Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models.

Human liver models that are three-dimensional (3D) in architecture are indispensable for compound metabolism/toxicity screening, to model liver diseases for drug discovery, and for cell-based therapies; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months. Therefore, here we determined how microscale 3D collagen I presentation and fibroblast interaction affect the longevity of PHHs. High-throughput droplet microfluidics was utilized to generate reproducibly sized (∼300-µm diameter) microtissues containing PHHs encapsulated in collagen I ± supportive fibroblasts, namely, 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as controls. Hepatic functions and gene expression were subsequently measured for up to 6 weeks. We found that microtissues placed within multiwell plates rescued PHH functions at 2- to 30-fold higher levels than spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Importantly, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically relevant manner to drug-mediated cytochrome P450 induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display high hepatic functions for 6+ weeks and are useful for assessing drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find utility for modeling liver diseases and as building blocks for cell-based therapies.

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles.

We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern. Graphical Abstract ᅟ.

In Vivo Confocal Microscopy 1 Year after Autologous Cultured Limbal Stem Cell Grafts.

PURPOSE: To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation. DESIGN: Prospective, interventional, noncomparative, masked case series. PARTICIPANTS: Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye). METHODS: Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patient's injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohen's κ coefficient. MAIN OUTCOME MEASURES: Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface). RESULTS: We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea. CONCLUSIONS: Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft.

Cultivation and phenotypic characterization of rabbit epithelial cells expanded ex vivo from fresh and cryopreserved limbal and oral mucosal explants.

PURPOSE: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies. MATERIALS AND METHODS: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin-eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco's Modified Eagle's Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures. RESULTS: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture. CONCLUSIONS: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells.

Microfabrication of a platform to measure and manipulate the mechanics of engineered microtissues.

Engineered tissues can be used to understand fundamental features of biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged tissue in vivo. However, a key limitation is an inability to test the wide range of parameters that might impact the engineered tissue in a high-throughput manner and in an environment that mimics the three-dimensional (3D) native architecture. We developed a microfabricated platform to generate arrays of microtissues embedded within 3D micropatterned matrices. Microcantilevers simultaneously constrain microtissue formation and report forces generated by the microtissues in real time, opening the possibility to use high-throughput, low-volume screening for studies on engineered tissues. Thanks to the micrometer scale of the microtissues, this platform is also suitable for high-throughput monitoring of drug-induced effect on architecture and contractility in engineered tissues. Moreover, independent variations of the mechanical stiffness of the cantilevers and collagen matrix allow the measurement and manipulation of the mechanics of the microtissues. Thus, our approach will likely provide valuable opportunities to elucidate how biomechanical, electrical, biochemical, and genetic/epigenetic cues modulate the formation and maturation of 3D engineered tissues. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated tissue gauges.

Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency.

PURPOSE: Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation. METHODS: Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63α and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas. RESULTS: Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface. CONCLUSIONS: Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

Geometry as a factor for tissue growth: towards shape optimization of tissue engineering scaffolds.

Scaffolds for tissue engineering are usually designed to support cell viability with large adhesion surfaces and high permeability to nutrients and oxygen. Recent experiments support the idea that, in addition to surface roughness, elasticity and chemistry, the macroscopic geometry of the substrate also contributes to control the kinetics of tissue deposition. In this study, a previously proposed model for the behavior of osteoblasts on curved surfaces is used to predict the growth of bone matrix tissue in pores of different shapes. These predictions are compared to in vitro experiments with MC3T3-E1 pre-osteoblast cells cultivated in two-millimeter thick hydroxyapatite plates containing prismatic pores with square- or cross-shaped sections. The amount and shape of the tissue formed in the pores measured by phase contrast microscopy confirms the predictions of the model. In cross-shaped pores, the initial overall tissue deposition is twice as fast as in square-shaped pores. These results suggest that the optimization of pore shapes may improve the speed of ingrowth of bone tissue into porous scaffolds.

Measurement of mechanical tractions exerted by cells in three-dimensional matrices.

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.

Type of cell death induced by seven metals in cultured mouse osteoblastic cells.

