Renal cement embolism during percutaneous vertebroplasty

Percutaneous vertebroplasty (PVP) is an effective treatment for lesions of the vertebral body that involves a percutaneous injection of polymethylmethacrylate (PMMA). Although PVP is considered to be minimally invasive, complications can occur during the procedure. We encountered a renal embolism of PMMA in a 57-year-old man that occurred during PVP. This rare case of PMMA leakage occurred outside of the anterior cortical fracture site of the L1 vertebral body, and multiple tubular bone cements migrated to the course of the renal vessels via the valveless collateral venous network surrounding the L1 body. Although the authors could not explain the exact cause of the renal cement embolism, we believe that physicians should be aware of the fracture pattern, anatomy of the vertebral venous system, and careful fluoroscopic monitoring to minimize the risks during the PVP.
Introduction
Percutaneous vertebroplasty (PVP) is an effective, minimally invasive procedure in which polymethylmethacrylate (PMMA) cement is injected into a diseased vertebral body. This technique provides pain relief and strengthens weakened vertebral bodies. Vertebroplasty has gained widespread popularity for the treatment of benign or malignant compression fractures since its initial description 21 years ago [9]. However, complications can still occur during the procedure even though PVP is considered to be minimally invasive. The reported complication rates range from 1 to 10%, but are generally minor [3]. This paper reports our experience of a renal cement embolism during PVP, with a review of the literature.
Case report
A 57-year-old man, who had fallen from a 2.4 m ladder 4 months earlier, complained of severe back pain. He had been a healthy farmer prior to the accident. However, since his fall, he had suffered from debilitating back pain refractory to conservative treatment for 2 months. In particular, he complained of activity-related pain corresponding to the level of a compression fracture. The outside computed tomography (CT) scans demonstrated a compression fracture of the L1 body without destruction of the posterior wall and significant vertebral collapse. The magnetic resonance imaging (MRI) showed a slight step-off of the anterior cortical margin of the compressed L1 vertebral body, with lower marrow signal intensity on T1-weighted image (Fig. 1).
Fig. 1The T1 weighted sagittal magnetic resonance image of the lumbar spine demonstrates a slight step-off of the anterior cortical margin of the compressed L1 vertebral body, with decreased marrow signal intensity
It was decided to proceed with PVP of the L1. The patient was placed in the prone position. An 11-gauge needle was advanced into the vertebral body via the left unilateral transpedicular approach using sterile technique and fluoroscopic guidance under local anesthesia. The PMMA (Zimmer Inc., IN, USA) cement has two components: an ampule of liquid and a fine powder of PMMA containing barium sulfate, e.g., 10 ml ampoule of liquid to 20 g of powder. Under careful fluoroscopic visualization, the mixture of PMMA cement and barium sulfate powder having a consistency similar to that of toothpaste was slowly injected into the vertebral body, diffusing throughout the intertrabecular marrow space. Before its injection, the mixture was loaded into several 1 ml syringes. However, when approximately 8 cc of PMMA had been injected unilaterally, multiple bone cement was detected at the bilateral renal fossae. The procedure was immediately stopped. After the procedure, the back pain had improved by more than 50%, but he still complained of severe right flank pain and fever. His renal function was impaired and the BUN to Cr ratio was high (27.2/1.3). On the same day, both kidneys were swollen (13 cm), and were found to contain a large amount of hyperechoic materials on the abdominal ultrasound (Fig. 2). The DMSA (99mTc dimercaptosuccinic acid) scan has been found to be quite useful for examining the bilateral kidney damage in detail, but the patient refused this. The CT scans showed the origin of the leak arising at the level of the vertebroplasty, and showed cement in the left anterior external venous plexus draining into both renal veins. Multiple tubular opacities, which corresponded to the course of the renal vessels, were detected, with some of the cement scattered through the left posterior external venous plexus on the CT scans (Fig. 3). The patient was diagnosed with a renal embolism and was treated conservatively. On the sixth day after the procedure, the BUN to Cr ratio had returned to normal, and the patient was released from hospital without pain. A follow-up ultrasound 8 months later revealed prominent atrophy of the right upper renal cortex with compensatory hypertrophy of the left kidney. However, the patient is currently doing well and has no subjective symptoms.
Fig. 2Both kidneys are swollen (13 cm), and the abdominal ultrasound shows large amounts of hyperechoic materials (a, right kidney; b, left kidney)Fig. 3Computed tomography scans show the origin of the leak arising at the level of the vertebroplasty (a), revealing cements in the left anterior external venous plexus draining into both renal veins (b)
Discussion
Image-guided percutaneous vertebral augmentation or PVP, was first performed in France in 1984, when Deramond and Galibert et al. [9] injected PMMA into the C2 vertebra, which had been partially destroyed by an aggressive hemangioma. Over the next 15 years, many groups advocated expanding the indications for PVP to include osteoporotic compression fractures, traumatic compression fractures, and painful vertebral metastasis [2, 6, 7, 11, 18, 19]. Our case had the anterosuperior fracture of the L1 body without destruction of the posterior wall and significant vertebral collapse so that it might not lead to a technically difficult vertebroplasty procedure; or might not constitute relative contraindications [6].
Even though PVP has many advantages including its simplicity, easy approach, and minimally invasiveness, many authors have reported several complications. The reported complication rates range from 1 to 10%, with a higher incidence of complications in cases with metastatic lesions [3]. However, complications are rare (1~2%) with osteoporosis, and are generally asymptomatic and transient [2, 11, 19]. The reported complications associated with these procedures include hypotension, rib fractures [18], dural tear [1], pulmonary cement embolism [1, 4, 14], adult respiratory distress syndrome [18], cerebral cement embolism [15], root compression due to intraforaminal cement leakage [6], paraplegia due to spinal cord [6] and cauda equina compression [16], intravascular extension of cement [1], infection, and cement toxicity [12].
The vascular leakage of PMMA, although rare, might have disastrous consequences. The viscosity of the PMMA cement is a crucial aspect during the procedure, and progressively increases as a result of methyl methacrylate polymerization. The rate of polymerization depends on several difficult-to-evaluate factors, including the ambient temperature and the quantity of the solvent. A paste is preferred to a liquid consistency because the latter may cause leakage of the PMMA into the venous system, particularly when the lesion is a highly vascular or extensive osteolytic tumor [5, 6]. However, it is not always possible to maintain the appropriate viscosity of PMMA at all times prior to the injection because the viscosity of the PMMA changes over time. The possibility that the renal cement embolism was caused by insufficient polymerization of the PMMA at the time of injection could not be excluded.An adequate amount of the infusing PMMA is also recommended. Cotten et al. [6] reported that pain relief does not appear to be proportional to the degree of lesion filling by the PMMA. In addition, Martin et al. [13] reported that complications were mainly related to an excessive PMMA injection.Good-quality fluoroscopy is essential for the early detection of a minimal cement leakage into the perivertebral vein. Jensen et al. [11] recommended a barium/tungsten combination in order to allow for adequate visualization of venous flow as well as early detection of venous PMMA migration during fluoroscopy. In our case, renal cement embolism caused by perivertebral venous migration was not early recognized. Injection was performed under lateral fluoroscopy, paying particular attention to the epidural space, the spinal canal, and the perivertebral veins. But real-time detection of laterovertebral leakage remained difficult owing to overlap of the cement filling vertebral body, even though intermittent anteroposterior fluoroscopy could not overcome this problem. Gangi et al. [8] recommended that CT guidance increases precision, improves the results, and reduces complications. But this is also the lack of real-time visualization of cement leak. The vertebral venography should be carried out before the PMMA injection. However, its value is also contentious. Jensen et al. [11] advocated the use of antecedent venography to decrease the incidence of complications associated with the needle placement within the basivertebral venous plexus and to delineate the route of cement egress. However, Deramond et al. [7] reported that the lesion is stained by the injection of contrast media in the case of spinal tumors, which interrupts the early venous leakage, and that the different flow characteristics of the contrast material and PMMA hamper the predictive value of venography [14]. Venography is not usually performed at our center. In our case, the PMMA leaked outside of the left anterior cortical fracture site of the L1 vertebral body and multiple tubular bone cement migrated to the course of the renal vessels via the anterior external vertebral venous plexus. Anatomically, the vertebral venous system is a large valveless collateral venous network within and around the vertebral column, extending from the sacral hiatus along the entire length of the vertebral column up to the foramen magnum [10]. There are also a large number of connections to the subcutaneous and vertebral veins as well as the sacral venous plexus. We do not know whether the cause of the renal cement embolism was via the vertebral venous network or the inappropriate PMMA viscosity. The patient’s position is also clinically important. Vogelsang reported that the intraosseous pressure recorded in the lumbar spinous process increased by compressing the inferior vena cava, which he called “retrograde congestion” [17]. Under this condition, the vertebral pressure is increased, which might reduce the risk of fat, bone marrow, air and bone cement extrusion into the IVVP and EVVP. Our procedure used a hand injection method for the PMMA in order to prevent an increase in the injection velocity.We could not determine the precise cause after a lengthy review of the whole procedure, but it is possible that there might have been some vascular anomaly, such as congenital communication, between the vertebral venous system and renal vessels.
Conclusion
The authors encountered an unexpected renal embolism during PVP. We could not explain the exact cause of the renal cement embolism, but we believe that physicians should be aware of the fracture pattern, anatomy of the vertebral venous system, and careful fluoroscopic monitoring to minimize the risk during the PVP.

embolism
percutaneous vertebroplasty
bone cement
vertebral venous system


The art of cellular communication: tunneling nanotubes bridge the divide

The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes.
Introduction
Communication with the environment is a basic principle of any biological system. With the increasing complexity of higher organisms, cells had to evolve diverse mechanisms to exchange spatial and temporal information crucial for tissue organization and the maintenance of the organism as a whole. Certainly, tissues are not just a loose accumulation of individual cells, but a highly organized population of interacting cells. Thus, in the various types of animal and plant tissues, cell-to-cell communication is important for maintaining a supracellular organization. Probably the best-characterized example of such an organization is given by the animal central nervous system, which is composed of a complex network of interconnected neuron and glial cells. It is widely accepted that the complexity of the brain reflects its enormous number of intercellular links provided by synaptic connectivity (including gap junctions) between axons and dendrites. Gap junctions, through which molecules <1 kDa can penetrate, are also important players in establishing supracellular organization in other tissues containing electrically excitable cells as heart and smooth muscle cells. Furthermore, these proteinaceous channels interconnect cells of the multicellular layer forming epithelial tissues. Finally, cell-to-cell coupling via gap junctions is a general phenomenon during early embryogenesis, where most cells are electrically coupled. Perhaps due to the presence of a thick cell wall, plant cells have evolved thin membrane channels referred to as plasmodesmata (PD) to allow direct connections over longer distances (Baluška et al. 2004a; Cilia and Jackson 2004; Gallagher and Benfey 2005). Because these bridges provide both membrane and cytoplasmic connectivity between cells, plants are regarded as a supracellular assembly (Baluška et al. 2004b, c). PD, in addition to the functions known for gap junctions in animals, facilitate the transfer of ribonucleoparticles, transcription factors and viruses (Ruiz-Medrano et al. 2004).
