UNC5B Receptor Deletion Exacerbates Tissue Injury in Response to AKI

Netrin-1 regulates cell survival and apoptosis by activation of its receptors, including UNC5B. However, the in vivo role of UNC5B in cell survival during cellular stress and tissue injury is unknown. We investigated the role of UNC5B in cell survival in response to stress using mice heterozygously expressing the UNC5B gene (UNC5B^−/flox) and mice with targeted homozygous deletion of UNC5B in kidney epithelial cells (UNC5B^{−/flox/GGT-cre}). Mice were subjected to two different models of organ injury: ischemia reperfusion injury of the kidney and cisplatin-induced nephrotoxicity. Both mouse models of UNC5B depletion had normal organ function and histology under basal conditions. After AKI, however, UNC5B^{−/flox/GGT-cre} mice exhibited significantly worse renal function and damage, increased tubular apoptosis, enhanced p53 activation, and exacerbated inflammation compared with UNC5B^−/flox and wild-type mice. shRNA-mediated suppression of UNC5B expression in cultured tubular epithelial cells exacerbated cisplatin-induced cell death in a p53-dependent manner and blunted Akt phosphorylation. Inhibition of PI3 kinase similarly exacerbated cisplatin-induced apoptosis; in contrast, overexpression of UNC5B reduced cisplatin-induced apoptosis in these cells. Taken together, these results show that the netrin-1 receptor UNC5B plays a critical role in cell survival and kidney injury through Akt-mediated inactivation of p53 in response to stress.AKI is a life-threatening disorder that has been increasing in incidence. About 6% of hospitalized patients and more than 30% of patients in the intensive care unit develop AKI, which can lead to organ failure and death.^1–3 Currently, there are effective therapies available to treat AKI. Several in vivo studies suggest that administration of netrin-1 suppresses inflammation and apoptosis in the kidney and other organs caused by different insults.^4–10 Moreover, netrin-1 is highly induced in tubular epithelial cells and secreted into the tubular lumen within hours after ischemia reperfusion.¹¹ Overexpression of netrin-1, specifically in proximal tubular epithelial cells, suppressed ischemia reperfusion and nephrotoxin-induced kidney injury and dextran sulfate sodium-induced colon injury by suppressing apoptosis.^12,13 However, the receptor that mediates the protective effects of netrin-1 and the mechanism of protection against cell death are unknown.Netrin-1 is a laminin-related secreted molecule identified as a neuronal guidance cue that directs neurons and their axons to targets during development of the nervous system.¹⁴ Although netrin-1 is primarily thought of as an axon guidance cue, this function is unlikely to be its only function, because expression studies have shown that netrins are widely expressed outside the nervous system, including in vascular endothelial cells as well as the liver, lung, colon, heart, and kidney.^4,15 In fact, the kidney was shown to have the highest level of netrin-1 mRNA compared with any other tissues studied so far.¹⁵ Netrin-1 mediates its biologic activity by binding to two vertebrate families of receptors: the Deleted in Colorectal Cancer (DCC) family comprising DCC and neogenin receptors and the uncoordinated 5 (UNC5) family UNC5A-D receptors.¹⁶ Both DCC and UNC5 family members are called dependence receptors because of the dependence of cell survival on the presence of the netrin-1 ligand, and the absence of these receptors is known to induce apoptosis.^17–19 However, the in vivo relevance of this dependence receptor hypothesis is not clear. For example, very little netrin-1 protein is expressed in normal kidney tubular epithelial cells, but UNC5B expression was found in the same cells. If dependence receptor hypothesis is functioning, then we would expect to see a large number of apoptotic cells in the normal kidney, which was not the case. Therefore, we hypothesized that UNC5B receptor signaling is critical for cell survival in response to injurious stimulus. In the absence of the UNC5B receptor, cells and tissues are highly prone to injury. Previous studies from our laboratory showed that the UNC5B receptor is highly expressed in proximal tubular epithelial cells of the kidney in vivo and in vitro, whereas DCC expression is very low or negligible. Moreover, UNC5C expression is restricted to distal tubules where UNC5A and UNC5D are not expressed in the kidney.²⁰ In addition, netrin-1–induced epithelial cell proliferation and migration in vitro were mediated through UNC5B, and in vivo antibody-based neutralization of UNC5B function abolished netrin-1 protective effects against ischemia reperfusion injury of the kidney.^10,21 These data led us to investigate the critical role of UNC5B in renal epithelial survival in response to AKI.In this study, we investigated our hypothesis using tissue-specific UNC5B knockout animals as well as animal with partial deficiency of UNC5B receptors. We have generated kidney proximal tubular epithelial (TKPTS) cell-specific UNC5B knockout mice using Cre-lox technology. These mice were subjected to ischemia reperfusion injury and cisplatin-induced kidney injury. Here, we report that deletion of the netrin-1 receptor UNC5B rendered the mice highly susceptible to ischemic and toxic kidney injury. The exacerbated kidney injury is caused by increased apoptosis, p53 activation, and inflammatory response in the epithelial cells of the kidney compared with wild-type (WT) or heterozygous knockout mice. Moreover, this activation of p53 was caused by defective activation of protein kinase B (Akt) pathways in UNC5B knockout epithelial cells.     

