Gianluca Toraldo

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

The Dynamic Structure of the Estrogen Receptor

The estrogen receptor (ER) mediates most of the biological effects of estrogens at the level of gene regulation by interacting through its site-specific DNA and with other coregulatory proteins. In recent years, new information regarding the dynamic structural nature of ER has emerged. The physiological effects of estrogen are manifested through ERs two isoforms, ERα and ERβ. These two isoforms (ERα and ERβ) display distinct regions of sequence homology. The three-dimensional structures of the DNA-binding domain (DBD) and ligand-binding domain (LBD) have been solved, whereas no three-dimensional natively folded structure for the ER N-terminal domain (NTD) is available to date. However, insights about the structural and functional correlations regarding the ER NTD have recently emerged. In this paper, we discuss the knowledge about the structural characteristics of the ER in general and how the structural features of the two isoforms differ, and its subsequent role in gene regulation.

Doxycycline reduces fibril formation in a transgenic mouse model of AL amyloidosis

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic λ6 LC using the cytomegalovirus (CMV) promoter to circumvent the disruption of B cell development by premature expression of recombined LC. The CMV-λ6 transgenic mice develop neurologic dysfunction and Congophilic amyloid deposits in the stomach. Amyloid deposition was inhibited in vivo by the antibiotic doxycycline. In vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant LC fibrils. Furthermore, treatment of ex vivo LC amyloid fibrils with doxycycline reduced the number of intact fibrils and led to the formation of large disordered aggregates. The CMV-λ6 transgenic model replicates the process of AL amyloidosis and is useful for testing the antifibril potential of orally available agents.

Topical androgen antagonism promotes cutaneous wound healing without systemic androgen deprivation by blocking β‐catenin nuclear translocation and cross‐talk with TGF‐β signaling in keratinocytes

Orchidectomy in rodents and lower testosterone levels in men are associated with improved cutaneous wound healing. However, due to the adverse effects on skeletal and sexual tissues, systemic androgen blockade is not a viable therapeutic intervention. Accordingly, we tested the hypothesis that topical application of an androgen antagonist would elicit accelerated wound healing without systemic androgen antagonism. Full‐thickness cutaneous wounds were created on adult C57BL6/J mice. Daily topical application of androgen receptor antagonist, flutamide, resulted in improved gap closure similar to orchiectomized controls and faster than orchidectomized mice treated with topical testosterone. In vivo data showed that the effects of androgen antagonism on wound closure primarily accelerate keratinocytes migration without effecting wound contraction. Consequently, mechanisms of testosterone action on reepithelialization were investigated in vitro by scratch wounding assays in confluent keratinocytes. Testosterone inhibited keratinocyte migration and this effect was in part mediated through promotion of nuclear translocation of β‐catenin and by attenuating transforming growth factor‐β (TGF‐β) signaling through β‐catenin. The link between Wnt and TGF beta signaling was confirmed by blocking β‐catenin and by following TGF‐β‐induced transcription of a luciferase reporter gene. Together, these data show that blockade of β‐catenin can, as a potential target for novel therapeutic interventions, accelerate cutaneous wound healing.

Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy

Because of its anabolic effects on muscle, testosterone is being explored as a function‐promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterones adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterones promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth‐promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone‐responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α‐difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterones effects on prostate, but did not affect testosterones anabolic effects on muscle. Co‐administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate‐sparing anabolic therapy.

Loss of muscle strength during sepsis is in part regulated by glucocorticoids and is associated with reduced muscle fiber stiffness

Sepsis is associated with impaired muscle function but the role of glucocorticoids in sepsis-induced muscle weakness is not known. We tested the role of glucocorticoids in sepsis-induced muscle weakness by treating septic rats with the glucocorticoid receptor antagonist RU38486. In addition, normal rats were treated with dexamethasone to further examine the role of glucocorticoids in the regulation of muscle strength. Sepsis was induced in rats by cecal ligation and puncture, and muscle force generation (peak twitch and tetanic tension) was determined in lower extremity muscles. In other experiments, absolute and specific force as well as stiffness (reflecting the function of actomyosin cross bridges) were determined in isolated skinned muscle fibers from control and septic rats. Sepsis and treatment with dexamethasone resulted in reduced maximal twitch and tetanic force in intact isolated extensor digitorum longus muscles. The absolute and specific maximal force in isolated muscle fibers was reduced during sepsis together with decreased fiber stiffness. These effects of sepsis were blunted (but not abolished) by RU38486. The results suggest that muscle weakness during sepsis is at least in part regulated by glucocorticoids and reflects loss of contractility at the cellular (individual muscle fiber) level. In addition, the results suggest that reduced function of the cross bridges between actin and myosin (documented as reduced muscle fiber stiffness) may be involved in sepsis-induced muscle weakness. An increased understanding of mechanisms involved in loss of muscle strength will be important for the development of new treatment strategies in patients with this debilitating consequence of sepsis.

