Glucose transport by epithelia prepared from harvested enterocytes

Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer ¹⁴C d-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell–cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5′-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of ¹⁴C d-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.     

No differential effects of divergent isocaloric supplements on signaling for muscle protein turnover during recovery from muscle-damaging eccentric exercise

Unaccustomed high-intensity eccentric exercise (ECC) can provoke muscle damage including several days of muscle force loss. Post-exercise dietary supplementation may provide a strategy to accelerate rate of force regain by affecting mechanisms related to muscle protein turnover. The aim of the current study was to investigate if protein signaling mechanisms involved in muscle protein turnover would be differentially affected by supplementation with either whey protein hydrolysate and carbohydrate (WPH+CHO) versus isocaloric carbohydrate (CHO) after muscle-damaging ECC. Twenty-four young healthy participants received either WPH+CHO (n = 12) or CHO supplements (n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to, at 3 h and at 24, 48, 96 and/or 168 h post-exercise, muscle strength, muscle soreness, and Akt-mTOR and FOXO signaling proteins, were measured in an ECC exercising leg and in the contralateral non-exercise control leg (CON). After ECC, muscle force decreased by 23–27 % at 24 h post-exercise, which was followed by gradual, although not full recovery at 168 h post-exercise, with no differences between supplement groups. Phosphorylation of mTOR, p70S6K and rpS6 increased and phosphorylation of FOXO1 and FOXO3 decreased in the ECC leg, with no differences between supplement groups. Phosphorylation changes were also observed for rpS6, FOXO1 and FOXO3a in the CON leg, suggesting occurrence of remote tissue effects. In conclusion, divergent dietary supplementation types did not produce differences in signaling for muscle turnover during recovery from muscle-damaging exercise.     

Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.     

Hydrophilic carotenoids: surface properties and aggregation behavior of a highly unsaturated carotenoid lysophospholipid

The water dispersibility of a hydrophobic carotenoid has been greatly enhanced by using it as the acyl part in the synthesis of a highly unsaturated lysophospholipid. Dynamic light scattering has revealed the formation of stable aggregates with an average hydrodynamic radius of a few nanometers, and absorption spectra show that the aggregates can withstand the addition of ethanol or acetonitrile until the volume fraction of water falls below 70 and 62%, respectively. The properties of the carotenoid phospholipids have been characterized by determining surface tension, critical micelle concentration, surface concentration, molecular area, free energy of adsorption and micellation, adsorption-micellar energy relationship, and equilibrium constants.     

Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways

Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.Virus recognition is essential for activation of innate antiviral immune defense and the subsequent induction of acquired immunity. Conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), are recognized by pattern recognition receptors (PRRs). Virus-recognizing PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and a number of intracellular DNA receptors. Several TLRs have been attributed roles in the recognition of virus. TLR2 and TLR4 recognize viral surface structures (3, 6, 18, 31), TLR3 recognizes double-stranded RNA (dsRNA) (2), and TLR7/8 and TLR9 function as signaling receptors for viral single-stranded RNA (ssRNA) (8, 11, 21) and CpG DNA (12, 20), respectively.Within the cell, cytoplasmic RLRs RIG-I and MDA5 both recognize accumulation of virus-derived dsRNA; in addition, RIG-I recognizes 5′-triphosphated RNA (14, 27, 39, 40). In addition to the RLRs, a number of receptors recognize foreign DNA. Presently, three DNA receptors have been identified: Z-DNA binding protein 1 (ZBP-1, or DAI) (36) and RNA polymerase III (Pol III) (1, 4) both mediate interferon (IFN) and cytokine production, whereas the AIM2 inflammasome is involved in caspase 1 activation in response to cytoplasmic dsDNA (13).Herpes simplex virus type 1 (HSV-1) and HSV-2 are two closely related human DNA viruses associated with a number of serious diseases, including orofacial infections, encephalitis, and genital infections (34). Macrophages play an important role in the first line of defense against viral infection via production of IFNs, cytokines, and chemokines that regulate the progress of the virus infection and activate and support appropriate defense mechanisms (9, 10, 24).TLR2, TLR3, and TLR9 have been identified as mediators of proinflammatory cytokine production during HSV infections. TLR2 mediates an overzealous inflammatory cytokine response following HSV-1 infection in mice, promoting mononuclear cell infiltration of the brain and development of encephalitis (18). TLR3 mediates type I and III IFN production in human fibroblasts (41). TLR9 recognizes genomic DNA from HSV-1 and HSV-2 in murine plasmacytoid dendritic cells (DCs) (17, 20) and mediates tumor necrosis factor alpha (TNF-α) and CCL5 production in murine macrophages (22). Both TLR2 and TLR9 mediate recognition of HSV and cytokine production in murine conventional DCs (35). HSV is recognized by an RLR/MAVS-dependent mechanism in murine macrophages and mouse embryonic fibroblasts (MEFs) (5, 29, 30). Recent data suggest that RNA Pol III mediates IFN production following HSV-1 infection and transfection with HSV-1 DNA in macrophage-like RAW 264.7 cells (4). Finally, murine L929 fibroblast-like cells are moderately inhibited in their ability to produce IFN after HSV-1 infection when ZBP-1 is knocked down (19, 36). Thus, several PRRs have been reported to recognize HSV-1 in murine cells and different cell lines, but the pathways responsible for sensing this virus in human primary macrophages and their impact on cytokine expression have not previously been described.In this work, we investigate the recognition pathways underlying HSV-induced cytokine and chemokine expression in human primary macrophages. We demonstrate that HSV-1-induced IFN and cytokine expression is independent of TLR2 and TLR9 but highly dependent on virus replication and intracellular nucleotide recognition systems. Specifically, induction of IFNs is dependent on MAVS and MDA5, whereas TNF-α is induced by a novel intracellular nucleotide-sensing system.     