抑制素B βB亚单位在不同病理分型的无精子症患者睾丸组织中的表达

目的:探讨抑制素B(INH B)βB亚单位在不同生精功能状态的人睾丸组织中的表达情况。方法:对83例无精子症患者进行睾丸组织病理检查诊断,根据病理形态的不同分为:唯支持细胞综合征型(n=21);生精功能低下型(n=20);生精阻滞型(n=24);生精功能基本正常型(n=18)。选择上述各型结构完整的睾丸组织,分别应用免疫组化法(SP)对血清INH B βB亚单位在不同生精功能状态的睾丸组织,进行定位研究。结果:各型睾丸组织内均存在血清INH B βB的表达,其分布特点为:间质细胞(Leydig cell)和早期生精细胞多为强阳性表达,呈深棕黄色;支持细胞(Sertoli cell)多为阳性表达;而晚期精子细胞和成熟精子未见表达;生精小管管周类肌细胞呈弱阳性表达。结论:INH B可能是睾丸Sertoli细胞和早期生精细胞的一个联合产物。     

Experimental Disc Herniation in the Rat Causes Downregulation of Serotonin Receptor 2c in a TNF-dependent Manner

Background

During recent decades, the knowledge of the pathophysiology of disc herniation and sciatica has drastically improved. What previously was considered a strict biomechanical process is now considered a more complex interaction between leaked nucleus pulposus and the tissue in the spinal canal. An inflammatory reaction, with tumor necrosis factor (TNF) playing an essential role, has been demonstrated. However, the exact mechanisms of the pathophysiology of disc herniation remain unknown.

Questions/purposes

In this study we use an animal model to investigate (1) if and/or how experimental disc herniation affects gene expression in the early phase (24 hours postsurgery) in the dorsal root ganglion; and (2) if TNF inhibition can reduce any observed changes.

Methods

A rat model of disc herniation was used. Twenty rats were evenly divided into four groups: naïve, sham, disc herniation, and disc herniation with TNF inhibition. The dorsal root ganglion of the affected nerve root was harvested 24 hours after surgery and analyzed with a TaqMan Low Density Array^® quantitative polymerase chain reaction assay. Gene expression levels in sham were compared with disc herniation to assess question 1 and disc herniation to disc herniation with TNF inhibition to assess question 2.

Results

Experimental disc herniation caused a decrease in the expression of the serotonin receptor 2c gene (p = 0.022). TNF inhibition was found to reduce the observed decrease in expression of serotonin receptor 2c (p = 0.037).

Conclusions

Our results suggest that a decrease in the expression of the serotonin receptor 2c gene may contribute to the pathophysiology of disc herniation. Further research on its involvement is warranted.

Clinical Relevance

This pilot study gives a brief insight into cellular changes that may contribute to the pathophysiology of disc herniation. This knowledge may contribute to the development of more and better treatment options for patients with disc herniation and sciatica.     

Effects of the Serotonin sub 2 Receptor Agonist DOI on Skeletal Muscle Specimens from Malignant Hyperthermia-susceptible Patients

Background: Administration of serotonin2 (5-HT2) receptor agonists in pigs triggers malignant hyperthermia (MH) and psychotic-like behavior. Both can be reduced by 5-HT2 receptor antagonists. Furthermore, an increase in the plasma concentration of 5-HT has been found during onset of halothane-induced MH in pigs. Therefore, in this study, the in vitro effects of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) were investigated in muscle specimens from MH-susceptible (MHS) and -negative (MHN) patients.

Methods: After MH classification using the caffeine-halothane contracture test (CHCT), surplus muscle specimens from 23 MHS and 17 MHN patients were used to examine the effects of DOI. In the first study, DOI was added to the bath in a concentration of 0.02 mM. In a second experiment, muscles were preincubated for 60 min with 0.02 mM DOI, and subsequently, halothane was added incrementally to the organ bath (0.11-0.22-0.44 mM) for 15 min according to the CHCT protocol. The in vitro effects of DOI on contracture development and muscle twitch were measured for 120 min in both investigations.