The use of dental metal alloys in the daily clinic makes it necessary to evaluate the cytotoxicity of eluted metal components against oral cells. However, the cytotoxic mechanism and the type of cell death induced by dental metals in osteoblasts have not been well characterized. This study investigated the cytotoxicity of seven metals against the mouse osteoblastic cell line MC3T3-E1. alpha-MEM was used as a culture medium, since this medium provided much superior proliferation of MC3T3-E1 cells over DMEM. Ag (NH(3))(2)F was the most cytotoxic, followed by CuCl>CuCl(2) >CoCl(2), NiCl(2)>FeCl(3) and FeCl(2) (least toxic). None of the metals showed any apparent growth stimulating effect (so-called 'hormesis') at lower concentrations. A time course study demonstrated that two hours of contact between oral cells and Ag (NH(3))(2)F, CuCl, CoCl(2) or NiCl(2) induced irreversible cell death. Contact with these metals induced a smear pattern of DNA fragmentation without activation of caspase-3. Preincubation of MC3T3-E1 cells with either a caspase inhibitor (Z-VAD-FMK) or autophagy inhibitors (3-methyladenine, bafilomycin) failed to rescue them from metal cytotoxicity. These data suggest the induction of necrotic cell death rather than apoptosis and autophagy by metals in this osteoblastic cell line.

Quantification of enhanced osteoblastic adhesion to ultraviolet-treated titanium plate.

Although the advantage of ultraviolet (UV) irradiation of titanium plates for the attachment of osteoblast is known, the details of the experimental conditions have not been described in previous literature. We established optimal conditions of UV irradiation of titanium plate for the adhesion of mouse osteoblast MC3T3-E1 cells. The viable cell number was determined by MTT method. UV irradiation at two different wavelengths (253.7 and 365 nm) enhanced the cell attachment on titanium plate to comparable extents. The optimal UV exposure duration was 20 minutes and prolonged irradiation slightly reduced cell attachment. The attached cells proliferated during 24 hours, accompanied by the enhanced consumption of extracellular glutamine and arginine. The present study supports the previous reports of the efficacy of UV irradiation, and this simple and rapid assay system may be applicable for the study of the interaction of osteoblast and UV-activated titanium plates.

A microfluidic magnetic bead impact generator for physical stimulation of osteoblast cell.

We developed a novel microfluidic cell culture device in which magnetic beads repetitively collide with osteoblast cells, MC3T3-E1, owing to attractive forces generated by pulsed electromagnetic fields and consequently the cells were physically stimulated by bead impacts. Our device consists of an on-chip microelectromagnet and a microfluidic channel which were fabricated by a microelectromechanical system technique. The impact forces and stresses acting on a cell were numerically analyzed and experimentally generated with different sizes of bead (4.5, 7.6 and 8.4 mum) and at various pulse frequencies (60 Hz, 1 kHz and 1 MHz). Cells were synchronized at each specific phase of the cell cycle before stimulation in order to determine the most susceptible phase against bead impacts. The cells were stimulated with different sizes of bead at various pulse frequencies for 1 min at G1, S and G2 phases, respectively, and then counted immediately after one doubling time. The growth rate of cells was highly accelerated when they were stimulated with 4.5 mum beads at G1 phase and a pulse frequency of 1 MHz. Almost all of the cells were viable after stimulation, indicating that our cell stimulator did not cause any cellular damage and is suitable for use in new physical stimulus modalities.

PTHrP 1-141 and 1-86 increase in vitro bone formation.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has anabolic effects in bone, which has led to the clinical use of N-terminal fragments of PTHrP and PTH. Since 10% to 20% of fractures demonstrate healing complications and osteoporosis continues to be a debilitating disease, the development of bone-forming agents is of utmost importance. Due to evidence that regions of PTHrP other than the N-terminus may have bone-forming effects, this study was designed to compare the effects of full-length PTHrP 1-141 to N-terminal PTHrP 1-86 on in vitro bone formation. MATERIALS AND METHODS: MC3T3-E1 pre-osteoblasts were treated once every 6 d for 36 d with 5, 25, and 50 pM of PTHrP 1-141 or 1-86 for 1 or 24 h. Cells were also treated after blocking the N-terminus, the nuclear localization sequence (NLS), and the C-terminus of PTHrP, individually and in combination. Area of mineralization, alkaline phosphatase (ALP), and osteocalcin (OCN) were measured. RESULTS: PTHrP 1-141 and 1-86 increased mineralization after 24-h treatments, but not 1-h. PTHrP 1-141 was more potent than 1-86. Treatment with PTHrP 1-141 for 24-h, but not 1-86, resulted in a concentration-dependent increase in ALP, with no effect after 1-h. Exposure to both peptides for 1- or 24-h induced a concentration-dependent increase in OCN, with 24-h exceeding 1-h. Antibody blocking revealed that the NLS and C-terminus are anabolic. CONCLUSIONS: Both PTHrP 1-141 and 1-86 increased in vitro bone formation; however, PTHrP 1-141 was more effective. The NLS and C-terminus have anabolic effects distinct from the N-terminus. This demonstrates the advantage of PTHrP 1-141 as a skeletal anabolic agent.