In 2004, a new principle of cell-to-cell communication between animal cells, based on the formation of thin membrane channels, was reported (Rustom et al. 2004). These channels, referred to as tunneling nanotubes (TNT), were initially found in cultures of rat pheochromocytoma (PC12) cells. As PD, they were shown to mediate membrane continuity between connected cells. TNT permit the direct intercellular transfer of organelles, cytoplasmic molecules, and membrane components (Gerdes et al. 2007). Subsequent studies on other cell types revealed morphologically similar structures directing intercellular transfer of cargo, including pathogens. It thus becomes apparent that nanotubular bridges provide an important and general mechanism of cell-to-cell communication between animal cells. In the following, we summarize the current knowledge and development in the field of nanotubular communication. As awareness of the potential physiological implications of these structures, we will give a short overview on published data describing long and thin cellular protrusions in tissue implicated in cellular communication.
Diversity of TNT-like structures
TNT connecting PC12 cells are typically 50–200 nm in diameter and can reach lengths up to several cell diameters. They are stretched, interconnecting cells at their nearest distance (Fig. 1) (Hodneland et al. 2006; Rustom et al. 2004). Their structural integrity is sensitive to mechanical stress, chemical fixation, and even to prolonged light exposure. The feature that most strikingly distinguishes TNT from other cellular protrusions is that they hover in the medium and have no contact to the substratum (Fig. 1a, a1). A structural characterization of TNT between PC12 cells revealed that they contain F-actin as a prominent cytoskeleton element but no microtubules (Table 1). Further characterization at the ultra-structural level showed a seamless transition of the surface membrane of the TNT with the plasma membranes of the connected cells (Fig. 1b, b1, b2). In addition, a continuous membrane with both connected cells was evident in transmission electron micrographs (Fig. 2a, a1, a2) (Table 1). These observations, together with the documentation of a limited lateral diffusion of fluorescent membrane proteins occurring between the plasma membranes of TNT-connected cells, led to the proposal that TNT mediate intercellular membrane continuity (Fig. 3a) (Gerdes et al. 2007; Rustom et al. 2004).
Fig. 1Architecture of TNT between cultured PC12 cells. (a) 3D fluorescence image ((x−y)-maximum projection of 40 consecutive 400 nm sections) of a wheat germ agglutinin-stained TNT connecting two live PC12 cells. (a1) (x−z)-projection in the plane of the TNT indicated in (a). (b) Scanning electron micrograph (SEM) showing the ultra-structure of a TNT between two PC12 cells. The boxed areas are shown as higher magnification images (b1, b2). Modified from Rustom et al. (2004) Science 303:1007–1010. Scale bars, a, a1, b, 5 μm; b1, b2, 500 nmTable 1TNT-like structures in vitroaCell type TNT-like structures Cytoskeletal components Membrane continuity/ “open-ended” Cargo PC12 cells (Rustom et al. 2004) F-actin, myosin Va (+)b Endosome-related organelles, lipid-anchored proteins (EGFP-f), EGFP-actin NRK cells (Rustom et al. 2004)c (F-actin, myosin Va)c ND Endosome-related organelles EBV-transformed human B cell line (721.221) (Önfelt et al. 2004) ND ND GPI-GFP, (HLA-Cw6-GFP, in coculture with human peripheral blood Natural Killer cells)d Between neonatal rat CM and adult human EPC (Koyanagi et al. 2005) ND ND Mitochondria, soluble proteins (GFP) Primary cultures of rat astrocytes (Zhu et al. 2005) F-actin, myosin Va ND ND DC (Watkins and Salter 2005) ND (+)e Calcium fluxes, surface receptors (HLA-A,B,C class I MHC)d THP-1 monocytes (Watkins and Salter 2005) F-actin (+)e Calcium fluxes, the fluid phase marker lucifer yellow, surface receptors (HLA-A,B,C class I MHC)d Between DC and THP-1 monocytes (Watkins and Salter 2005) ND (+)e Calcium fluxes Human monocyte-derived macrophages (bridges with diameter ≥0.7 μm) (Önfelt et al. 2006) F-actin, microtubules (+)f Mitochondria, endosome-related organelles, lysosomes Human monocyte-derived macrophages (nanotubes with diameter <0.7 μm) (Önfelt et al. 2006) F-actin ND Surfing Mycobacterium bovis bacillus Calmette-Guérin Cos-1, XC and HEK 293 cells (Sherer et al. 2007) F-actin (−)b MLV Jurkat T cells (Sowinski et al. 2008) F-actin (−)b,e HIV-1 protein Gag aOnly those publications that fulfill at least one of the listed criteria are shownbAccessed by electron microscopycGurke, S., Barroso, J., Bukoreshtliev, N., Gerdes, H.-H., unpublished datadThese molecules were shown to localize in TNT-like structures, but their intercellular transfer was not proveneAccessed by the measurement of calcium fluxesfProposed from the observation of a seamless transition between microtubules of the bridge with microtubular networks of both connected cellsND: not determinedFig. 2Transmission electron micrographs (TEM) showing the ultra-structure of distinct TNT-like bridges in different cell types. (a) Open-ended TNT connecting two PC12 cells reconstructed from images of two consecutive 80 nm sections. The boxed areas are shown as higher magnification images (a1, a2). A continuous membrane is observed between the nanotube and the plasma membrane of the two connected cells. Modified from Rustom et al. (2004) Science 303:1007–1010. (b) Close-ended TNT-like bridge connecting two T cells and displaying a junctional border, reconstructed from images of 13 consecutive 60 nm sections. The boxed areas are shown as higher magnification images (b1, b2). The nanotube formed by one cell (b2) protrudes into an invagination (arrowhead) of the connected cell (b1). Modified from Sowinski et al. (2008) Nat Cell Biol 10:211–219. Scale bars, a, b, 1 μm; a1, a2, b1, b2, 500 nmFig. 3Schematic representations of three distinct nanoscaled cellular protrusions and proposed modes of cell-to-cell communication. (a) A TNT-mediating membrane continuity between cells. (a1) Organelles like endocytic vesicles and mitochondria are transported uni-directionally between cells by an actin-dependent mechanism. (b) Nanotubular bridge between cells displaying a junctional border. (b1) Distinct viral particles are transported either at the surface of the nanotube by a receptor-dependent mechanism using actin retrograde flow or inside the cellular nanotube by an actin-dependent mechanism. (c) Cellular nanotube (cytoneme) extending toward a target cell by chemotaxis. (c1) Signaling molecules secreted by the target cell are proposed to be endocytosed by a receptor-mediated mechanism at the tip of the cytoneme and transported in a retrograde manner toward the cell body of the receiving cell. The arrows (a–c) indicate the direction of transfer
Based on the morphological criteria defined for PC12 cells, similar TNT-like connections were subsequently identified for several permanent cell lines and primary cultures (for a comprehensive overview see (Gerdes et al. 2007)) (Table 1). TNT-like bridges between dendritic cells (DC) (Watkins and Salter 2005) and perhaps also those connecting neonatal rat cardiomyocytes (CM) and adult human endothelial progenitor cells (EPC) (Koyanagi et al. 2005), appear to be most closely related to the TNT described for PC12 cells. The flow of cytoplasmic molecules suggests membrane continuity between connected cells (Table 1). This contrasts the TNT-like bridges between T cells, which have been characterized lately in considerable detail (Sowinski et al. 2008). The observed T-cell nanotubes have an average length of ∼20 μm, a diameter of 180–380 nm, and are not tethered to the substratum. Moreover, as in PC12 cells, only F-actin but not microtubules were detected in T-cell nanotubes (Fig. 3). However, despite the overall similarity in architecture and capacity to facilitate intercellular transfer of cargo between nanotubes of PC12 cells, DC, CM/EPC and T-cells, the latter do not mediate membrane continuity between connected cells (Fig. 2b, b1, b2) (Table 1). This was concluded from the observation that nanotubes between T-cells (i) do not facilitate the transfer of cytoplasmic molecules, (ii) do not permit free diffusion of fluorescent plasma membrane components and (iii) display a junctional border between the nanotube and the connected T-cell at the ultra-structural level (Fig. 2b1). Accordingly, they were classified as “not open-ended” cellular nanotubes (Fig. 3b). Other identified close-ended nanotubular bridges that share some morphological features with TNT are murine leukemia virus-induced “filopodial bridges” (Sherer et al. 2007) (Table 1).
Interestingly, it has been shown that different classes of nanotubes exist even within a single cell type. In the case of macrophages, not only thin, F-actin-containing bridges similar to other TNT-like structures were detected, but also a thicker type of cellular connection (≥0.7 μm diameter), which contained both F-actin and microtubules (Önfelt et al. 2006). This thicker type of connection was suggested to be open ended due to a seamless transition of the microtubules inside the bridge with the microtubular networks of both connected cells (Önfelt et al. 2006) (Table 1). Thin, and thicker microtubule-containing connections were also found between prostate cancer cells (Vidulescu et al. 2004).
Finally, another type of long and thin F-actin-containing cellular protrusions that share striking features with TNT-like intercellular bridges, are cytonemes. These membrane nanotubes were discovered in Drosophila imaginal wing discs, emanating from the periphery of the columnar cell sheet toward the signaling center associated with the anterior/posterior border (Ramírez-Weber and Kornberg 1999). They are thought to extend toward the target cells by chemotaxis (Ramírez-Weber and Kornberg 1999) (Fig. 3c) to accomplish the receptor-mediated uptake of the secreted morphogen Decapentaphlegic (Dpp) during spatial patterning (Hsiung et al. 2005). Once endocytosed, the morphogen is thought to be delivered to the cell body by retrograde transport along the cytoneme (Fig. 3c1). Thus, these structures are proposed to fulfill an important task in long-range cell-to-cell signaling during embryonic development. In contrast to TNT-like structures, cytonemes have not been shown to physically bridge cells. Nevertheless, functionally similar structures, emanating from tracheal cells in response to Branchless (Bnl)-Fibroblast Growth Factor (FGF) signaling, appear to do so (Sato and Kornberg 2002).
The identification of TNT-like structures in cell cultures has been mainly based on the morphological criteria defined for PC12 cells (Rustom et al. 2004). In some cases, a proper comparison was hindered by their limited characterization. Nonetheless, heterogeneity regarding formation, structure, and functional properties across cell types and even within a given cell type has emerged (Table 1). Certainly, more detailed information is necessary for a proper classification of all these structures and thus, as things are now, we shall refer to them as TNT-like structures, irrespective of whether membrane continuity was observed.