Results: Muscle specimens from all patients developed contractures after administration of DOI, characterized by a significantly earlier development of contracture in MHS (16.8+/-1.7 min) than in MHN (66.3+/-5.8 min) muscles (P < 0.05). There was no overlap between the groups in the range of times. The onset of contracture development after DOI was prolonged by halothane in specimens from MHN patients (89.7 +/-5.6 min) but not MHS patients. Preincubation with DOI increased the halothane-induced contractures in specimens from MHS patients compared to the results of the CHCT. The contracture development in specimens from MHS patients was larger than from MHN patients. At the end of the experiment, contractures had reached a maximum of 12.9+/-1.1 mN in specimens from MHS and 5.3+/-0.6 mN in MHN patients (P < 0.05). The additional administration of halothane led to significantly increased contractures in specimens from MHS individuals (15.9+/- 0.9 mN) at 120 min. However, the contracture development decreased significantly to 3.1+/-0.4 mN in MHN muscles. Muscle twitch after DOI administration was reduced significantly in specimens from MHS and MHN patients.     

Gene Deletion of the Kinin Receptor B1 Attenuates Cardiac Inflammation and Fibrosis During the Development of Experimental Diabetic Cardiomyopathy

OBJECTIVE

Diabetic cardiomyopathy is associated with increased mortality in patients with diabetes. The underlying pathology of this disease is still under discussion. We studied the role of the kinin B1 receptor on the development of experimental diabetic cardiomyopathy.

RESEARCH DESIGN AND METHODS

We utilized B1 receptor knockout mice and investigated cardiac inflammation, fibrosis, and oxidative stress after induction of streptozotocin (STZ)-induced diabetes. Furthermore, the left ventricular function was measured by pressure-volume loops after 8 weeks of diabetes.

RESULTS

B1 receptor knockout mice showed an attenuation of diabetic cardiomyopathy with improved systolic and diastolic function in comparison with diabetic control mice. This was associated with a decreased activation state of the mitogen-activated protein kinase p38, less oxidative stress, as well as normalized cardiac inflammation, shown by fewer invading cells and no increase in matrix metalloproteinase-9 as well as the chemokine CXCL-5. Furthermore, the profibrotic connective tissue growth factor was normalized, leading to a reduction in cardiac fibrosis despite severe hyperglycemia in mice lacking the B1 receptor.

CONCLUSIONS

These findings suggest that the B1 receptor is detrimental in diabetic cardiomyopathy in that it mediates inflammatory and fibrotic processes. These insights might have useful implications on future studies utilizing B1 receptor antagonists for treatment of human diabetic cardiomyopathy.Diabetic cardiomyopathy, as it occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality (1). Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricular systolic and diastolic dysfunction. Accumulation of cardiac fibrosis with distinct changes in the regulation of the extracellular matrix (2,3), excessive generation of reactive oxygen species (4), and cardiac inflammation (5,6), characterized by increased levels of proinflammatory cytokines and transendothelial migration of immunocompetent cells, plays a role in the manifestation of diabetic cardiomyopathy. Experimental stimulation of the local tissue kallikrein-kinin system has been shown to be beneficial in different forms of cardiomyopathies (7–11). Most of these effects are attributed to the kinin B2 receptor (B2R), while the role of the kinin B1 receptor (B1R) in cardiac failure is still under discussion. In contrast to the B2R, which is constitutively expressed in the cardiac tissue, the B1R is expressed at very low levels under basal conditions. Nevertheless, it is highly inducible under pathological conditions by pathological mediators such as bacterial lipopolysaccharide (12), cytokines (13), and ischemia but also by hyperglycemia (14), as can be shown in different animal models of cardiomyopathy. Also, in endomyocardial biopsies of patients with end-stage heart failure, this upregulation could be demonstrated and correlated with increased expression of proinflammatory cytokines in those patients (15). Whether B1R upregulation is cardioprotective, parallel to that of the B2R (16,17), or is cardiotoxic (13,18,19) remains debated. To further clarify the role of the B1R in the pathogenesis of diabetic cardiomyopathy, we investigated the left ventricular function in an animal model of streptozotocin (STZ)-induced type 1 diabetes using B1R knockout mice. Furthermore, changes in the left ventricular remodeling, inflammation, and oxidative stress were analyzed.     