Chloride channel ClC-3 promotion of osteogenic differentiation through Runx2.

ClC-3 chloride channel has been speculated to contribute to the acidification of synaptic vesicles and endosomes. However, the biological function of ClC-3 in osteogenesis remains to be determined. In this study, we first analyzed ClC-3 expression in MC3T3-E1 cells and primary mouse osteoblasts and then performed the osteoinductive procedure to determine the effects on gene expression. Subsequently, we transiently transfected ClC-3 cDNA or ClC-3-siRNA into MC3T3-E1 cells to determine the changed phenotype and gene expression. Lastly, we assessed the underlying mechanism responsible for ClC-3-induced osteodifferentiation. We found that ClC-3 mRNA was expressed in primary mouse osteoblasts and MC3T3-E1 cells and induced by using an osteoinductive procedure. We also found that overexpression of ClC-3 contributed to osteodifferentiation, such as increase in the expression of osteogenic markers [alkaline phosphatase (Alp), osteocalcin (Oc), bone sialoprotein (Bsp), osterix (Osx), and runt-related transcription factor 2 (Runx2)], morphological changes, and mineralized nodules in MC3T3-E1 cells. ClC-3 gene silencing suppressed gene expression of these osteogenic markers. Moreover, overexpressed ClC-3 protein co-localized with TGF-beta1 in intracellular organelles, inhibited TGF-beta1 protein expression and induced endosomal acidification. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC-3 in osteodifferentiation and expression of osteogenic markers. The data from the current study suggest that the function of ClC-3 in osteodifferentiation may be through the Runx2 pathway.

Poly(ethylene glycol)-grafted poly(propylene fumarate) networks and parabolic dependence of MC3T3 cell behavior on the network composition.

We present a method to modify poly(propylene fumarate) (PPF), an injectable biomaterial for bone-tissue-engineering applications, by photo-crosslinking it with methoxy poly(ethylene glycol) monoacrylate (mPEGA) at various mPEGA compositions of 0-30%. The bulk properties such as thermal and rheological properties of uncrosslinked mPEGA/PPF blends and the mechanical properties of photo-crosslinked mPEGA/PPF blends were also investigated and correlated with surface characteristics to elaborate on the modulation of mouse MC3T3 cell adhesion, spreading, proliferation and differentiation through controlled physicochemical properties. Unlike PPF crosslinked with PEG dimethacrylate, mPEGA chains tethered on the surface of crosslinked PPF did not influence the swelling ratio in water while increased surface hydrophilicity greatly. Meanwhile, surface frictional coefficient and the capability of adsorbing proteins from cell culture medium decreased continuously with increasing the mPEGA composition in mPEGA/PPF networks. Demonstrating cell repulsive effect at the mPEGA compositions higher than 7%, the modified surfaces improved MC3T3 cell attachment, proliferation and differentiation, which reached maxima at the mPEGA composition of 5-7%. Besides revealing that mPEGA pendant chains could enhance cell responses by increasing hydrophilicity when their fraction on the hydrophobic surface was small, the present study also offered a new method of improving the wettability and performance of the scaffolds made from PPF for bone repair.

Differential effect of saturated and unsaturated free fatty acids on the generation of monocyte adhesion and chemotactic factors by adipocytes: dissociation of adipocyte hypertrophy from inflammation.

OBJECTIVE: Obesity is associated with monocyte-macrophage accumulation in adipose tissue. Previously, we showed that glucose-stimulated production by adipocytes of serum amyloid A (SAA), monocyte chemoattractant protein (MCP)-1, and hyaluronan (HA) facilitated monocyte accumulation. The current objective was to determine how the other major nutrient, free fatty acids (FFAs), affects these molecules and monocyte recruitment by adipocytes. RESEARCH DESIGN AND METHODS: Differentiated 3T3-L1, Simpson-Golabi-Behmel syndrome adipocytes, and mouse embryonic fibroblasts were exposed to various FFAs (250 micromol/l) in either 5 or 25 mmol/l (high) glucose for evaluation of SAA, MCP-1, and HA regulation in vitro. RESULTS: Saturated fatty acids (SFAs) such as laurate, myristate, and palmitate increased cellular triglyceride accumulation, SAA, and MCP-1 expression; generated reactive oxygen species (ROS); and increased nuclear factor (NF) kappaB translocation in both 5 and 25 mmol/l glucose. Conversely, polyunsaturated fatty acids (PUFAs) such as arachidonate, eicosapentaenate, and docosahexaenate (DHA) decreased these events. Gene expression could be dissociated from triglyceride accumulation. Although excess glucose increased HA content, SFAs, oleate, and linoleate did not. Antioxidant treatment repressed glucose- and palmitate-stimulated ROS generation and NFkappaB translocation and decreased SAA and MCP-1 expression and monocyte chemotaxis. Silencing toll-like receptor-4 (TLR4) markedly reduced SAA and MCP-1 expression in response to palmitate but not glucose. DHA suppressed NFkappaB translocation stimulated by both excess glucose and palmitate via a peroxisome prolifterator-activated receptor (PPAR) gamma-dependent pathway. CONCLUSIONS: Excess glucose and SFAs regulate chemotactic factor expression by a mechanism that involves ROS generation, NFkappaB, and PPARgamma, and which is repressed by PUFAs. Certain SFAs, but not excess glucose, trigger chemotactic factor expression via a TLR4-dependent pathway.

