Issue published June 1, 2007

I nherited and acquired diseases of the hematopoietic system can be cured by allogeneic hematopoietic stem cell transplantation. This treatment strategy is highly successful when an HLA-matched sibling donor is available, but if not, few therapeutic options exist. Gene-modified, autologous bone marrow transplantation can circumvent the severe immunological complications that occur when a related HLA-mismatched donor is used and thus represents an attractive alternative. In this review, we summarize the advantages and limitations associated with the use of gene therapy to cure SCID. Insertional mutagenesis and technological improvements aimed at increasing the safety of this strategy are also discussed.

T he transfusion of lymphocytes, referred to as adoptive T cell therapy, is being tested for the treatment of cancer and chronic infections. Adoptive T cell therapy has the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease. This form of personalized medicine is now in various early- and late-stage clinical trials. These trials are currently testing strategies to infuse tumor-infiltrating lymphocytes, CTLs, Th cells, and Tregs. Improved molecular biology techniques have also increased enthusiasm and feasibility for testing genetically engineered T cells. The current status of the field and prospects for clinical translation are reviewed herein.

I n this issue of the JCI, Wang, Clemens, and colleagues demonstrate that hypoxia-inducible factor α (HIFα) signaling in bone-building osteoblasts is central to the coupling of angiogenesis and long bone development in mice (see the related article beginning on page 1616). They show that bone formation controlled by osteoblast HIFα signaling is not cell autonomous but is coupled to skeletal angiogenesis dependent upon VEGF signaling. Thus, strategies that promote HIFα signaling in osteoblasts may augment bone formation and accelerate fracture repair.

A new study by Galeano and colleagues in this issue of the JCI reports the first glomerular disease caused by a genetic defect in sialic acid biosynthesis (see the related article beginning on page 1585). Mice that harbor mutations in the Gne/Mnk gene produce lower amounts of sialic acid, suffer from hematuria, proteinuria, and structural defects in the glomerulus and die within days after birth. Remarkably, the lesion can be reversed through dietary addition of N-acetylmannosamine, a sialic acid precursor, raising the intriguing possibility that this approach might have therapeutic benefit in patients with glomerular disease.

W hile the term neuroinflammation often conjures up images of cellular damage, mounting evidence suggests that certain proinflammatory molecules, such as the cytokine IL-1β, may have beneficial and protective effects. In a report in this issue of the JCI, Shaftel and coworkers have generated an elegant mouse model in which local hippocampal overexpression of IL-1β in an Alzheimer disease (AD) transgenic mouse model resulted not in the expected exacerbation of the amyloid β plaque deposition common to AD, but instead in plaque amelioration (see the related article beginning on page 1595). Thus, manipulation of the immune system may be a potential therapeutic approach to protect against AD, although further studies are needed to understand all of the downstream effects of this manipulation.

T hrombin is clearly a key trigger of thrombosis, the proximal cause of most morbidity and mortality in atherosclerotic cardiovascular disease. Might thrombin also contribute to longer-term, structural changes in the arterial wall that promote narrowing and clotting? A study in this issue of the JCI argues that it can. Aihara et al. report that haploinsufficiency of heparin cofactor II, a glycosaminoglycan-dependent thrombin inhibitor, exacerbates injury- or hyperlipidemia-induced arterial lesion formation in mice, possibly by excessive thrombin signaling through protease-activated receptors (see the related article beginning on page 1514).

A ctivation of the transcription factor NF-κB/Rel has been shown to be involved in inflammatory disease. Here we studied the role of RelA/p65, the main transactivating subunit, during acute pancreatitis using a Cre-loxP strategy. Selective truncation of the rela gene in pancreatic exocrine cells led to both severe injury of the acinar cells and systemic complications including lung and liver damage. Our data demonstrated that expression and induction of the protective pancreas-specific acute phase protein pancreatitis-associated protein 1 (PAP1) depended on RelA/p65. Lentiviral gene transfer of PAP1 cDNA reduced the extent of necrosis and infiltration in the pancreata of mice with selective truncation of RelA/p65. These results provide in vivo evidence for RelA/p65 protection of acinar cell death via upregulation of PAP1. Moreover, our data underscore the pancreas-specific role of NF-κB/Rel and suggest multidimensional roles of NF-κB/Rel in different cells and contexts during inflammation.