Metabolic phenotype in an AL amyloidosis transgenic mouse model

In an attempt to elucidate the in vivo process of protein aggregation and mechanisms of amyloid organ disease, we have engineered a genetically defined mouse model of AL amyloidosis. These transgenic mice broadly expressing a human amyloidogenic lambda 6 immunoglobulin light chain (LC) using a cytomegalovirus (CMV) promoter have circulating LC and develop typical Congo red-positive amyloid deposits in the stomach, previously described at the XIth International Symposium on Amyloidosis in Woods Hole [1]. The CMV-lambda 6 transgenic mice display neurologic and metabolic phenotypes. The transgenic mice are larger and have metabolic dysregulation, accompanied by a decreased respiratory exchange ratio, indicating preferential lipid oxidation. With age, the mice develop hyperglycemia upon glucose challenge. We hypothesize that this may be due to a non-fibril-dependent effect of overexpression of LC in tissues, perhaps pancreas. Introduction: We have engineered a transgenic mouse model of AL amyloidosis, expressing a human amyloidogenic lambda 6 light chain (LC) cloned from a patient with aggressive, multi-organ amyloidosis. The LC transgene is expressed using a CMV promoter, and human LC protein is detected mainly in epithelial cells of several organs, such as the kidney, pancreas, lungs, stomach, intestines, and subsets of cells in the brain, spinal cord, and heart. Human LC is detected in the blood, urine, and CSF. CMV-lambda 6 transgenic mice have a variety of phenotypes and here we report upon their body composition and metabolic dysregulation. Methods: Body composition was quantitated by magnetic resonance to directly measure total body fat mass and total body lean mass on unanesthetized mice of various ages (EchoMRI-900, Echo Medical Systems). Three independent transgenic lines were generated, with different random integration of the transgene in the genome. All lines had alterations in the body composition. A single line (line 55) was chosen for additional study. In order to examine if the changes in body composition were associated with the metabolic alterations, indirect calorimetry was utilized to examine substrate utilization. Age-matched, old, and young male transgenic mice were observed for 48 hours, first fed (24 hours) and then fasted (24 hours), in the Oxymax open circuit calorimeter (Columbus Instruments). The amount of heat produced per liter of O2 consumed depends on whether the body is utilizing protein, carbohydrate, or fat as the primary source. The respiratory exchange ratio (RER) calculated from the ratio of the measured volume of gas produced (VCO2) to the measured volume of gas consumed (VO2). Exercise capacity of transgenic and age-matched wild-type friend virus B-type (FVB) mice (Jackson Labs) was assessed by running on a variable speed, multilane treadmill at a 108angle (Exer-6M, Columbus Instruments). Food consumption was measured using a feeding jar. Glucose challenge was performed by injecting 1 g/kg of 10% glucose intraperitoneally (IP) into fasted mice and measuring blood sugars by glucometer at 30, 60, and 120 minutes. Results and discussion: The CMV-lambda 6 transgenic mice displayed significantly different body composition compared with wild-type littermates. Transgenic mice (n1⁄4 13) were heavier than their wild-type counterparts (n1⁄4 12) (Figure 1), but did not consume more food than their non-transgenic counterparts (data not shown), nor did they have any apparent abnormality of digestion or stool consistency. The transgenic mice had an increase in both fat (average 6.1 g vs. 4.3 g, p1⁄4 0.043) and lean (muscle) (average 21.6 g vs. 20.1 g, p1⁄4 0.0037) mass. Furthermore, their exercise capacity was significantly diminished. RER was decreased in both young (3 months) and old (9 months) transgenic Figure 1. Three-month-old transgenic CMV-lambda 6 mice (triangles) have increased body mass compared to non-transgenic controls (circles), (p1⁄40.0085, t-test with unequal variances). The bar marks the average mass. 40

Joint dysfunction and functional decline in middle age myostatin null mice

Since its discovery as a potent inhibitor for muscle development, myostatin has been actively pursued as a drug target for age- and disease-related muscle loss. However, potential adverse effects of long-term myostatin deficiency have not been thoroughly investigated. We report herein that male myostatin null mice (mstn(-/-)), in spite of their greater muscle mass compared to wild-type (wt) mice, displayed more significant functional decline from young (3-6months) to middle age (12-15months) than age-matched wt mice, measured as gripping strength and treadmill endurance. Mstn(-/-) mice displayed markedly restricted ankle mobility and degenerative changes of the ankle joints, including disorganization of bone, tendon and peri-articular connective tissue, as well as synovial thickening with inflammatory cell infiltration. Messenger RNA expression of several pro-osteogenic genes was higher in the Achilles tendon-bone insertion in mstn(-/-) mice than wt mice, even at the neonatal age. At middle age, higher plasma concentrations of growth factors characteristic of excessive bone remodeling were found in mstn(-/-) mice than wt controls. These data collectively indicate that myostatin may play an important role in maintaining ankle and wrist joint health, possibly through negative regulation of the pro-osteogenic WNT/BMP pathway.