Formation of TNT-like structures
TNT-like structures form de novo between cells on a time-scale of several minutes by apparently two distinct mechanisms. With respect to the first mechanism, initial studies on PC12 cells showed that TNT could be established by a seemingly directed outgrowth of swaying filopodia-like protrusion(s) toward a neighboring cell. Once contact is made, a single dilated and bended bridge is often observed along with the degeneration of remainder protrusions. This bended structure is then remodeled into a straight and thin bridge showing the characteristic morphology of typical TNT (Rustom et al. 2004). One can speculate that the seemingly directed filopodia outgrowth preceding TNT formation is under control of chemotactic guidance. Evidence for this is provided for related processes like the formation of cytonemes toward a Bnl-FGF gradient in in vitro cultures of Drosophila cells (Ramírez-Weber and Kornberg 1999) (Fig. 3c) or murine leukemia virus (MLV)-induced nanotubular bridges emanating from non-infected fibroblast cells toward infected cells (Sherer et al. 2007). The observation that TNT-like bridges also emerge when attached PC12 (Rustom et al. 2004) or immune cells (Önfelt et al. 2004, 2006; Sowinski et al. 2008) depart from each other, led to the proposal of a second mechanism of TNT formation (Gerdes et al. 2007; Önfelt et al. 2004), which a priori excludes any dependence on chemotactic guidance of filopodia. Subsequent detailed studies on T cells revealed that cell-to-cell interaction for at least a few minutes before dislodging is required for successful nanotubular bridge formation (Sowinski et al. 2008). It is of note that, in contrast to PC12 cells, a filopodium-dependent mechanism of nanotubular bridge formation between immune cells was not reported to date (Önfelt et al. 2004, 2006; Sowinski et al. 2008).
Actin polymerization is thought to be a key event for both mechanisms of TNT formation (Gerdes et al. 2007). It drives filopodia outgrowth important in the first mechanism and is most probably important for the stabilization of TNT-like structures emerging by the second mechanism. In support of this, TNT formation is not observed in the presence of actin-depolymerizing drugs (Rustom et al. 2004), and TNT-like structures between T cells only form if the cells diverge with a speed below that of processes driven by actin polymerization (Cameron et al. 1999; Sowinski et al. 2008). Zhu et al. (2005) obtained further evidence that actin polymerization and TNT formation are linked by demonstrating their concurrent induction by H2O2 in primary cultures of rat astrocytes.
TNT-like structures have a dynamic nature leading to only transient bridges as observed in in vitro cultures. The nanotubular bridges between T cells (Sowinski et al. 2008), PC12 cells or normal rat kidney (NRK) cells (Bukoreshtliev, N., Gerdes, H.-H., unpublished data) have variable lifetimes, ranging from a few minutes to less than 60 min for the former and even up to several hours for the latter two. These considerable differences in lifetime, even at the cellular level, may reflect the existence of different subclasses of TNT-like structures. It is interesting to speculate that the fusion of the nanotube with the plasma membrane of the connected cell is part of a time-dependent maturation step leading to such heterogeneity.
Studies on model artificial membrane tubes such as those created by pulling tethers from synthetic lipid vesicles or cellular plasma membrane provide a complementary approach to obtain mechanistic insights into the formation of TNT-like structures. The morphology of such nanotubes resembles that of TNT-like structures and it seems plausible that the same physical laws govern the formation and architecture of both structures. The exploitation of such in vitro models revealed that the elongation of plasma membrane nanotubes requires a membrane flow from the cell plasma membrane into the growing tube. It is suggested that cells maintain a membrane reservoir (e.g. ruffles, invaginations), controlled by the cytoskeleton, to provide a buffer against membrane tension over several micrometers of tube elongation (Raucher and Sheetz 1999; Sun et al. 2005). Thus, the available membrane reservoir may restrict the number, total length, and lifetime of TNT-like connections for a given cell.
TNT-like structures are conduits for the delivery of cargo
An obvious advantage of establishing direct bridges between cells is an improvement in selective communication even over long distances. In agreement with this assumption, the common feature of all TNT-like structures characterized in more detail, is the transfer of cargo between connected cells (Table 1).
Cellular components
Direct evidence for the intercellular exchange of cargo via TNT-like structures was obtained by video-microscopic studies. By employing fluorescent dyes, organelles belonging to the endosomal/lysosomal system (Rustom et al. 2004) (Gurke, S., Gerdes, H.-H., unpublished data) as well as mitochondria (Koyanagi et al. 2005) were shown to traffic uni-directionally along TNT-like structures between cells over long distances (Fig. 3a1) (Table 1). The occurrence of an intercellular transfer of organelles raises the question as to what kind of information is transferred and how this information is integrated in the target cell. In the case of the endosomal system, early endocytic vesicles are particularly interesting candidates for TNT-mediated delivery. These organelles are one of the major reloading points for a variety of signaling complexes resulting from the endocytosis of activated cell surface receptors (Miaczynska et al. 2004). Once delivered to the target cells, the transferred endocytic vesicles are able to fuse with their counterparts (Rustom et al. 2004), and provide a potential way to integrate information. In essence, the transfer of endocytic organelles propagates signaling information from the single cell to a larger community, which may lead to coordinated cell behavior. Mitochondria were shown to transfer via TNT from neonatal rat CM to adult human EPC and it was suggested that this event could contribute to the acquisition of a cardiomyogenic phenotype by the progenitor cells (Koyanagi et al. 2005) (Fig. 4). Moreover, the active transfer of this organelle from adult stem cells and somatic cells to mammalian cells with non-functional mitochondria was shown to rescue aerobic respiration in the latter (Csordás 2006; Spees et al. 2006). The obtained data are consistent with a TNT-related delivery, although alternative mechanisms were not excluded.
Fig. 4Emerging physiological implications of TNT-like structures
In addition to the transfer of organelles, plasma membrane components such as lipid-anchored proteins can laterally transfer along the TNT-like bridge into the plasma membrane of connected cells. This was demonstrated for both the inner and outer leaflets of the plasma membrane by using glycosylphosphatidylinositol conjugated to GFP (GPI)-GFP (Önfelt and Davis 2004; Önfelt et al. 2004) and the farnesylation signal of c-Ha-Ras fused to the C-terminus of EGFP (EGFP-f) (Rustom et al. 2004), respectively (Table 1). Such a transport is consistent with membrane continuity between connected cells. Surface receptors could also be transferred directly by lateral diffusion in the plane of the cell surface, in addition to an endosome-dependent delivery. Interestingly, the transfer of membrane proteins between myeloid cells was found to involve scavenger receptors (Dr. R. D. Salter, Univ. of Pittsburgh, personal communication). It is of note that major histocompatibility complex (MHC) class I receptors were detected at the entire length of TNT-like bridges connecting immune cells (Önfelt et al. 2004; Watkins and Salter 2005). This points to a key role of TNT in a faster and more efficient presentation of antigens, in particular at the immunological synapse (Groothuis et al. 2005; Williams et al. 2007) (Fig. 4), defined as a highly specialized interface between immune cells (Norcross 1984; Paul and Seder 1994). In this case, the lateral transfer of only a few molecules can fulfill important roles in a coordinated immune response. Interestingly, in analogy to TNT, membrane continuity was found at the immunological synapse (Stinchcombe et al. 2001). The characterized “membrane bridges” had a diameter of 50–95 nm as observed by electron microscopy (Stinchcombe et al. 2001).
Finally, the proposed model of membrane continuity suggests a cytoplasmic bridge in analogy to PD, which permits the free flow of soluble molecules up to a certain size limit defined by the free space along the interior of the nanotube. In support of this view, EGFP-actin was transferred between TNT-connected PC12 cells (Rustom et al. 2004), GFP between CM and EPC (Koyanagi et al. 2005), and the fluid phase marker Lucifer yellow between THP-1 monocytes (Watkins and Salter 2005) (Table 1). Notably, both GFP and the small dye molecule calcein, with a molecular weight of 400 Da, were apparently impeded to diffuse passively between TNT-connected PC12 cells (Rustom et al. 2004). Thus, TNT-like structures, like PD, may have a variable size exclusion limit depending on the cell type (Gerdes et al. 2007). With regard to soluble markers, certainly the most physiologically relevant finding was the TNT-dependent propagation of calcium signals between THP-1 monocytes as well as from DC to THP-1 monocytes (Watkins and Salter 2005) (Table 1). Stimulation by contact or exogenous soluble stimulators of both DC and THP-1 monocytes led to a calcium flux that propagated through a network of TNT-connected cells up to 500 μm away from the point of stimulation. The elicited calcium wave propagated uni-directional with an initial speed of 35 μm/s that slowed down rapidly to 10–15 μm/s (Watkins and Salter 2005). This rather low and rapidly decreasing speed supports a TNT-mediated signal spread along the cellular network, which is driven by a chemical gradient rather than an action potential. Such a view also corroborates a TNT-mediated membrane continuity providing a cytoplasmic bridge between connected cells (Table 1). Most remarkably, the transfer of calcium ions through TNT led to the flattening and membrane extension of the receiving cells, as occurs during physiological responses in preparation for phagocytosis (Watkins and Salter 2005). Thus, this study provided the first evidence for functional connectivity accomplished by TNT and suggests a crucial role of these structures in immune defense (Fig. 4).
Pathogens
An emerging topic is the efficient spread of pathogens, like bacteria and mammalian viruses, along cell membrane protrusions (Figs. 3, 4). By using established or induced nanotubular connections between cells, pathogens circumvent the rate-limiting step of diffusion and efficiently accelerate their spreading (Sherer et al. 2007; Sowinski et al. 2008). Among the viruses shown to exploit cellular protrusions to transfer from cell-to-cell are the vaccinia virus (Cudmore et al. 1995,. 1996), pseudorabies virus (Favoreel et al. 2005, 2006), the herpes simplex virus (La Boissière et al. 2004), MLV (Sherer et al. 2007) and human immunodeficiency virus type 1 (HIV-1) (Sowinski et al. 2008). In the following, we will focus on the latter two, which were characterized in much detail and gave rise to two different mechanisms of virus transmission.
Sowinski et al. (2008) proposed membrane nanotubes between T cells as a new route for HIV-1 transmission (Fig. 3b1), in addition to cellular contact points known as virological synapses (Jolly et al. 2004; Jolly and Sattentau 2004; Sol-Foulon et al. 2007). Using a recombinant HIV-1 expressing Gag-GFP, they demonstrated its uni-directional move through innate nanotubular bridges and subsequent transmission into connected T cells (Sowinski et al. 2008) (Table 1). Furthermore, the requirement of cell surface receptors for viral transmission implied, in agreement with ultra-structural data, a junctional border between the nanotube and the connected T cell. It should be emphasized that the transmission of HIV-1 through nanotubular bridges can minimize the exposure of the virus to extracellular antibodies or complement. However, a nanotube-dependent spread may open new avenues in the development of antiviral drugs.
