The role of caloric load and mitochondrial homeostasis in the regulation of the NLRP3 inflammasome

Sterile inflammation is a cornerstone of immune activation in obesity and type 2 Diabetes Mellitus. The molecular underpinnings of this inflammation include nutrient excess-mediated activation of the innate immune NLRP3 inflammasome. At the same time, disruption of mitochondrial integrity is emerging as an integral control node in NLRP3 inflammasome activation and is also associated with caloric overload conditions including obesity and diabetes. Conversely, caloric restriction and fasting mimetic interventions alleviate these caloric excess-linked diseases and reduce inflammation and the NLRP3 inflammasome. The objective of this review is to integrate the findings linking mitochondrial integrity to the activation of the NLRP3 inflammasome and to evaluate how caloric restriction or caloric restriction mimetic compounds may play a role in attenuating the NLRP3 inflammasome and sterile inflammation.     

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H⁺ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS^?/? conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS^?/?) or with compound heterozygous loss-of-function mutations (mutCTNS^?/?). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS^?/? ciPTEC by 56%?±?10.5, P?<?0.0001; mutCTNS^?/? by 26%?±?4.3, P?<?0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.     

E3 ubiquitin ligase RNF13 involves spatial learning and assembly of the SNARE complex

Changes in the structure and number of synapses modulate learning, memory and cognitive disorders. Ubiquitin-mediated protein modification is a key mechanism for regulating synaptic activity, though the precise control of this process remains poorly understood. RING finger protein 13 (RNF13) is a recently identified E3 ubiquitin ligase, and its in vivo function remains completely unknown. We show here that genetic deletion of RNF13 in mice leads to a significant deficit in spatial learning as determined by the Morris water maze test and Y-maze learning test. At the ultrastructral level, the synaptic vesicle density was decreased and the area of the active zone was increased at hippocampal synapses of RNF13-null mice compared with those of wild-type littermates. We found no change in the levels of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex proteins in the hippocampus of RNF13-null mice, but impaired SNARE complex assembly. RNF13 directly interacted with snapin, a SNAP-25-interacting protein. Interestingly, snapin was ubiquitinated by RNF13 via the lysine-29 conjugated polyubiquitin chain, which in turn promoted the association of snapin with SNAP-25. Consistently, we found an attenuated interaction between snapin and SNAP-25 in the RNF13-null mice. Therefore, these results suggest that RNF13 is involved in the regulation of the SNARE complex, which thereby controls synaptic function.     

Megalin mediates plasma membrane to mitochondria cross-talk and regulates mitochondrial metabolism

Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-β protein from the brain. Megalin mutations underlie the pathogenesis of Donnai–Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-β to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.     

SIRT1 modulates MAPK pathways in ischemic–reperfused cardiomyocytes

SIRT1, an ubiquitous NAD(+)-dependent deacetylase that plays a role in biological processes such as longevity and stress response, is significantly activated in response to reactive oxygen species (ROS) production. Resveratrol (Resv), an important activator of SIRT1, has been shown to exert major health benefits in diseases associated with oxidative stress. In ischemia-reperfusion (IR) injury, a major role has been attributed to the mitogen-activated protein kinase (MAPK) pathway, which is upregulated in response to a variety of stress stimuli, including oxidative stress. In neonatal rat ventricular cardiomyocytes subjected to simulated IR, the effect of Resv-induced SIRT1 activation and the relationships with the MAPK pathway were investigated. Resv-induced SIRT1 overexpression protected cardiomyocytes from oxidative injury, mitochondrial dysfunction, and cell death induced by IR. For the first time, we demonstrate that SIRT1 overexpression positively affects the MAPK pathway-via Akt/ASK1 signaling-by reducing p38 and JNK phosphorylation and increasing ERK phosphorylation. These results reveal a new protective mechanism elicited by Resv-induced SIRT1 activation in IR tissues and suggest novel potential therapeutic targets to manage IR-induced cardiac dysfunction.     