Nonirradiated human fibroblasts and irradiated 3T3-J2 murine fibroblasts as a feeder layer for keratinocyte growth and differentiation in vitro on a fibrin substrate.

The standard method for producing graftable epithelia relies on the presence of a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (Rheinwald and Green technique). Here, we studied a new keratinocyte culture system, which envisages the utilization of nonirradiated human fibroblasts embedded into a fibrin substrate, in cultures destined for a future clinical application. We tested this culture system using keratinocytes grown on a fibrin gel precoated with 3T3-J2 murine fibroblasts as a control. In order to evaluate the new technology, we compared the clonogenic potential and the proliferative, differentiative and metabolic characteristics of keratinocytes cultured on the fibrin gel under the two culture conditions. The results demonstrated that the proposed technology did not impair the behavior of cultured keratinocytes and revealed that cells maintained their proliferative potential and phenotype under the experimental conditions. In particular, the demonstration of stem cell maintenance under the adopted culture conditions is very important for acute burn treatment with skin substitutes. This work is a first step in the evaluation of a new keratinocyte culture system, which has been studied in order to take advantage of an additional human cell population (i.e. nonirradiated, growing fibroblasts) for future transplantation purposes in acute and chronic wounds. Additional research will allow us to attain (1) the removal of murine cells in the initial phase of keratinocyte cultures, and (2) the removal of other potentially dangerous animal-derived materials from the entire culture system.

Conjugated linoleic acid activates AMP-activated protein kinase and reduces adiposity more effectively when used with metformin in mice.

Trans-10, cis-12 (t10c12) conjugated linoleic acid (CLA) reduces lipid levels in adipocytes, but the mechanisms involved are still emerging. The hypotheses of this study were that t10c12 CLA treatment activated AMP-activated protein kinase (AMPK) and that the effectiveness of a low dose of t10c12 CLA would be increased when combined with an AMPK activator. We demonstrated t10c12 CLA, directly or indirectly, activated AMPK and increased the amount of phosphorylated acetyl-CoA carboxylase (ACC) in 3T3-L1 adipocytes. Compound C, a potent inhibitor of AMPK, attenuated the phosphorylation of ACC, integrated stress response (ISR), inflammatory response, reduction in key lipogenic transcription factors, and triglyceride (TG) reduction that otherwise occurred in t10c12 CLA-treated adipocytes. Treatment of adipocytes or mice with a low dose of t10c12 CLA in conjunction with the AMPK activator metformin resulted in more TG loss than treatment with the individual chemicals. Additionally, although an inflammatory response was required for robust TG reduction, the combination of t10c12 CLA with AMPK activators had a similar TG loss with a reduced inflammatory response. A microarray analysis of the transcriptional response to either t10c12 CLA, metformin, or the combination, indicated the responses were very similar, with a correlation coefficient of 0.91 or better for genes in the ISR or lipid-related pathways. Altogether, these results support our hypotheses that t10c12 CLA activates AMPK, directly or indirectly, and that metformin increases the effectiveness of t10c12 CLA in reducing TG amounts in adipocytes.

The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex.

The conserved murine gammaherpesvirus 68 ORF20 has recently been demonstrated to induce G2 cell cycle arrest followed by apoptosis in human and mouse cells. Here, we demonstrate that its homologues UL24 in HSV-1, ORF20 in KSHV and UL76 in HCMV are also inducers of cell cycle arrest followed by apoptosis in both human and mouse cells. The mechanism of action is similar to that reported for MHV-68 ORF20, inactivating the mitotic complex cyclinB/Cdc2.