A different, even though nanotube-dependent, mechanism of transmission was reported for MLV (Sherer et al. 2007). The cell-to-cell transmission of MLV was directly visualized by infecting cultured cells with a recombinant virus expressing the viral capsid protein Gag and the envelope protein Env as a fusion construct with fluorescent proteins. MLV induces the outgrowth of thin and long filopodia from uninfected cells toward infected cells (Sherer et al. 2007). These filopodia are proposed to stably anchor to the cell body of an infected cell by viral envelope glycoprotein (Env)/receptor interactions. Subsequently, MLV moves along the outer surface of these nanotubular bridges to reach the cell body of target cells, where it finally enters (Sherer et al. 2007) (Fig. 3b1). In addition to viruses, bacteria were reported to surf along TNT-like structures connecting macrophages (Önfelt et al. 2006) (Table 1).
Mechanism of transfer
It is possible that more than one transfer mechanism for shipping cargo via TNT-like bridges has evolved considering their structural diversity and the multitude of transferred cargo. For organelles and viruses, an F-actin-dependent, uni-directional transport prevails (Fig. 3), with the exception of a microtubule-dependent, bi-directional movement of organelles in thick bridges connecting macrophages (Önfelt et al. 2006). In the case of endocytic organelles, an acto-myosin-dependent transport system is presumed (Fig. 3a1) due to the presence of the barbed-end directed actin-specific motor myosin Va in TNT (Rustom et al. 2004; Zhu et al. 2005) (Bukoreshtliev, N., Gerdes, H.-H., unpublished data) (Table 1) partially co-localizing with the respective organelles (Rustom et al. 2004). This model is supported by the low transport velocity in the range of actin-dependent transport (Rustom et al. 2004) and implies that the F-actin fibers inside the bridge have the same polarity. Also HIV-1 particles were suggested to move in an actin-dependent manner through nanotubular bridges (Sowinski et al. 2008) (Fig. 3b1). Interestingly, even in the case of MLV surfing along filopodia bridges with a speed ∼7 times slower as compared to that of HIV-1 within the bridges, an acto-myosin transfer mechanism appears to be in place (Sherer et al. 2007). This results in the uni-directional movement of all viral particles from infected to target cells, presumably using myosin II controlled retrograde flow of actin and cognate Env-receptor interactions (Fig. 3b1). In contrast to organelles and viruses, the uni-directional propagation of calcium waves is likely to be driven by a chemical gradient rather than an active transport mechanism. Passive diffusion is likely to control the transfer of other small cytoplasmic molecules and also some plasma membrane components, if membrane continuity is present.
Regarding the modality by which shipped cargo enters the target cell, two different scenarios are conceivable. In the case of membrane continuity as observed in PC12 cells, an open-ended transport along cytoskeleton elements without border crossing is envisaged, consistent with the observed uniform movement of transferred organelles (Rustom et al. 2004). In analogy to PD, gating mechanisms may exist to control this open-ended transport (Gerdes et al. 2007). In the case of close-ended cell-to-cell bridges containing junctional borders, as are the innate nanotubes connecting T cells and the MLV-induced cellular bridges, the cargo has to traverse the plasma membrane boundary. This is consistent with the finding that a receptor-dependent transmission of HIV-1 Gag could be blocked by the HIV-1 fusion inhibitor T20 (Sowinski et al. 2008).
Ultra-fine cellular extensions in tissue
Does intercellular communication mediated by TNT-like structures play a physiological role in tissue? This is probably the most interesting and important question that needs to be addressed in the near future. The absence of a TNT-specific marker does not allow an explicit answer to this question yet. However, studies employing cell cultures already point to important physiological implications of TNT-like structures in intercellular communication (reviewed in Gerdes et al. (2007)) (Fig. 4). It could be argued that cell culture models are rough representations of physiological systems and that TNT-like structures are the sole result of stress conditions in this in vitro situation. However, the widely documented richness of cellular protrusions in tissue implicated in intercellular communication (Rørth 2003) (Table 2) disagrees with such a possibility and instead supports an in vivo function. Cellular extensions have been documented in diverse tissues and during various physiological processes such as embryogenesis, stem cell differentiation, cell migration, and wound healing.
Table 2Selected examples of cell protrusions in tissueProtrusions Tissue Cytoskeletal components Cargo Length [μm] Diameter [nm] Growth speed [μm/min] Thin, elongated, active filopodia and lamellipodia Primary mesenchyme sea urchin embryos (Gustafson and Wolpert 1967; Miller et al. 1995) F-actinaND 80a200–400b25a Cytonemes Drosophila wing imaginal discs (Ramírez-Weber and Kornberg 1999) F-actin ND <700 200 15 Cytoneme-like Mouse limb bud cells (Ramírez-Weber and Kornberg 1999); Drosophila wing imaginal discs (Jacinto et al. 2000) F-actinbND 10bND 1bApical peripodial extensions Drosophila wing/eye imaginal discs (Gibson and Schubiger 2000) Microtubules Mitochondriac 5–30 ND ND Myopodia Drosophila embryonic muscle cells (Ritzenthaler et al. 2000; Ritzenthaler and Chiba 2003) F-actin ND 40 ND ND Myopodia-like Mouse transverse muscle of thorax (Misgeld et al. 2002); Xenopus (Kullberg et al. 1977) ND ND 10dND ND Cellular extensions Drosophila eye imaginal discs (Chou and Chien 2002) F-actin Scabrous vesicles 40 ND ND Apical and lateral cell protrusion Drosophila wing imaginal discs (Demontis and Dahmann 2007) F-actin GFP-Rab5c, GFP-Tkvc1–10 200 ND aData from Miller et al. (1995)bData from Jacinto et al. (2000)cThese molecules were shown to localize in cell protrusions, but their intercellular transfer was not provendData from Misgeld et al. (2002)ND: not determined
The formation of cellular extensions (“pseudopods”) during embryogenesis became first evident by studying sea urchin morphogenesis (Gustafson and Wolpert 1967; Miller et al. 1995), suggesting a function of these structures in force generation or cell recognition. During subsequent studies over the last decades, a variety of similar cellular extensions were found to be associated with developmental processes. Interestingly, most of them appear to be actin-rich as TNT-like structures. The Drosophila wing imaginal disc provides an attractive system for studying cell protrusions within a developing tissue. The first described example of such protrusions were the F-actin-containing cytonemes extending from the periphery of the columnar cell sheet toward the signaling center within this layer (Ramírez-Weber and Kornberg 1999). This discovery inspired further studies describing other cellular protrusions in the wing disc extending not only within (Chou and Chien 2002; Demontis and Dahmann 2007) but also between cell layers (Demontis and Dahmann 2007; Gibson and Schubiger 2000). In a very recent study on live wing discs, Demontis and Dahmann (2007) observed two new types of cellular extensions, i.e. lateral protrusions interconnecting distant cells of the columnar epithelium, and protrusions of the apical surface connecting to the squamous epithelium. Furthermore, ectopically expressed GFP-actin and the early endosome marker GFP-Rab 5 were detected inside these protrusions, resembling the finding made for TNT-like structures between cultured cells. These different protrusions may be involved in the intercellular signaling necessary for growth and patterning of wing imaginal discs (Demontis and Dahmann 2007).
Thin, actin-based membrane extensions were also found to be crucial for embryonic dorsal closure in Drosophila (Jacinto et al. 2000). Data obtained by confocal imaging of living fly embryos suggest that these long protrusions participate in both the mechanics of epithelial adhesion and in the search for a correct partner within the opposing epithelium. Likewise, related fundamental processes such as gastrulation, neural crest closure (Bard 1992), and wound healing (Wood et al. 2002) seem to involve similar actin-rich membrane extensions.
Other examples of actin-based cellular extensions during development are the so-called myopodia in Drosophila embryonic muscle cells that can be observed in whole mount. These dynamic postsynaptic microprocesses, with lengths up to 40 μm and highly sensitive to photoillumination, cluster at the site of motoneuron innervation while interacting with presynaptic filopodia. It was proposed that they play a role in the process of synaptic target recognition by contributing to direct long-distance cellular communication (Ritzenthaler et al. 2000; Ritzenthaler and Chiba 2003). Importantly, similar structures have been described in Xenopus (Kullberg et al. 1977) and mouse (Misgeld et al. 2002).
Finally, long, thin cellular extensions are also associated with guided cellular migration during development (Ribeiro et al. 2002; Sato and Kornberg 2002), immune defense such as the migration of macrophages through the endothelial cell layer (Blue and Weiss 1981) and tumor cell invasion (Vignjevic and Montagnac 2008). Certainly, there are many more examples of long cellular nanotubes in tissue involved in cellular communication besides the ones listed here (Table 2). This suggests that extensions in tissue are a rather common feature and that cells in culture preserve the ability to form similar, physiologically relevant structures. But, an important question remains: Do TNT-like structures, observed in cell culture models, have mechanistically and/or functionally related counterparts in live tissue? Furthermore, with respect to TNT, which have been found to mediate membrane continuity between cultured cells, it will be of foremost interest to find out, if this type of connection also exists in vivo.

tunneling nanotube
tnt
intercellular transfer
cytoneme
viral transmission


Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity

Aims/hypothesis Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake.
Introduction
Type 2 diabetes mellitus is a multifactorial disease with a complex pathophysiology that includes defects in insulin production and a failure in peripheral tissues to take up glucose in response to insulin. Signalling via phosphatidylinositol 3-kinase (PI3K) is a key pathway in the regulation of glucose uptake by insulin [1]. Activation of PI3K via insulin receptor substrate-1 causes PI3K to phosphorylate inositol-containing phospholipids at the 3′-position of the inositol ring, increasing the levels of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), among others. PIP3 activate a phosphoinositide kinases, which in turn phosphorylate and activate protein kinase B (PKB), also known as Akt, a key effector kinase for many of the downstream metabolic effects initiated by insulin via PI3K. The phosphatase and tensin homologue (PTEN) is a phosphatase which recognises both protein and lipid substrates. As a lipid phosphatase, PTEN dephosphorylates PIP3 at the 3′-position of the inositol ring, thereby acting as an antagonist to PI3K signalling [2, 3]. PTEN was originally identified as a tumour suppressor gene, and is one of the most commonly mutated genes in human cancer [4–6]. Mutation or deletion of PTEN has been identified in many cancers including prostate, brain, breast, endometrium and skin [4–8].
