fat diet-induced glucose intolerance and insulin resistance compared with their wild type littermates. Obesity in the transgenic mice was attributed to impaired energy expenditure and reduced thermogenesis. Histological examination of interscapular brown adipose tissue (BAT) in the transgenic mice demonstrated a markedly increased size of lipid droplets and decreased mitochondrial density in adipocytes, a phenotype similar to that in white adipose tissue. These changes in BAT of the transgenic mice were accompanied by decreased mitochondrial biogenesis and reduced expression of key thermogenic genes. In the transgenic mice, angiogenesis in BAT was decreased but was little affected in white adipose tissue. These findings support an indispensable role of HIF1α in maintaining the thermogenic functions of BAT, possibly through promoting angiogenesis and mitochondrial biogenesis in this tissue.     

FGFR1 is essential for N-acetyl-seryl-aspartyl-lysyl-proline regulation of mitochondrial dynamics by upregulating microRNA let-7b-5p

Fibroblast growth factor receptor (FGFR) 1 plays a key role in endothelial homeostasis by inducing microRNA (miR) let-7. Our previous paper showed that anti-fibrotic effects of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) were associated with restoring diabetes-suppressed expression of FGFR1 and miR let-7, the key contributor of mitochondrial biogenesis, which is regulated by mitochondrial membrane GTPase proteins (MFN2 and OPA1). Here, we found that the FGFR1 signaling pathway was critical for AcSDKP in maintaining endothelial mitochondrial biogenesis through induction of miR let-7b-5p. In endothelial cells, AcSDKP restored the triple cytokines (TGF-β2, interleukin-1β, tumor necrosis factor-α)-suppressed miR let-7b-5p and protein levels of the mitochondrial membrane GTPase. This effect of AcSDKP was lost with either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1-treated cells. Similarly, AcSDKP had no effect on the miR let-7b-5p inhibitor-suppressed GTPase levels in endothelial cells. In addition, a miR let-7b-5p mimic restored the levels of FRS2 siRNA-reduced GTPases in endothelial cells. These findings were also confirmed using MitoTracker Green and an immunofluorescence assay. Our results demonstrated that the AcSDKP-FGFR1 signaling pathway is critical for maintaining mitochondrial dynamics by control of miR let-7b-5p in endothelial cells.     

siRNA repositioning for guide strand selection by human Dicer complexes

The human ribonuclease Dicer and its double-stranded RNA (dsRNA)-binding protein (dsRBP) partners TRBP and PACT play important roles in the biogenesis of regulatory RNAs. Following dicing, one dsRNA product strand is preferentially assembled into an RNA-induced silencing complex (RISC). The mechanism of strand selection in humans and the possible role of Dicer in this process remain unclear. Here we demonstrate that dsRNAs undergo significant repositioning within Dicer complexes following dicing. This repositioning enables directional binding of RNA duplexes, thereby biasing their orientation for guide strand selection according to the thermodynamic properties of the helix. Our findings indicate that Dicer is itself capable of sensing siRNA thermodynamic asymmetry regardless of the dsRBP to which it is bound. These results support a model in which Dicer employs two distinct RNA-binding sites-one for dsRNA processing and the other for sensing of siRNA thermodynamic asymmetry-during RISC loading in humans.     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3' UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2' O-methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)-mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3' UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia.     

Trib1 deficiency causes brown adipose respiratory chain depletion and mitochondrial disorder

Tribbles homolog 1 (TRIB1) belongs to the Tribbles family of pseudokinases, which plays a key role in tumorigenesis and inflammation. Although genome-wide analysis shows that TRIB1 expression is highly correlated with blood lipid levels, the relationship between TRIB1 and adipose tissue metabolism remains unclear. Accordingly, the aim of the present study was to explore the role of TRIB1 on mitochondrial function in the brown adipose tissue (BAT). Trib1-knockout mice were established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The metabolic function of the BAT was induced by a β3-adrenoceptor agonist and the energy metabolism function of mitochondria in the BAT of mice was evaluated. Trib1-knockout mice exhibited obesity and impaired BAT thermogenesis. In particular, Trib1 knockout reduced the ability of the BAT to maintain body temperature, inhibited β3-adrenoceptor agonist-induced thermogenesis, and accelerated lipid accumulation in the liver and adipose tissues. In addition, Trib1 knockout reduced mitochondrial respiratory chain complex III activity, produced an imbalance between mitochondrial fusion and fission, caused mitochondrial structural damage and dysfunction, and affected heat production and lipid metabolism in the BAT. Conversely, overexpression of Trib1 in 3T3-L1 adipocytes increased the number of mitochondria and improved respiratory function. These findings support the role of Trib1 in regulating the mitochondrial respiratory chain and mitochondrial dynamics by affecting mitochondrial function and thermogenesis in the BAT.Subject terms: Energy metabolism, Obesity     