Degradation of the mitochondrial complex I assembly factor TMEM126B under chronic hypoxia

Cell stress such as hypoxia elicits adaptive responses, also on the level of mitochondria, and in part is mediated by the hypoxia-inducible factor (HIF) 1α. Adaptation of mitochondria towards acute hypoxic conditions is reasonably well understood, while regulatory mechanisms, especially of respiratory chain assembly factors, under chronic hypoxia remains elusive. One of these assembly factors is transmembrane protein 126B (TMEM126B). This protein is part of the mitochondrial complex I assembly machinery. We identified changes in complex I abundance under chronic hypoxia, in association with impaired substrate-specific mitochondrial respiration. Complexome profiling of isolated mitochondria of the human leukemia monocytic cell line THP-1 revealed HIF-1α-dependent deficits in complex I assembly and mitochondrial complex I assembly complex (MCIA) abundance. Of all mitochondrial MCIA members, we proved a selective HIF-1-dependent decrease of TMEM126B under chronic hypoxia. Mechanistically, HIF-1α induces the E3-ubiquitin ligase F-box/WD repeat-containing protein 1A (β-TrCP1), which in turn facilitates the proteolytic degradation of TMEM126B. Attenuating a functional complex I assembly appears critical for cellular adaptation towards chronic hypoxia and is linked to destruction of the mitochondrial assembly factor TMEM126B.     

Alpha-synuclein elicits glucose uptake and utilization in adipocytes through the Gab1/PI3K/Akt transduction pathway

Insulin is the main glucoregulator that promotes the uptake of glucose by tissues and the subsequent utilization of glucose as an energy source. In this paper, we describe a novel glucoregulator, the alpha-synuclein (SNCA) protein, that has previously been linked to Parkinson’s disease. Treatment with recombinant SNCA promotes glucose uptake in vitro in preadipocytes and in vivo in the adipose tissues and skeletal muscles of mice through the LPAR2/Gab1/PI3K/Akt pathway; these effects occur independently of the insulin receptor. This function of SNCA represents a new mechanistic insight that creates novel avenues of research with respect to the process of glucose regulation.     

Mitochondrial network remodeling: an important feature of myogenesis and skeletal muscle regeneration

The remodeling of the mitochondrial network is a critical process in maintaining cellular homeostasis and is intimately related to mitochondrial function. The interplay between the formation of new mitochondria (biogenesis) and the removal of damaged mitochondria (mitophagy) provide a means for the repopulation of the mitochondrial network. Additionally, mitochondrial fission and fusion serve as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been characterised in multiple tissue- and cell-types, and under various conditions. In skeletal muscle, the robust remodeling of the mitochondrial network is observed, particularly after injury where large portions of the tissue/cell structures are damaged. The significance of mitochondrial remodeling in regulating skeletal muscle regeneration has been widely studied, with alterations in mitochondrial remodeling processes leading to incomplete regeneration and impaired skeletal muscle function. Needless to say, important questions related to mitochondrial remodeling and skeletal muscle regeneration still remain unanswered and require further investigation. Therefore, this review will discuss the known molecular mechanisms of mitochondrial network remodeling, as well as integrate these mechanisms and discuss their relevance in myogenesis and regenerating skeletal muscle.

     

Sphingolipid metabolism and its role in the skeletal tissues

The regulators affecting skeletal tissue formation and its maintenance include a wide array of molecules with very diverse functions. More recently, sphingolipids have been added to this growing list of regulatory molecules in the skeletal tissues. Sphingolipids are integral parts of various lipid membranes present in the cells and organelles. For a long time, these macromolecules were considered as inert structural elements. This view, however, has radically changed in recent years as sphingolipids are now recognized as important second messengers for signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death. In the current review, we discuss the available data showing the roles of various sphingolipids in three different skeletal cell types—chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We provide an overview of the biology of sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 is localized in the plasma membrane and has been shown to cleave sphingomyelin to generate ceramide, a bioactive lipid second messenger, and phosphocholine, an essential nutrient. SMPD3 deficiency in mice impairs the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities. A detailed understanding of SMPD3 function may provide a novel insight on the role of sphingolipids in the skeletal tissues.     

Heterogeneous bioenergetic behaviour of subsarcolemmal and intermyofibrillar mitochondria in fed and fasted rats

This study was designed to examine energetic behaviour of skeletal muscle subsarcolemmal and intermyofibrillar mitochondrial populations. The data show that subsarcolemmal mitochondria exhibited a lower degree of coupling and efficiency than intermyofibrillar ones, and can therefore be considered less efficient at producing ATP. In addition, subsarcolemmal mitochondria showed an increased sensitivity to palmitate-induced uncoupling, in line with high adenine nucleotide translocator content and decreased oxidative damage. We then determined the effect of 24 h fasting on energetic characteristics of skeletal muscle mitochondrial populations. We found that fasting enhanced proton leak and decreased the degree of coupling and efficiency, both in the absence and in the presence of palmitate only in subsarcolemmal mitochondria. Moreover, this mitochondrial population showed lower oxidative damage, probably due to a counter-regulatory mechanism mediated by uncoupling protein 3. Subsarcolemmal and intermyofibrillar mitochondria appear to exhibit different energetic characteristics and can be differently affected by physiological stimuli. Received 28 September 2005; received after revision 9 November 2005; accepted 28 November 2005     

Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopolysaccharide ofPorphyromonas gingivalis

Porphyromonas gingivalis 381 lipopolysaccharide (LPS) definitely exhibited mitogenic activity in purified B-cells, separated from spleens of LPS-responsive C3H/HeN mice and LPS-non-responsive C3H/HeJ mice by using a magnetic cell sorting system. The mitogenic activity induced byP. gingivalis LPS was incompletely inhibited by polymyxin B.P. gingivalis LPS also induced a higher production of interleukin-6 (IL-6) in splenic B-cells of C3H/HeN mice as compared withEscherichia coli LPS. Furthermore,P. gingivalis LPS, but notE. coli LPS, induced definite IL-6 production in C3H/HeJ mice.P. gingivalis LPS increased tyrosine, serine/threonine phosphorylation of proteins with various major induced bands in splenic B-cells of both C3H/HeN and C3H/HeJ mice. Additionally, radioiodinatedP. gingivalis LPS, similarly toE. coli LPS, bound to a 73-kDa protein on C3H/HeJ as well as C3H/HeN B-cells. ThusP. gingivalis LPS may activate B-cells of C3H/HeJ as well as C3H/HeN mice via the LPS-specific binding protein on the cells.     

Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.     

Tumor suppressor role of protein 4.1B/DAL-1

Protein 4.1B/DAL-1 is a membrane skeletal protein that belongs to the protein 4.1 family. Protein 4.1B/DAL-1 is localized to sites of cell–cell contact and functions as an adapter protein, linking the plasma membrane to the cytoskeleton or associated cytoplasmic signaling effectors and facilitating their activities in various pathways. Protein 4.1B/DAL-1 is involved in various cytoskeleton-associated processes, such as cell motility and adhesion. Moreover, protein 4.1B/DAL-1 also plays a regulatory role in cell growth, differentiation, and the establishment of epithelial-like cell structures. Protein 4.1B/DAL-1 is normally expressed in multiple human tissues, but loss of its expression or prominent down-regulation of its expression is frequently observed in corresponding tumor tissues and tumor cell lines, suggesting that protein 4.1B/DAL-1 is involved in the molecular pathogenesis of these tumors and acts as a potential tumor suppressor. This review will focus on the structure of protein 4.1B/DAL-1, 4.1B/DAL-1-interacting molecules, 4.1B/DAL-1 inactivation and tumor progression, and anti-tumor activity of the 4.1B/DAL-1.     

Maternal vitamin A restriction alters biochemical development of the brain in rats

Summary The biochemical development of the fetal brain in relation to maternal vitamin A restriction was studied in rats. The vitamin A status of pregnant rats was varied by supplying low, medium and adequate amounts (6, 40, and 100 g retinol/day/kg body weight, respectively) of vitamin A during pregnancy and suckling. The maternal vitamin A restriction caused an altered brain development in terms of tissue weight, DNA, RNA and protein levels, and biosynthesis of DNA and protein from [³H]-thymidine and [³H]-leucine, respectively. A dose-dependent effect of maternal vitamin A restriction on the metabolism of DNA, RNA and protein was noticed in the developing fetal brain of rats.     

Syncoilin, an intermediate filament-like protein linked to the dystrophin associated protein complex in skeletal muscle

Syncoilin is a member of the intermediate filament protein family, highly expressed in skeletal and cardiac muscle. Syncoilin binds α-dystrobrevin, a component of the dystrophin associated protein complex (DAPC) located at the muscle cell membrane, and desmin, a muscle-specific intermediate filament protein, thus providing a link between the DAPC and the muscle intermediate filament network. This link may be important for muscle integrity and force transduction during contraction, a theory that is supported by the reduced force-generating capacity of muscles from syncoilin-null mice. Additionally, syncoilin is found at increased levels in the regenerating muscle fibres of patients with muscular dystrophies and mouse models of muscle disease. Therefore, syncoilin may be important for muscle regeneration in response to injury. The aims of this article are to review current knowledge about syncoilin and to discuss its possible functions in skeletal muscle. Received 21 May 2008; received after revision 10 July 2008; accepted 18 July 2008