A ctivation of the inhibitor of NF-κB kinase/NF-κB (IKK/NF-κB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell–specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell–specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-κB target genes, including mediators of the inflammatory response such as TNF-α and ICAM-1. Indeed, inhibition of TNF-α activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-κB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.

H eparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII^–/– mice were embryonically lethal. In HCII^+/– mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor–1–positive (PAR-1–positive) cells was increased in the thickened vascular wall of HCII^+/– mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII^+/– mice. The intimal hyperplasia in HCII^+/– mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance.

E ndothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

S T2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II– and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-κB, it inhibited angiotensin II– and phenylephrine-induced phosphorylation of inhibitor of NF-κBα (IκBα) and NF-κB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myocardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2^–/– mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening, more fibrosis, and impaired survival compared with WT littermates. Furthermore, recombinant IL-33 treatment reduced hypertrophy and fibrosis and improved survival after TAC in WT mice, but not in ST2^–/– littermates. Thus, IL-33/ST2 signaling is a mechanically activated, cardioprotective fibroblast-cardiomyocyte paracrine system, which we believe to be novel. IL-33 may have therapeutic potential for beneficially regulating the myocardial response to overload.

B cells from patients with common variable immunodeficiency (CVID) who are heterozygous for transmembrane activator and CAML interactor (TACI) mutation C104R, which abolishes ligand binding, fail to produce Igs in response to TACI ligand. It is not known whether this is due to haploinsufficiency or dominant interference. Using in vitro transfection assays, here we demonstrate that C104R and the corresponding murine TACI mutant, C76R, which also does not bind ligand, dominantly interfere with TACI signaling. This effect was dependent on preassociation of the mutants with WT TACI in the absence of ligand. The mutants did not interfere with ligand binding by WT TACI, suggesting that they may act by disrupting ligand-induced receptor rearrangement and signaling. This work demonstrates that TACI preassembles as an oligomeric complex prior to ligand binding and provides a mechanistic insight into how the heterozygous C104R TACI mutation can potentially lead to CVID.

Determination of the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that, despite being derived from healthy people, antibodies from B cells that have class switched to IgD via genetic recombination (and thus become class switched to Cδ [Cδ-CS] cells) are highly reactive to self antigens. Over half of the antibodies from Cδ-CS B cells bind autoantigens on human epithelioma cell line 2 (HEp-2) cells or antinuclear antigens, and a quarter bind double-stranded DNA; both groups of antibodies are frequently polyreactive. Intriguingly, some Cδ-CS B cells have accumulated basic residues in the antibody variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, while other Cδ-CS B cells are naturally autoreactive. Though the total percentage was appreciably less than for Cδ-CS cells, a surprising 31% of IgG memory cell antibodies were somewhat autoreactive, and as expected, about 24% of naive cell antibodies were autoreactive. We interpret these findings to indicate either that autoreactive B cells can be induced to class switch to IgD or that autoreactive B cells that use IgD as the B cell receptor are not effectively deleted. Determination of the mechanism by which the majority of Cδ-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight into the enigmatic function of the IgD antibody.

T he ileal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive E. coli (AIEC) that are able to adhere to and invade intestinal epithelial cells. Here, we show that CD-associated AIEC strains adhere to the brush border of primary ileal enterocytes isolated from CD patients but not controls without inflammatory bowel disease. AIEC adhesion is dependent on type 1 pili expression on the bacterial surface and on carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6) expression on the apical surface of ileal epithelial cells. We report also that CEACAM6 acts as a receptor for AIEC adhesion and is abnormally expressed by ileal epithelial cells in CD patients. In addition, our in vitro studies show that there is increased CEACAM6 expression in cultured intestinal epithelial cells after IFN-γ or TNF-α stimulation and after infection with AIEC bacteria, indicating that AIEC can promote its own colonization in CD patients.