Effects of ovariectomy and 17-β estradiol replacement on rat brown adipose tissue mitochondrial function

Taking into account the sexual dimorphism previously reported regarding mitochondrial function and biogenesis in brown adipose tissue, the aim of the present study was to go further into these differences by investigating the effect of ovariectomy and 17-β estradiol (E2) replacement on brown adipose tissue mitochondrial function. In this study, fourteen-week-old control female and ovariectomized female Wistar rats were used. Rats were ovariectomized at 5 weeks of age and were treated every 2 days with placebo (OVX group) or E2 (10 μg/kg) (OVX+E2 group) for 4 weeks before sacrifice. We studied the levels of oxidative capacity, antioxidant defence and oxidative damage markers in brown adipose tissue. Moreover, the levels of key elements of mitochondrial biogenesis as well as UCP1 protein levels, as an index of mitochondrial thermogenic capacity, were also determined. In response to ovariectomy, mitochondrial proliferation increased, resulting in less functional mitochondria, since oxidative capacity and antioxidant defences decreased. Although E2 supplementation was able to restore the serum levels of E2 shown by control rats, the treatment reverted the effects of the ovariectomy only in part, and oxidative and antioxidant capacities in OVX+E2 rats did not reach the levels shown by control females. Taking these results into account, we suggest that ovarian hormones are responsible, at least in part, for the sexual dimorphism in BAT mitochondrial function. However, other signals produced by ovary, rather than E2, would play an important role in the control of mitochondrial function in BAT.     

Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

PITPNC1 promotes the thermogenesis of brown adipose tissue under acute cold exposure

Brown adipose tissue(BAT) plays an essential role in non-shivering thermogenesis. The phosphatidylinositol transfer protein,cytoplasmic 1(PITPNC1) is identified as a lipid transporter that reciprocally transfers phospholipids between intracellular membrane structures. However, the physiological significance of PITPNC1 and its regulatory mechanism remain unclear. Here,we demonstrate that PITPNC1 is a key player in thermogenesis of BAT. While Pitpnc1^-/-mice do not differ with wildtype m...     

Lipocalin 2 Regulates Brown Fat Activation via a Nonadrenergic Activation Mechanism

In this study, we report that lipocalin 2 (Lcn2), a recently characterized adipokine/cytokine, is a novel regulator of brown adipose tissue (BAT) activation by modulating the adrenergic independent p38 MAPK-PGC-1α-UCP1 pathway. Global Lcn2 knock-out (Lcn2^−/−) mice have defective BAT thermogenic activation caused by cold stimulation and decreased BAT activity under high fat diet-induced obesity. Nevertheless, Lcn2^−/− mice maintain normal sympathetic nervous system activation as evidenced by normal catecholamine release and lipolytic activity in response to cold stimulation. Further studies showed that Lcn2 deficiency impairs peroxisomal and mitochondrial oxidation of lipids and attenuates cold-induced Pgc1a and Ucp1 expression and p38 MAPK phosphorylation in BAT. Moreover, in vitro studies showed that Lcn2 deficiency reduces the thermogenic activity of brown adipocytes. Lcn2^−/− differentiated brown adipocytes have significantly decreased expression levels of brown fat markers, decreased p38 MAPK phosphorylation, and decreased mitochondrial oxidation capacity. However, Lcn2^−/− brown adipocytes have normal norepinephrine-stimulated p38 MAPK and hormone-sensitive lipase phosphorylation and Pgc1a and Ucp1 expression, suggesting an intact β-adrenergic signaling activation. More intriguingly, recombinant Lcn2 was able to significantly stimulate p38 MAPK phosphorylation in brown adipocytes. Activating peroxisome proliferator-activated receptor γ, a downstream effector of PGC-1α, by thiazolidinedione administration fully reverses the BAT function of Lcn2^−/− mice. Our findings provide evidence for the novel role Lcn2 plays in oxidative metabolism and BAT activation via an adrenergic independent mechanism.