As many of the metabolic outcomes of insulin are achieved through recruitment of PI3K and the subsequent rise in PIP3 levels, PTEN may have a critical role in modulating sensitivity to insulin-stimulated glucose uptake. Recent studies in which Pten was ablated specifically in liver, adipose tissue and muscle in mice by Cre recombinase-based strategies have shown a role for Pten in the regulation of insulin sensitivity in those organs, and highlighted the contributions of those organs to glucose homeostasis of the whole animal [9–11]. However, the effect of global reduction of Pten levels on glucose uptake and metabolism has not yet been shown in an in vivo mouse model. Unfortunately, experimental genetic inactivation of both Pten alleles (Pten−/−) results in early embryonic lethality [6, 12]. Thus mice harbouring inactivation of one Pten allele (haploinsufficiency, Pten+/−) are an important experimental model for studying the role of Pten in vivo. Haploinsufficiency for Pten has important consequences for cell proliferation and tumorigenesis in mice [6, 12–14]. Pten+/− mice exhibit defective apoptosis in T cells, B cells and macrophages and spontaneous neoplasms in various tissues including prostate, liver, endometrium and others [12–14]. Here we show that Pten haploinsufficiency results in insulin hypersensitivity and enhanced insulin-mediated glucose uptake.
Materials and methods
Experimental animals All laboratory animals were cared for and used according to guidelines of the Canadian Council on Animal Care. Pten+/− mice were generated by R. Parsons [12]. These mice were backcrossed with C57BL6 mice for more than ten generations. The genotypes of the mice were determined as described [12].
Blood glucose determination For determination of blood glucose levels, glucose levels in blood samples taken from tail vein were determined using a One Touch Ultra blood glucose monitor (LifeScan Inc., Milpitas, CA, USA). For the glucose tolerance test, mice were fasted overnight (15 h) prior to i.p. injection of glucose (2 mg/g body weight). Blood glucose levels were then determined at the indicated time-points following glucose injection. For the insulin challenge test, mice were fasted for 15 h prior to i.p. injection of 0.6 mU insulin/g body weight. Blood glucose levels were then determined at the indicated time-points following insulin injection.
Determination of insulin in plasma Mice were fasted for 15 h, then approximately 100 μl of blood was collected from the tail vein using pipette tips precoated with heparin. The mice were then injected i.p. with 2 mg glucose/g body weight, and a second blood sample was obtained 15 min following injection. The blood samples were centrifuged at 805 g in a table-top centrifuge, and insulin in the plasma fraction was determined using an insulin ELISA system (ALPCO Diagnostics, Salem, NH, USA) according to the manufacturer’s instructions.
Isolation of pancreatic islets and glucose-stimulated insulin release Islets were isolated from pancreata of mice as described [15]. Pancreata from three mice were pooled. After 24 h in culture, islets were preincubated in Krebs–Ringer bicarbonate buffer plus 0.1% BSA (KRBB-BSA) and 1.67 mmol/l glucose. The islet cells were incubated for 1 h in 1 ml KRBB-BSA containing the desired concentration of glucose (1.67 or 16.7 mmol/l). The incubation medium was then collected, and centrifuged and the supernatant fractions were used for assay of insulin content by ELISA. Total extractable insulin in islets was determined by adding 0.5 ml lysis buffer (1 mol/l acetic acid with 0.1% BSA) to the islet pellet. Cell debris was pelleted by centrifugation, and the supernatant fraction was used for insulin assay.
Fluorescent immunohistochemistry of pancreatic islets Immunocytochemistry has been described [16]. Pancreata from wild-type and Pten+/− mice were embedded in paraffin. Five-micron sections were incubated for 1 h at room temperature with guinea-pig anti-insulin antibody (1:100 dilution; Dako, Mississagua, ON, Canada) and rabbit anti-glucagon antibody (Dako; 1:75 dilution). Sections were visualised by incubation with Alexa–Fluor 488 goat anti-guinea-pig (Invitrogen, Molecular Probes, Burlington, ON, Canada; 1:100 dilution) and Texas Red donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:100 dilution). Sections were examined by fluorescence microscopy.
Beta cell mass calculation Following insulin immunostaining, pancreatic sections were captured under an FITC filter using a Zeiss Axioplan 2 microscope equipped for epifluorescence and Pathvysion imaging software. The insulin immunopositive area (as a proportion of pancreatic area) was determined in at least six fields per section using Zeiss AxioVision software, performed on eight pancreatic sections per mouse. Beta cell mass was calculated as pancreatic mass times the percent beta cell area for each mouse.
2-[18F]fluoro-2-deoxyglucose uptake and microPET scanning Mice were fasted for 15 h prior to i.p. injection with 0.6 mU insulin/g body weight. Twenty minutes after insulin injection, mice were injected with 2-[18F]fluoro-2-deoxyglucose (18FDG) (4.1 MBq) via the tail vein, and whole-body distribution of 18FDG was monitored by positron emission tomography (PET) using a microPET R4 tomograph (Concorde Microsystems, Knoxville, TN, USA). The image data were attenuation-corrected using a 68Ge rod source. Wild-type and Pten+/− mice were positioned side-by-side and scanned simultaneously for 60 min after 18FDG injection. Data were analysed using the manufacturer’s software, ASIPro VM 5.0.1.0. For data analysis, regions of interest (ROIs) were specified in the coronal section of a hindlimb for each mouse. The 18FDG activities in each ROI were summed across all coronal planes, and expressed as a proportion of 18FDG in the whole mouse (total 18FDG activity was also taken as the sum of activity across coronal planes).
Primary myocyte culture Primary myocytes were cultured from fore- and hindlimbs from wild-type and Pten+/− mice according to a protocol adapted from Springer et al. [17]. Skeletal muscle was excised and minced and then incubated at 37°C for 2 h with 2.4 units/ml dispase II. Dispase digestion was halted by addition of 10 ml F-10-based myocyte medium (400 ml F-10 nutrient mixture, 100 ml fetal bovine serum, 25 μg basic fibroblast growth factor, penicillin/streptomycin). The tissue slurry was then strained through a fine metal mesh and centrifuged at 500 g for 5 min. The supernatant fraction was removed to a fresh centrifuge tube and the pellet was resuspended and plated to a 5-cm collagen-coated culture dish in a total of 4 ml F-10-based medium. The supernatant fraction was re-centrifuged and the pellet was pooled with the first. The cells were passaged when they reached 70–80% confluence. To enrich for myocytes, the old medium was removed by aspiration, the cells washed with PBS, then incubated in a film of this buffer for 15 min. Myocytes were loosened from the dish by striking the dish several times sharply on the bench top. Cells were washed from the dish with PBS. The PBS incubation was repeated and the cells were pooled. Cells kept in culture for more than 5–6 days began to exhibit the elongated morphology typical of myotubes [18]. Cells in these older cultures also exhibited spontaneous twitching. All experiments were performed with cells on days 2–3 of culture.
2-Deoxy[3H]glucose uptake Experiments were performed on wild-type or Pten+/− cells from passage two to eight, grown on 24-well (1-cm diameter wells) plates. Cells were preincubated in serum-free medium for 12 h prior to experiments. The cells were washed twice with HEPES-buffered saline (140 mmol/l NaCl, 20 mmol/l HEPES, 2.5 mmol/l MgSO4, 1 mmol/l CaCl2, 5 mmol/l KCl, pH 7.4), then incubated with 10 μmol/l 2-deoxy[3H]glucose (0.037 MBq/ml) for 5, 10, 20 or 30 min, in the absence or presence of 0.1 μmol/l insulin. At the end of the incubation period, the radioactive substrate was removed, and the cells were washed three times with ice-cold 20 mmol/l glucose. Cells were lysed with 0.2 ml 2% SDS, and the lysates were used for scintillation counting. Non-specific diffusion of 2-deoxy[3H]glucose was determined by incubating cells in the presence of 2-deoxy[3H]glucose and 10 μmol/l cytochalasin B, a specific inhibitor of glucose uptake [19].
Determination of cell-surface GLUT1 and GLUT4 levels Primary myocytes were grown in 10-cm culture dishes. The cells were rinsed with PBS, and incubated with 0.5 mg/ml sulpho-NHS-LC-biotin (Pierce Biotechnology, Rockford, IL, USA) in PBS for 20 min. Cells are impermeable to this reagent and it labels the amine groups of cell-surface proteins. Following the incubation, the cells were washed three times with PBS, then lysed with 0.5 ml lysis buffer (1% NP-40, 150 mmol/l NaCl, 50 mmol/l Tris pH 7.5, including protease and phosphatase inhibitors). Protein was quantified with a BCA protein assay kit (Pierce). Streptavidin-agarose beads (Pierce) were pre-blocked by incubation with 2% BSA in lysis buffer. Each sample (1.2 mg protein) was incubated with the streptavidin beads (100 μl, 50% bead slurry) for 1 h at room temperature. The beads were washed five times, for 30–60 min each wash, using lysis buffer containing 0.1% SDS. After the final wash, 50 μl SDS sample-loading buffer were added, and heated at 95°C for 10 min, then vortexed to elute the biotinylated proteins. The proteins in the supernatant fractions were analysed by SDS-PAGE, then transferred to nitrocellulose. Proteins were probed using polyclonal antibodies against GLUT1 and GLUT4 (also known as SCLA2A1 and SCLA2A4, respectively).
Western immunoblotting for PKB, phospho-PKB and phospho-glycogen synthase kinase 3 (GSK3)-α/β Total protein extracts of primary myocytes were prepared by lysing cells in NP-40-based lysis buffer. Thirty to fifty micrograms of protein per sample were analysed by SDS-PAGE, then transferred to nitrocellulose. Rabbit polyclonal anti-PKB, anti-phospho-PKB (Ser473) and anti-phospho-GSK3-α/β (Ser21/9) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-vinculin antibody was obtained from Sigma (St Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Dako. Protein bands were visualised using an enhanced chemiluminescence system and autoradiography by exposure to X-ray film.
Statistical analysis Statistical analysis was performed using Student’s t test. Significance was assessed at the 95% confidence level.
Results
As glucose metabolism may be altered due to a change in insulin sensitivity in Pten+/− mice, blood glucose levels in wild-type and Pten+/− mice were determined. Slight but statistically significant hypoglycaemia was observed in Pten+/− mice during ad libitum feeding (11.3% lower blood glucose in Pten+/− mice) (Table 1). Fasting caused a drop in glucose levels in both groups, with slight but significant hypoglycaemia in the Pten+/− group (10.8% lower blood glucose) relative to wild-type mice. An insulin challenge test revealed hypersensitivity to insulin in Pten+/− mice (Fig. 1a). Following i.p. insulin injection (0.6 mU/g body weight), blood glucose levels in Pten+/− mice remained depressed at all time-points measured (up to 120 min). In contrast, wild-type mice started to recover after 30 min, and glucose levels returned to fasting levels 120 min after injection. A glucose tolerance test showed that Pten+/− mice had greater glucose tolerance than wild-type mice (Fig. 1b). After i.p. glucose injection, blood glucose levels in Pten+/− mice returned to fasting levels in less than 60 min, approximately twice as rapidly as in wild-type mice.