H oloprosencephaly (HPE) is a clinically heterogeneous developmental anomaly affecting the CNS and face, in which the embryonic forebrain fails to divide into distinct halves. Numerous genetic loci and environmental factors are implicated in HPE, but mutation in the sonic hedgehog (Shh) gene is an established cause in both humans and mice. As growth arrest–specific 1 (Gas1) encodes a membrane glycoprotein previously identified as a Shh antagonist in the somite, we analyzed the craniofacial phenotype of mice harboring a targeted Gas1 deletion. Gas1^–/– mice exhibited microform HPE, including midfacial hypoplasia, premaxillary incisor fusion, and cleft palate, in addition to severe ear defects; however, gross integrity of the forebrain remained intact. These defects were associated with partial loss of Shh signaling in cells at a distance from the source of transcription, suggesting that Gas1 can potentiate hedgehog signaling in the early face. Loss of a single Shh allele in a Gas1^–/– background significantly exacerbated the midline craniofacial phenotype, providing genetic evidence that Shh and Gas1 interact. As human GAS1 maps to chromosome 9q21.3–q22, a region previously associated with nonsyndromic cleft palate and congenital deafness, our results establish GAS1 as a potential locus for several human craniofacial malformations.

M utations in the key enzyme of sialic acid biosynthesis, uridine diphospho–N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne^M712T/M712T) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne^M712T/M712T muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne^M712T/M712T pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne^M712T/M712T knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

N euroinflammation is a conspicuous feature of Alzheimer disease (AD) pathology and is thought to contribute to the ultimate neurodegeneration that ensues. IL-1β has emerged as a prime candidate underlying this response. Here we describe a transgenic mouse model of sustained IL-1β overexpression that was capable of driving robust neuroinflammation lasting months after transgene activation. This response was characterized by astrocytic and microglial activation in addition to induction of proinflammatory cytokines. Surprisingly, when triggered in the hippocampus of the APPswe/PS1dE9 mouse model of AD, 4 weeks of IL-1β overexpression led to a reduction in amyloid pathology. Congophilic plaque area fraction and frequency as well as insoluble amyloid beta 40 (Aβ40) and Aβ42 decreased significantly. These results demonstrate a possible adaptive role for IL-1β–driven neuroinflammation in AD and may help explain recent failures of antiinflammatory therapeutics for this disease.

< i>Tsc22d3 coding for glucocorticoid-induced leucine zipper (GILZ) was initially identified as a dexamethasone-responsive gene involved in the control of T lymphocyte activation and apoptosis. However, the physiological role of this molecule and its function in the biological activity of glucocorticoids (GCs) has not been clarified. Here, we demonstrate that GILZ interacts directly with Ras in vitro and in vivo as shown by GILZ and Ras coimmunoprecipitation and colocalization upon PMA activation in primary mouse spleen T lymphocytes and thymus cells. The analysis of GILZ mutants showed that they bound Ras through the tuberous sclerosis complex box (TSC) and, depending on the Ras activation level, formed a trimeric complex with Ras and Raf, which we previously identified as a GILZ binder. As a consequence of these interactions, GILZ diminished the activation of Ras and Raf downstream targets including ERK1/2, AKT/PKB serine/threonine kinase, and retinoblastoma (Rb) phosphorylation and cyclin D1 expression, leading to inhibition of Ras- and Raf-dependent cell proliferation and Ras-induced NIH-3T3 transformation. GILZ silencing resulted in an increase in concanavalin A–induced T cell proliferation and, most notably, inhibition of dexamethasone antiproliferative effects. Together, these findings indicate that GILZ serves as a negative regulator of Ras- and Raf-induced proliferation and is an important mediator of the antiproliferative effect of GCs.

S keletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxia-inducible factor α (HIFα) pathway. Here we provide evidence that HIFα promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIFα in osteoblasts through selective deletion of the von Hippel–Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIFα pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone.