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P₂] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by 20% in modestly, and 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P₂ analogue or a PtdIns(4,5)P₂-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P₂. It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization. cell swelling; kinase; phospholipid phosphatidylinositol-(4,5)-bisphosphate; cytoskeleton     

Regulation of the Pleuronectes americanus Na+/H+ exchanger by osmotic shrinkage, β-adrenergic stimuli, and inhibition of Ser/Thr protein phosphatases

The ubiquitous Na⁺/H⁺ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, β-adrenergic stimuli, and the Ser/Thr protein phosphatase PP1 and PP2A inhibitor calyculin A (CLA) (Pedersen et al. [2003] Am. J. Physiol. 284, C1561–C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by osmotic cell swelling. Activation by the β-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic shrinkage or CLA. IP-mediated, but neither shrinkage-nor CLA-mediated paNHE1 activation were associated with an increase in cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A (PKA) inhibitor H89 (10μM), wherease shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected by inhibitors of protein kinase C (calphostin C, 5 μM) and protein kinase G (KT5823, 10 μM) as well as of myosin light chain kinase (ML-7, 10 μM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially convergent signaling pathways, whereas activation of paNHE1 by β-adrenergic stimuli is mediated by a separate pathway.     

Mice with targeted disruption of the acyl-CoA binding protein display attenuated urine concentrating ability and diminished renal aquaporin-3 abundance

The acyl-CoA binding protein (ACBP) is a small intracellular protein that specifically binds and transports medium to long-chain acyl-CoA esters. Previous studies have shown that ACBP is ubiquitously expressed but found at particularly high levels in lipogenic cell types as well as in many epithelial cells. Here we show that ACBP is widely expressed in human and mouse kidney epithelium, with the highest expression in the proximal convoluted tubules. To elucidate the role of ACBP in the renal epithelium, mice with targeted disruption of the ACBP gene (ACBP(-/-)) were used to study water and NaCl balance as well as urine concentrating ability in metabolic cages. Food intake and urinary excretion of Na(+) and K(+) did not differ between ACBP(-/-) and (+/+) mice. Interestingly, however, water intake and diuresis were significantly higher at baseline in ACBP(-/-) mice compared with that of (+/+) mice. Subsequent to 20-h water deprivation, ACBP(-/-) mice exhibited increased diuresis, reduced urine osmolality, elevated hematocrit, and higher relative weight loss compared with (+/+) mice. There were no significant differences in plasma concentrations of renin, corticosterone, and aldosterone between mice of the two genotypes. After water deprivation, renal medullary interstitial fluid osmolality and concentrations of Na(+), K(+), and urea did not differ between genotypes and cAMP excretion was similar. Renal aquaporin-1 (AQP1), -2, and -4 protein abundances did not differ between water-deprived (+/+) and ACBP(-/-) mice; however, ACBP(-/-) mice displayed increased apical targeting of pS256-AQP2. AQP3 abundance was lower in ACBP(-/-) mice than in (+/+) control animals. Thus we conclude that ACBP is necessary for intact urine concentrating ability. Our data suggest that the deficiency in urine concentrating ability in the ACBP(-/-) may be caused by reduced AQP3, leading to impaired efflux over the basolateral membrane of the collecting duct.