Table 1Blood glucose levels in wild-type and Pten+/− mice during ad libitum feeding and after a 15-h fast Mean blood glucose±SD (mmol/l)p value (independent Student’s t test)Wild-typePten+/−Ad libitum feeding7.1 ± 0.4 (n = 5)6.3 ± 0.2 (n = 4)0.006Fasting6.5 ± 0.3 (n = 5)5.8 ± 0.3 (n = 5)0.008Fig. 1Insulin hypersensitivity in Pten+/−mice. a Insulin challenge test. Mice were fasted for 15 h prior to i.p. injection of insulin (0.6 mU/g body weight). Blood glucose levels were determined at the indicated times. Means±SEM for six wild-type mice (white circles) and six Pten+/− mice (black circles). b Glucose tolerance test. Mice were fasted for 15 h prior to i.p. injection of glucose at 2 mg/g body weight. Means±SEM for six wild-type mice (white circles) and seven Pten+/− mice (black circles)
The observed difference in ability to normalise blood glucose levels could be due to differences in insulin sensitivity or islet function and insulin production. We therefore investigated insulin levels in the mice, as well as properties of pancreatic cell morphology and function. Fasting plasma insulin levels were slightly but significantly lower in Pten+/− mice (Table 2), which is consistent with increased peripheral insulin sensitivity. Fifteen minutes after i.p. glucose administration (2 mg glucose/g body weight), similar increases in plasma insulin were observed in the two groups (Table 2), suggesting that there is no marked difference in insulin production in the Pten+/− mice. Insulin released from islets isolated from Pten+/+ and Pten+/− mice was also examined. Islets were cultured as described in Materials and methods, and incubated in the presence of low (1.67 mmol/l) or high (16.7 mmol/l) glucose. Insulin released into the medium was determined by ELISA and calculated as percentage of total insulin content in the islets. Per cent of total insulin release (presented as mean value±SD) was similar under conditions of low glucose (Pten+/+, 3.3 ± 0.4% vs Pten+/−, 2.8 ± 0.3%; p > 0.05 no significant difference, n = 3 per group) and high glucose (Pten+/+, 4.2 ± 0.7% vs Pten+/−, 3.7 ± 0.2%; p > 0.05 no significant difference, n = 3 per group).
Table 2Plasma insulin levels in wild-type and Pten+/− mice after fasting, and 15 min after i.p. glucose administration (2 mg glucose/g body weight) Mean plasma insulin±SD (ng/ml)p value (independent Student’s t test)Wild-type (n = 7)Pten+/− (n = 6)Fasting0.50 ± 0.040.36 ± 0.100.05Post-glucose challenge1.8 ± 0.52.4 ± 0.4>0.05 (not significant)
Immunofluorescence of pancreatic sections revealed similar morphology of islets of wild-type and Pten+/− mice (Fig. 2), suggesting Pten haploinsufficiency has no marked effect on islet morphology. Accordingly, beta cell mass was not found to be significantly different between wild-type and Pten+/− mice (Table 3).
Fig. 2Pancreatic islet morphology and immunohistochemistry for insulin (green) and glucagon (red) in wild-type (a) and Pten+/− (b) mice. Pancreatic sections were immunostained as described in Materials and methods and visualised by fluorescence microscopy. Original magnification ×40Table 3Pancreatic beta cell mass of Pten+/+ and Pten+/− littermates Mean beta cell mass±SE (mg)Pten+/+1.24 ± 0.30 (n = 3)Pten+/−1.29 ± 0.15 (n = 3)
Skeletal muscle is an important tissue involved in insulin-stimulated glucose uptake [10, 20]. In vivo glucose uptake was assessed by monitoring distribution of 18FDG into muscles in real-time by PET. 18FDG is transported into cells by glucose transporters and is phosphorylated by hexokinase, but is not a substrate for further metabolism in the glycolytic pathway [21]. To compare insulin-stimulated glucose uptake in wild-type and Pten+/− mice, the mice were fasted for 15 h, then injected i.p. with insulin (0.6 mU/g body weight) 20 min prior to injection with 18FDG via the tail vein. Mice were imaged for 18FDG uptake into the hindlimb for 60 min (Fig. 3a). 18FDG activity in hindlimbs was analysed as a proportion of total body activity, and was found to be higher in Pten+/− than in wild-type mice at all time-points (Fig. 3b).
Fig. 3Insulin-stimulated uptake of 18FDG in wild-type and Pten+/− mice. Mice were fasted for 15 h prior to i.p. injection of 0.6 mU insulin/g body weight. Twenty minutes after insulin injection, mice were injected with 18FDG via a tail vein, and whole-body distribution of 18FDG was monitored by microPET for 60 min. The experiment was repeated three times with similar results. Typical results are shown. a–f Imaging of 18FDG distribution in coronal section; wild-type mouse is on the left and Pten+/− mouse is on the right (a 5 min; b 15 min; c 25 min; d 35 min; e 45 min; f 55 min). Hindlimb muscles were highlighted as regions of interest, and 18FDG activity in each region of interest quantified across coronal planes at each time-point, as described in Materials and methods. g Graphical representation of relative 18FDG activity. Data are shown as proportion of 18FDG activity in hindlimbs relative to activity in whole body for wild-type mice (circles) and Pten+/− mice (triangles) (mean±SD; n = 3 per point)
Tissues from Pten+/− mice exhibited slightly reduced levels of Pten protein relative to wild-type mice [14, 22–24]. In order to examine cellular glucose uptake and intracellular signalling events, primary myocytes were cultured from hindlimb muscle of wild-type and Pten+/− mice. In the absence of insulin, cells from Pten+/− mice showed increased 2-deoxy[3H]glucose uptake at all time-points measured, to a maximum increase of twofold relative to wild-type cells at the 3 h time-point (Fig. 4a). This result, together with the lower fasting glucose level of Pten+/− mice, suggest that the PI3K/PKB pathway was chronically activated in Pten+/− animals. In the presence of insulin, Pten+/− cells exhibited a further increase in glucose uptake, up to threefold greater than wild-type cells. This observation is consistent with the above results of the insulin and glucose challenge tests and 18FDG uptake experiment in showing a hypersensitivity to insulin due to Pten haploinsufficiency.
Fig. 4a 2-Deoxy[3H]glucose uptake in wild-type and Pten+/− myocytes. Myocytes were serum-starved for 12 h prior to incubation with 10 μmol/l 2-deoxy[3H]glucose (0.037 MBq/μmol/l) for the indicated times with 0 or 1 μmol/l insulin. Following incubation cells were lysed and aliquots of lysates were taken for determination of radioactivity by scintillation counting. Means±SD for three separate determinations. White circles, wild-type cells 0 μmol/l insulin; black circles, wild-type cells 1 μmol/l insulin; white triangles, Pten+/− cells 0 μmol/l insulin; black triangles, Pten+/− cells 1 μmol/l insulin. b Cell-surface GLUT1 and GLUT4 levels. Levels of cell-surface GLUT1 and GLUT4 proteins in wild-type (WT) and Pten+/− cells were determined as described in Materials and methods
Glucose transport into skeletal muscle occurs by a facilitated diffusion process mediated primarily by the glucose transporter proteins, GLUT1 and GLUT4. The level of GLUT4 at the plasma membrane is tightly regulated by insulin, whereas GLUT1 is found constitutively on the cell surface [25]. Since 2-deoxy[3H]glucose uptake was elevated in Pten+/− cells even in the absence of insulin (Fig. 4a), we analysed cell-surface GLUT4 and GLUT1 by western immunoblot as described in Materials and methods (Fig. 4b). This analysis showed increased GLUT4 at the cell surface in Pten+/− cells, while similar GLUT1 levels were observed in the two cell types.
As PKB is a key effector kinase that mediates many of the downstream cellular events induced by the PI3K signalling pathway, including insulin-stimulated glucose uptake [26, 27], we determined the phosphorylation status of PKB in wild-type and Pten+/− myocytes. Little to no phosphorylation was observed in the absence of insulin, and phosphorylation at Ser473 was observed upon addition of insulin (Fig. 5). Whereas PKB phosphorylation started to decrease after 4 h in wild-type cells in the presence of insulin, phosphorylation was sustained in Pten+/− cells during the course of the experiment (up to 6 h). Immunoblotting for total PKB protein showed that PKB levels were consistent in Pten+/− and Pten+/+ cells (Fig. 5). Immunoblotting for vinculin was included as a protein-loading control. An immediate downstream substrate of PKB is GSK3β. This enzyme becomes inactivated upon phosphorylation by PKB, in turn allowing its substrate, glycogen synthase, to mediate glycogen synthesis and thus promote glucose storage. Consistent with the observed enhancement of PKB phosphorylation in Pten+/− cells, these cells also exhibited a higher degree of GSK3β phosphorylation in comparison with Pten+/+ cells (Fig. 5).
Fig. 5Phosphorylation of PKB and GSK3β. Myocytes were serum-starved for 12 h prior to incubation with 1 μmol/l insulin for the indicated times. Cell lysates were prepared, and proteins were separated by PAGE followed by western immunoblot analysis for vinculin, total PKB, phospho-PKB (p-PKB S473) and phospho-GSK3β (p-GSK3β) as indicated. This experiment was repeated three times with typical results shown. WT, wild-type
Discussion
Recent studies utilising tissue-specific deletion of Pten in liver, adipose and muscle have shown that these organs make important contributions to the glucose homeostasis of the animal and clearly establish Pten as a negative regulator of insulin signalling in vivo [9–11]. In a separate report, in vivo antisense treatment resulted in 90% reduction of Pten levels in the liver of diabetic mice, normalising blood glucose levels and improving performance in an insulin tolerance test [28]. While the above studies focused on the role of Pten in specific tissues, the current work defines the effects of partial systemic reduction of Pten on the regulation of insulin sensitivity in the context of the whole animal.
PTEN expression/activity is tightly regulated by a complex mechanism that is currently poorly understood. PTEN is transcriptionally regulated by transcription factors such as p53, Egr-1, NFκB and SMADs, while protein levels and activity are modulated by phosphorylation, oxidation, subcellular localisation, phospholipid binding and protein stability [29]. Previous studies showed that Pten haploinsufficiency results in only a 15–50% reduction in Pten protein, leaving a substantial amount of Pten protein remaining in the tissues examined [14, 22–24, 30]. In our hands, western blot analysis indicated similar decreases in tissue Pten levels (data not shown). The present study provides the first evidence that Pten haploinsufficiency leads to insulin hypersensitivity in vivo, suggesting that even small changes in Pten levels or activity can lead to dramatic effects on insulin responses. Thus partial systemic inhibition of PTEN activity may represent a novel strategy to ameliorate the pathology of type 2 diabetes.