A ggrecan loss from cartilage in arthritis is mediated by aggrecanases. Aggrecanases cleave aggrecan preferentially in the chondroitin sulfate–2 (CS-2) domain and secondarily at the E³⁷³↓³⁷⁴A bond in the interglobular domain (IGD). However, IGD cleavage may be more deleterious for cartilage biomechanics because it releases the entire CS-containing portion of aggrecan. Recent studies identifying aggrecanase-2 (ADAMTS-5) as the predominant aggrecanase in mouse cartilage have not distinguished aggrecanolysis in the IGD from aggrecanolysis in the CS-2 domain. We generated aggrecan knockin mice with a mutation that rendered only the IGD resistant to aggrecanases in order to assess the contribution of this specific cleavage to cartilage pathology. The knockin mice were viable and fertile. Aggrecanase cleavage in the aggrecan IGD was not detected in knockin mouse cartilage in situ nor following digestion with ADAMTS-5 or treatment of cartilage explant cultures with IL-1α. Blocking cleavage in the IGD not only diminished aggrecan loss and cartilage erosion in surgically induced osteoarthritis and a model of inflammatory arthritis, but appeared to stimulate cartilage repair following acute inflammation. We conclude that blocking aggrecanolysis in the aggrecan IGD alone protects against cartilage erosion and may potentiate cartilage repair.

I nitial immune responses to allergens may occur before birth, thereby modulating the subsequent development of atopy. This paradigm remains controversial, however, due to the inability to identify antigen-specific T cells in cord blood. The advent of MHC tetramers has revolutionized the detection of antigen-specific T cells. Tetramer staining of cord blood after CMV infection has demonstrated that effective CD8⁺ antigen-specific immune responses can follow intrauterine viral infections. We hypothesized that sensitization to antigens occurs in utero in humans. We studied cord blood B and T cell immune responses following vaccination against influenza during pregnancy. Anti-Fluzone and anti-matrix protein IgM antibodies were detected in 38.5% (27 of 70) and 40.0% (28 of 70), respectively, of cord blood specimens. Using MHC tetramers, HA-specific CD4⁺ T cells were detected among 25.0% (3 of 12) and 42.9% (6 of 14) of cord blood specimens possessing DRB1*0101 and DRB1*0401 HLA types, respectively, and were detected even when the DRB1 HLA type was inherited from the father. Matrix protein–specific CD8⁺ T cells were detected among 10.0% (2 of 20) of HLA-A*0201⁺ newborns. These results suggest that B and T cell immune responses occur in the fetus following vaccination against influenza and have important implications for determining when immune responses to environmental exposures begin.

I n recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms of prostate-specific regulation and functional evolution of TRPM8 during PCa progression remain unclear. Here we show, for the first time to our knowledge, that only secretory mature differentiated human prostate primary epithelial (PrPE) luminal cells expressed functional plasma membrane TRPM8 (_PMTRPM8) channels. Moreover, PCa epithelial cells obtained from in situ PCa were characterized by a significantly stronger _PMTRPM8-mediated current than that in normal cells. This _PMTRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. However, we found that in contrast to _PMTRPM8, endoplasmic reticulum TRPM8 (_ERTRPM8) retained its function as an ER Ca²⁺ release channel, independent of cell differentiation. We hypothesize that the constitutive activity of _ERTRPM8 may result from the expression of a truncated TRPM8 splice variant. Our study provides insight into the role of TRPM8 in PCa progression and suggests that TRPM8 is a potentially attractive target for therapeutic intervention: specific inhibition of either _ERTRPM8 or _PMTRPM8 may be useful, depending on the stage and androgen sensitivity of the targeted PCa.

P PARγ is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPARγ in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPARγ-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPARγ following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPARγ in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

C irculating resistin stimulates endogenous glucose production (GP). Here, we report that bi-directional changes in hypothalamic resistin action have dramatic effects on GP and proinflammatory cytokine expression in the liver. The infusion of either resistin or an active cysteine mutant in the third cerebral ventricle (icv) or in the mediobasal hypothalamus stimulated GP independent of changes in circulating levels of glucoregulatory hormones. Conversely, central antagonism of resistin action markedly diminished the ability of circulating resistin to enhance GP. We also report that centrally mediated mechanisms partially control resistin-induced expression of TNF-α, IL-6, and SOCS-3 in the liver. These results unveil what we believe to be a novel site of action of resistin on GP and inflammation and suggest that hypothalamic resistin action can contribute to hyperglycemia in type 2 diabetes mellitus.