The PI3K/PKB signalling axis is central to maintenance of normal glucose homeostasis [1]. Pten-deficient cells typically have elevated levels of intracellular PIP3 [31, 32]. The sustained phosphorylation of PKB/Akt and a downstream effector, GSK3β, observed in the current study is consistent with previous reports of elevated PKB/Akt activation as a result of impaired desensitisation of PI3K-dependent signals under conditions of Pten inactivation or inhibition [9–11, 28]. A subtle reduction of Pten in the Pten+/− mice appears sufficient to achieve a prolonged and robust insulin signal. The roles of PIP3 and PKB/Akt in insulin-mediated glucose uptake have been well-established; therefore, our data collectively suggest that sustained PI3K/PKB/Akt/GSK3 signalling may, in part, contribute to insulin hypersensitivity in Pten+/− mice. However, other PKB/Akt-independent signalling pathways, as well as additional signals delivered by the protein phosphatase and/or adaptor functions of Pten may also contribute to the insulin hypersensitivity phenotype manifested by Pten haploinsufficiency.
GLUT1 glucose transporters are constitutively present at the cell surface, while GLUT4 cycles between intracellular vesicles and the cell surface in a manner controlled by insulin-mediated signalling mechanisms [25]. In unstimulated fat and muscle cells, the majority of GLUT4 exists in intracellular compartments, while the insulin signal stimulates the exocytosis of GLUT4 to the cell surface. Studies involving PKB/Akt-null animals and silencing of PKB/Akt by RNA interference have demonstrated that PKB/Akt is of primary importance in GLUT4 translocation [33–36]. In the current study, we observed an enhancement of 2-deoxyglucose uptake in Pten+/− cells even in the absence of insulin stimulation, which was consistent with our observation of increased biotinylation of GLUT4 at the cell surface, while GLUT1 levels were unaffected. However, we detected no increase in phosphorylation of PKB/Akt in the absence of insulin in Pten+/− cells. Consistent with these observations, a recent study in which Pten was specifically knocked down by RNA interference reported slight but significant enhancement of deoxyglucose uptake in adipocytes, but no apparent increase in phosphorylation of PKB/Akt [37]. These results could reflect a mechanism(s) affecting glucose transport involving Pten/PIP3 but independent of PKB/Akt. Such a mechanism could involve the atypical protein kinases C-λ and -ζ (PKC-λ and PKC-ζ), which, like PKB/Akt, are subject to control by PI3K via PIP3 and 3-phosphoinositide-dependent protein kinase-1 (PDK-1) [38–40]. A number of studies have implicated PKC-λ/ζ in insulin-stimulated glucose transport and GLUT4 translocation [38, 41–43]. These studies indicate a role for PKC-λ/ζ which appears to be complementary or parallel to that of PKB/Akt.
The importance of the PI3K pathway in insulin signalling has raised interest in developing pharmaceutical antagonists targeting PIP3 phosphatases such as PTEN or Sh2-containing inositol 5′-phosphatase 2 (SHIP2; also known as Inppl1) in an effort to enhance insulin responsiveness in type 2 diabetes [44]. While a study on Ship2-null mice initially implicated Ship2 as a key regulator of glucose uptake [45], it was subsequently recognised that these mice also had deleted a neighbouring gene, Phox2a, which may have contributed to the observed phenotype (corrigendum appears in Nature vol. 431, p. 878). A new gene-targeted mouse has since been generated in which only the gene encoding Ship2 is deleted [46]. Interestingly, this mouse does not exhibit insulin hypersensitivity but is protected against diet-induced obesity,  [46, 47]. In the light of these new findings, our observations of dramatic enhancement of insulin sensitivity and glucose uptake resulting from Pten haploinsufficiency suggests that PTEN plays a role in the desensitisation of insulin signalling, and strengthens the hypothesis that PTEN is a key regulator of insulin-stimulated glucose uptake.
Type 2 diabetes is associated with insulin deficiency and resistance to insulin signalling [48, 49]. The current study provides further support for PTEN as a candidate for drug intervention in treating type 2 diabetes. However, enthusiasm for systemic PTEN inhibitors as a therapeutic approach for treating type 2 diabetes is tempered by the potential side-effects of such agents. Most notably, a modest reduction of Pten in Pten+/− mice promotes tumour formation in a wide variety of tissue, suggesting that long-term chronic administration of systemic PTEN inhibitors may lead to enhanced cancer development and progression. Furthermore, the PI3K pathway is an important regulator of numerous normal cellular processes and systemic PTEN inhibition may cause dysfunction of many cells and tissues, leading to alterations in liver, kidney, brain function, etc. For example, hepatocyte-specific Pten-deficient mice exhibit hepatomegaly and liver steatosis [9]. Systemic inhibition of PTEN may therefore be unacceptable clinically because of these inherent potential toxicities [44]. However, targeted delivery of PTEN antagonists to muscle cells or adipocytes may overcome these limitations and they hold promise as clinically viable agents in treatment of type 2 diabetes [44]. This study shows that partial inhibition of Pten is sufficient to enhance insulin sensitivity. This observation is important from a therapeutic standpoint, as most drugs are unlikely to completely inhibit enzyme activity in vivo, nor would complete inhibition be desirable given the potential for cancer development resulting from chronic or overactivation of the PI3K/PKB pathway [12, 14, 23].
In conclusion, Pten haploinsufficiency results in insulin hypersensitivity, which underscores the importance of PTEN in attenuating insulin signals, and suggests that small-molecule drugs that antagonise PTEN activity may represent a novel class of insulin-sensitising agents and targeted delivery of these agents to muscle cells or adipocytes may have potential clinical utility in treating insulin resistance observed in type 2 diabetes.

insulin hypersensitivity
glucose uptake
insulin sensitivity
pten haploinsufficiency


Chitotriosidase deficiency is not associated with human hookworm infection in a Papua New Guinean population

Human chitotriosidase (CHIT1) is a chitinolytic enzyme with suggested anti-fungal properties. Previous studies have suggested that chitotriosidase may also protect individuals against filarial nematode infections and malaria. A mutant allele, which renders chitotriosidase unstable and enzymatically inactive, is found at a frequency of >20% in Caucasians and other populations. This allele is found at much lower frequency in parts of West Africa where malarial and intestinal helminth infections are endemic. Here, we investigate whether there is a significant association between chitotriosidase genotype and the intensity of hookworm infection in 693 individuals from five villages in Papua New Guinea. Individuals were genotyped for chitotriosidase using a PCR-based assay. There was no association between CHIT1 genotype and the intensity of hookworm infection as determined by faecal egg counts. The frequency of the mutant allele was 0.251, very similar to that found in non-endemic countries. The extent of geographical variation in allele frequencies across worldwide populations was not high (Fst = 0.11), and does not provide evidence for directional selection at this locus between different geographical areas. We conclude that the CHIT1 genotype does not play a crucial role in protection against hookworm infection. This does not correlate with a previous study that linked the mutant CHIT1 genotype to filariasis susceptibility. The possible reasons for this discrepancy are discussed.
1
Introduction
Chitotriosidase is a chitinolytic enzyme which was recently identified in man and is synthesised in large quantities by activated macrophages (Hollak et al., 1994). As humans lack endogenous chitin, a role for chitotriosidase is not fully understood. However the anti-fungal action of homologous plant chitinases has led to the suggestion that chitotriosidase may degrade chitin containing pathogens (Boot et al., 1998). The chitotriosidase gene (CHIT1) consists of 12 exons located on chromosome 1q31–32 (Boot et al., 1998). A 24 base pair duplication in exon 10 leads to the activation of a cryptic 3′ splice site that results in an abnormally spliced mRNA with an inframe 87 nucleotide deletion (Boot et al., 1998). The mutant protein lacks amino acids 344–372 that are required for the formation of the TIM-barrel catalytic core (Boot et al., 1998; Fusetti et al., 2002). Macrophages from chitotriosidase deficient individuals express only small amounts of mRNA and secrete virtually no chitotriosidase protein (Boot et al., 1998).
In Dutch and Ashkenazi Jewish populations 6% of individuals are homozygous for the mutant allele, whereas 35% and 34%, respectively, are heterozygous carriers (Boot et al., 1998) with a mutant allele frequency of 0.23. A similar allelic frequency is present in Portugal (0.22), Sicily (0.27) and Sardinia (0.21) (Malaguarnera et al., 2003). However in two West African countries, Benin and Burkina Faso, that are mesoendemic for Plasmodium falciparum malaria and endemic for gastrointestinal helminths, a total absence of the homozygous mutation and significant reduction in heterozygous individuals has been reported, with mutant allele frequency 0.00 and 0.02, respectively (Malaguarnera et al., 2003). This observation led to the hypothesis that chitotriosidase may be involved in resistance to protozoan or helminth infections common in tropical countries (Malaguarnera et al., 2003).
Parasitic nematodes are known to contain chitin, and are thus a potential target of human chitotriosidase. Chitin is found in the egg-shell of both free-living and parasitic nematodes, and in the microfilarial sheath surrounding the first-stage larvae of filarial nematodes (Fuhrman and Piessens, 1985). More recently, chitin has been demonstrated in the pharynx of both the free-living Caenorhabditis elegans (Zhang et al., 2005) and the gastrointestinal parasite Oesophagostomum dentatum (Neuhaus et al., 1997). There are two chitin synthase genes in C. elegans, chs-1 and chs-2, encoding eggshell and pharynx chitin, respectively, and chs-2 knockdown leads to lack of pharyngeal function and starvation (Zhang et al., 2005). This suggests that host chitotriosidase could potentially interfere with parasitic nematode feeding. Two studies to date have investigated associations between CHIT1 genotype and filarial nematode infection. Choi et al. (2001) found significant association between susceptibility to lymphatic filariasis (Wuchereria bancrofti) and homozygosity for the mutant allele in an Indian population, but in a similar study in Papua New Guinea, no association between CHIT1 genotype and lymphatic filariasis was observed (Hise et al., 2003).
There have been no previous studies investigating CHIT1 genotype and gastrointestinal nematode infection. Human hookworm infection is an abundant chronic gastrointestinal nematode infection in sub-tropical and tropical countries, causing significant morbidity, principally due to iron-deficiency anaemia (Brooker et al., 2004). Human hookworm burden (as assessed by faecal egg count) is known to be under host genetic control (Williams-Blangero et al., 1997), but the genes responsible have not been identified. The aims of the current study were (1) to investigate associations between CHIT1 genotype and human hookworm infection in an endemic population from Madang Province, Papua New Guinea and (2) to investigate evidence for directional selection at the CHIT1 locus, by comparing worldwide allele frequency variation at the CHIT1 locus to that observed at other loci across the genome.