I ncreased fat deposition in skeletal muscle is associated with insulin resistance. However, exercise increases both intramyocellular fat stores and insulin sensitivity, a phenomenon referred to as “the athlete’s paradox”. In this study, we provide evidence that augmenting triglyceride synthesis in skeletal muscle is intrinsically connected with increased insulin sensitivity. Exercise increased diacylglycerol (DAG) acyltransferase (DGAT) activity in skeletal muscle. Channeling fatty acid substrates into TG resulted in decreased DAG and ceramide levels. Transgenic overexpression of DGAT1 in mouse skeletal muscle replicated these findings and protected mice against high-fat diet–induced insulin resistance. Moreover, in isolated muscle, DGAT1 deficiency exacerbated insulin resistance caused by fatty acids, whereas DGAT1 overexpression mitigated the detrimental effect of fatty acids. The heightened insulin sensitivity in the transgenic mice was associated with attenuated fat-induced activation of DAG-responsive PKCs and the stress mediator JNK1. Consistent with these changes, serine phosphorylation of insulin receptor substrate 1 was reduced, and Akt activation and glucose 4 membrane translocation were increased. In conclusion, upregulation of DGAT1 in skeletal muscle is sufficient to recreate the athlete’s paradox and illustrates a mechanism of exercise-induced enhancement of muscle insulin sensitivity. Thus, increasing muscle DGAT activity may offer a new approach to prevent and treat insulin resistance and type 2 diabetes mellitus.

F atty acid oversupply is a key mediator of skeletal muscle insulin resistance in obesity, primarily via accumulation of fatty acid metabolites and activation of proinflammatory pathways. Herein, we demonstrate that fatty acid–induced insulin resistance in humans is completely prevented the day after 1 session of endurance exercise. Because skeletal muscle is the primary site for systemic glucose disposal and is highly susceptible to impaired insulin action by elevated fatty acid availability, we obtained skeletal muscle samples to investigate possible mechanisms mediating this protective effect of exercise. Prevention of fatty acid–induced insulin resistance after exercise accompanied enhanced skeletal muscle protein expression of key lipogenic enzymes and an increase in muscle triglyceride synthesis. Partitioning more fatty acids toward triglyceride synthesis within muscle reduced the accumulation of fatty acid metabolites and suppressed the proinflammatory response in skeletal muscle, as evidenced by decreased phosphorylation and activation of JNK and increased abundance of inhibitor of NF-κB α (IκB-α) and IκB-β. We believe this is the first study to demonstrate that 1 session of exercise completely reverses fatty acid–induced insulin resistance in humans. Reversal of insulin resistance accompanied enhanced lipogenic capacity within skeletal muscle, reduced accumulation of highly bioactive fatty acid metabolites, and suppressed activation of proinflammatory pathways known to impair insulin action.

S ingle gene mutations in β integrins can account for functional defects of individual cells of the hematopoietic system. In humans, mutations in β₂ integrin lead to leukocyte adhesion deficiency (LAD) syndrome and mutations in β₃ integrin cause the bleeding disorder Glanzmann thrombasthenia. However, multiple defects in blood cells involving various β integrins (β₁, β₂, and β₃) occur simultaneously in patients with the recently described LAD type III (LAD-III). Here we show that the product of a single gene, Ca²⁺ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), controlled the activation of all 3 integrins in the hematopoietic system. Neutrophils from CalDAG-GEFI^–/– mice exhibited strong defects in Rap1 and β₁ and β₂ integrin activation while maintaining normal calcium flux, degranulation, and ROS generation. Neutrophils from CalDAG-GEFI–deficient mice failed to adhere firmly to stimulated venules and to migrate into sites of inflammation. Furthermore, CalDAG-GEFI regulated the activation of β₁ and β₃ integrins in platelets, and CalDAG-GEFI deficiency caused complete inhibition of arterial thrombus formation in mice. Thus, mice engineered to lack CalDAG-GEFI have a combination of defects in leukocyte and platelet functions similar to that of LAD-III patients.

K -Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1^–/– mice, decreased in Kcc3^–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1^–/– or Kcc3^–/– mice were of normal density, rbc of Kcc1^–/–Kcc3^–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1^–/–Kcc3^–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K⁺ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

T his study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad^–/–) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad^–/– mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad^–/– mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-α–induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with N_ω-nitro-l-arginine methyl ester (l-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.