2
Materials and methods
2.1
Geographical location and study population
The study population consisted of five villages in lowland Madang Province, Papua New Guinea, where Necator americanus is the only hookworm species (Pritchard et al., 1990). Villages were censused and pedigree information collected in 1998, and sample containers for faecal collection offered to all individuals aged 4 years and above. Faecal egg counts were performed using a modified McMaster salt flotation method and results were expressed as eggs per gram (epg) of faeces (Quinnell et al., 2004). Venous blood was then collected, and all individuals were offered treatment with albendazole or pyrantel pamoate. DNA was extracted from buffy coats using phenol/chloroform. In September 2001, a second faecal sample was taken from three of the five villages and reinfection hookworm burden determined; some previously untreated individuals provided samples in 2001. Hookworm epg has been shown to be a heritable phenotype in the study population (Breitling and Quinnell, unpublished results). The study was approved by the Medical Research Advisory Committee of Papua New Guinea and informed consent was received from all subjects or their parents.
2.2
Chitotriosidase genotyping by PCR
Thermal cycling reactions consisted of Reddy Mix PCR master mix (ABgene), 4.5 pmol forward and 4.5 pmol reverse chitotriosidase primers (forward: AGCTATCTGAAGCAGAAG; reverse: GGAGAAGCCGGAAAGTC) (Sigma) and 2 μl genomic DNA in a final volume of 15 μl. Samples were run in 96 well PCR plates (ABgene) on a Peltier PTC-200 Thermal Cycler (MJ Research). The thermal cycling protocol was: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s; and 72 °C for 5 min. Thermal cycling products with 10 base pair markers (Promega) were separated on 4% high resolution agarose (Sigma) gels in 1× TBE buffer and ethidium bromide. Gels were viewed using the Gene Genius Bio-imaging System and Gene Snap software (Syngene). The size of wild type product is 75 base pairs whereas the mutant product is 99 base pairs due to the mutant allele containing a 24 base pair duplication in exon 10 (Boot et al., 1998).
2.3
Statistical analysis
Variation in faecal egg counts by CHIT1 genotype was analysed under both a codominant and recessive model. Faecal egg counts were highly overdispersed, so analysis was performed using a generalised linear model with a negative binomial error structure in Stata 9.1. This method has been shown to be more accurate than analysis of log-transformed parasite burden data (Wilson et al., 1996). Significant covariates included in the analysis were age, village, faecal consistency, anthelmintic treatment and the age × treatment and village × treatment interaction terms. Egg counts from both years were analysed together; since some individuals were sampled both pre- and post-treatment, standard errors were adjusted for the non-independence of samples from the same individual using the ‘cluster’ option. To control for non-independence of individuals due to genetic relatedness, further analysis was performed with the total test of association in a variance components framework using the programme QTDT (Abecasis et al., 2000). Finally, to control for potential population stratification, transmission disequilibrium testing was carried out using the orthogonal association model in QTDT. The phenotypic variable used for these analyses was the residuals from a negative binomial regression of egg counts against significant covariates; where two samples were taken from the same individual, the mean residual for that individual was calculated.
To quantify geographic variation in allele frequencies, Fst values were calculated according to Cockerham and Weir (1984) using the programme FSTAT 2.9.3 (Goudet, 1995). Allele frequencies were compared across five broad geographical areas, using the weighted mean allele frequency for each geographical area (Europe/Mediterranean, Africa, South Asia, East Asia and Papua New Guinea). To compare observed values of Fst to those expected for neutral markers, Fst was also calculated across comparable populations selected from the Human Genome Diversity Panel using published data for 210 genome-wide short insertion/deletion polymorphisms genotyped by the Mammalian Genotyping Service (data available from http://research.marshfieldclinic.org/genetics/) (Soranzo et al., 2005). The populations selected were European (French/Italian), African (Yoruba), South Asia (Sindhi), East Asia (Chinese Han) and Papuan.
3
Results
In total 693 individuals from Papua New Guinea were genotyped for the chitotriosidase mutation. The allelic frequency of the 24 bp duplication in 279 unrelated individuals, determined by PCR genotyping, was 0.251. The percentage of individuals who were homozygous mutant was 6% (16/279), heterozygous 39% (108/279) and homozygous wild type 56% (155/279). Genotype frequencies did not differ from those expected under Hardy–Weinberg equilibrium (χ2 = 0.25, d.f. = 1, P = 0.62).
Both egg counts and CHIT1 genotypes were available for a total of 602 individuals (574 sampled pre-treatment and 210 sampled post-treatment). The prevalence and intensity of infection (mean egg count) in these individuals were 81% (2574 epg) pre-treatment, and 92% (1727 epg) post-treatment. Egg counts were very similar across CHIT1 genotypes both pre-treatment and after reinfection (Table 2). Combined analysis of both years’ data revealed no significant relationship between egg count and genotype under either a codominant (χ2 = 0.03, d.f. = 1, p = 0.87) or recessive model (χ2 = 0.02, d.f. = 1, p = 0.90). Further analysis using the total test of association in QTDT, controlling for non-independence of individuals due to genetic relatedness, again showed no significant association between CHIT1 genotype and egg count (χ2 = 0.39, d.f. = 1, p = 0.53). Similarly, transmission disequilibrium testing, which controls for population stratification, was not significant (χ2 = 0.58, d.f. = 1, p = 0.45).
Published allele frequencies for CHIT1 in different geographical areas are shown in Table 1. Using these data and data from the current study, Fst values were calculated to measure variation in allele frequency across five broad geographical areas (Europe, Africa, South Asia, East Asia, and Papua New Guinea). The Fst across all populations for CHIT1 was 0.11. This was comparable to the median Fst of 0.12 for 210 indels typed in populations from similar geographical areas. Pairwise comparisons showed higher Fst values between Africa and East Asia (Fst = 0.69) and Africa and South Asia (Fst = 0.54), than those between other areas (Fst < 0.40). In each case the observed Fst was greater than that for all but 7 of 210 indels, giving an empirical P = 0.038 (8/210).
4
Discussion
The results from the present study in Papua New Guinea strongly suggest that chitotriosidase does not have a critical role in protection against N. americanus. The relative number of people who are wild type homozygous for chitotriosidase, heterozygous and homozygous mutant is similar to that seen in countries where this infection is not endemic, suggesting no advantage for the wild type allele in endemic areas. Moreover, there was no evidence for an association between CHIT1 genotype and hookworm burden in our study population.
The mutant chitotriosidase genotype has been associated with an increased susceptibility to W. bancrofti infection in a study of 216 individuals from South India (Choi et al., 2001). Previously it has been demonstrated that the microfilariae, oocytes and zygotes of the filarial nematode Brugia malayi contain chitin (Fuhrman and Piessens, 1985; Schraermeyer et al., 1987). In the rodent filarial nematode Acanthocheilonema vitae, there are distinctive chito-oligomeric N-glycans that contain projections of up to six GlcNAc residues that may be involved in parasite-host interactions (Haslam et al., 1999). Additionally chitin synthase is required for sheath development in microfilarial progeny. Chitin has also been identified in the cuticle of mature W. bancrofti (Araujo et al., 1993). The presence of chitin in the sheath and cuticle of filarial nematodes and the requirement for chitin synthase in reproduction show that chitin has an important role in the lifecycle of these species. If host-derived chitotriosidase can access this chitin, thus interfering with the parasite's growth and development, then it may explain why susceptibility to W. bancrofti is increased in individuals with the mutant genotype. However, a second study by Hise and coauthors (Hise et al., 2003) in 906 individuals in a region endemic for bancroftian filariasis in Papua New Guinea did not find any significant correlation with infection status or disease phenotype. Thus the issue is in further need of clarification.
In contrast to filarial worms, gastrointestinal nematodes such as hookworm do not have a microfilarial stage, and the chitin-containing eggs are not accessible to host chitotriosidase. However, chitin has recently been demonstrated in the pharynx of the free-living nematode C. elegans (Zhang et al., 2005). It is not known whether chitin occurs in the pharynx of N. americanus, but it has been demonstrated in the related strongyloid parasite O. dentatum (Neuhaus et al., 1997). Pharyngeal chitin provides a potential target for host chitotriosidase, both in tissue-dwelling larval stages, which may be accessible for macrophage or neutrophil-derived chitotriosidase, and blood-feeding adults. Chitotriosidase levels can be elevated in serum, e.g. in acute falciparum malaria (Barone et al., 2003), and as adult hookworms are thought to ingest 0.03–0.2 ml of blood daily, they may be exposed to high levels of chitotriosidase. However, the lack of an association between CHIT1 genotype and hookworm burden seen in the present study suggests that chitotriosidase is not an important effector mechanism against hookworm infection. Further studies would be useful to examine the bioavailability of nematode chitin, its accessibility for exogenous chitinases, or their effect on parasite viability.
The mutant allele frequency observed in the current study was higher than that in another Papuan population from East Sepik Province (Hise et al., 2003). Such variation within Papua New Guinea is not surprising, and differences between these populations have been observed at other loci (Main et al., 2001). Analysis of worldwide variation in allele frequencies showed that the degree of population differentiation at the CHIT1 locus (Fst = 0.11) was very similar to the median value for 210 indels across the genome. Though the populations sampled were not identical, this suggests that there has not been directional selection at this locus across different geographical areas, and that the worldwide variation in allele frequencies is consistent with genetic drift (Akey et al., 2002). There was some evidence for higher than average population differentiation at the CHIT1 locus between African and Asian populations, which may suggest some local adaptation, though this would not be significant after correction for multiple testing.
Malaguarnera et al. (2003) suggest that an intact CHIT1 genotype may be advantageous in protection against malaria, based on the very low frequency of the mutant genotype in Burkina Faso and Benin, where malaria is meso-endemic. However, our results are not consistent with the hypothesis that chitotriosidase is involved in protection against malaria, or any tropical disease. There is no evidence for directional selection worldwide, and the greatest difference in allele frequencies is observed between Africa and other tropical countries, also endemic for malaria (Choi et al., 2001; Chien et al., 2005). Although Barone et al. (2003) found that chitotriosidase levels are elevated in the serum in acute P. falciparum malaria, there is no evidence that elevated serum chitotriosidase levels have an impact on the severity and outcome of malaria, and no study has compared genotypes with disease status. Indeed, increased serum chitotriosidase may actually increase malaria transmission, by inhibiting the formation of the peritrophic membrane in the anopheline gut (Di Luca et al., 2007). As the study area in Papua New Guinea is endemic for malaria, we have performed an association analysis which showed no significant difference between chitotriosidase genotype and the presence of P. falciparum and P. vivax malaria infection (data not shown). The caveat of this analysis is that it was based on a single time point assessment of parasitaemia, rather than repeated measurements. As parasitaemia is very variable, we cannot make a reliable statement regarding the effect of the CHIT1 genotype on malarial infection, and further studies are needed.
Taken together, our data suggest that the chitotriosidase genotype does not play a major role in protection against hookworm infection, but this may be different in the case of lymphatic or cutaneous filariasis. However, there is no evidence for selection at this locus mediated by malaria or another tropical disease.

chitotriosidase
papua new guinea
malaria
necator americanus
chit1 mutation