结肠癌中P2X7 受体表达水平及其与患者预后的关系

目的:探讨结肠癌组织中P2X 亚单位受体(P2X7)表达与结肠癌肿瘤特征及患者预后的关系。方法:选取2017 年1 月—2018 年1 月在河北中石油中心医院手术治疗的120 例结肠癌患者,获取其结肠癌组织及癌旁组织标本,采用免疫组化染色检测P2X7 受体的阳性表达率,分析不同预后患者的病理学特征差异,同时分析P2X7 受体与患者3 年预后结局的关系。结果:结肠癌组织中,P2X7 受体低表达率为65.0%,高于癌旁组织的28.3%,差异有统计学意义(P <0.05);随访3 年,P2X7 受体低表达组患者的死亡率为52.6%,高于高表达组的33.3%,差异有统计学意义(P <0.05);生存分析结果显示,P2X7 受体低表达组患者的生存时间短于高表达组患者,差异有统计学意义(Log Rank χ2=4.652,P =0.031);Logistic 回归分析结果显示:TNM 分期高、切缘阳性、淋巴结转移阳性、脉管浸润、P2X7 受体低表达是结肠癌患者不良预后的独立危险因素(P <0.05)。结论:结肠癌组织中的P2X7 受体呈低表达,并且是结肠癌患者预后不良的独立危险因素。     

睾丸外组织雄激素受体的分布及检测

雄激素受体 (AR)是介导雄激素在靶细胞中发挥作用的关键大分子 ,主要存在于男性睾丸组织中。本文综述了AR及其mRNA在泌尿生殖系统的前列腺、附睾、阴茎皮肤等睾丸外组织 ,头皮、海马旁回、脂肪等非泌尿生殖系统组织及在胃癌、喉癌等睾丸外肿瘤组织中的分布情况 ,并对AR的检测方法进行了复习 ,以进一步研究循环血中雄激素对人体各系统的作用     

Alterations of Angiotensin II Receptor Levels in Sutured Wounds in Rat Skin

Angiotensin II receptor levels have been shown to vary with postoperative time in tissue harvested from full-thickness dermal excisional wounds on adult rats. This study examined the expression of AII receptors in a sutured wound model. Two full-thickness incisionul wounds were made in the dorsal skin of adult Sprague-Dawley rats and sutured immediately under general anesthesia. The wound tissues were harvested at 0, 0.5, 1, 2, 4, 24 h and on days 2, 3, 4. 5, 7, and 10 after the wounding. The levels of ¹²⁵I-Sar¹.lle⁸-AII bound to membrane preparations of the wound tissues decreased at early time points (from 0.5 to 4 h), increased from day 1 to day 7, and returned to nonsurgical levels by day 10. Competitive binding studies showed that the receptors were predominantly of the ATI receptor subtype. These results suggest that an immediate and transient reduction in AII receptor expression occurred after wounding, followed by an increase in the number of AII receptors that was maintained for 5 to 7 days postoperatively. Because these data are consistent with those observed after excisional wounding, temporal changes in AII receptor expression may be integral to the process of wound healing     

Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.     

Mouse Models of Breast Cancer Share Amplification and Deletion Events with Human Breast Cancer

Breast tumor heterogeneity has been well documented through the use of multiplatform –omic studies in human tumors. However, there is no integrative database to capture the heterogeneity within mouse models of breast cancer. This project identifies genomic copy number alterations (CNAs) in 600 tumors across 27 major mouse models of breast cancer through the application of a predictive algorithm to publicly available gene expression data. It was found that despite the presence of strong oncogenic drivers in most mouse models, CNAs are extremely common but heterogeneous both between models and within models. Many mouse CNA events are largely conserved in human tumors and in the mouse we show that they are associated with secondary tumor characteristics such as tumor histology, metastasis, as well as enhanced oncogenic signaling. These data serve as an important resource in guiding investigators when choosing a mouse model to understand the gene copy number changes relevant to human breast cancer.     

Allogeneic Parenchymal and Hematopoietic Tissues Differ in Their Ability to Induce Deletion of Donor-Reactive T Cells

The establishment of immune tolerance to self antigen expressed exclusively in the periphery is a crucial yet incompletely understood feature of the immune system. A dominant concept of peripheral tolerance has been that exposure of T cells to signal one, the TCR-MHC interaction, in the absence of signal two, or costimulation, is a major mechanism of peripheral tolerance. This model suggests that any cell type that expresses MHC-peptide complexes, be they of self or foreign origin, should have the capacity to tolerize antigen-specific T cells when critical costimulatory interactions are interrupted. However, a spectrum of responses, from permanent engraftment to rapid rejection, has been observed in various transplantation models utilizing costimulatory blockade. Therefore we undertook a series experiments to directly assess the tolerogenic potential of donor hematopoietic and parenchymal cells. We find that allogeneic tissues differ profoundly in their ability to promote peripheral tolerance concurrent with combined blockade of B7-CD28 and CD40-CD40L pathways. Non-vascularized and vascularized parenchymal grafts as well as donor-specific transfusions promote varying degrees of donor-specific hyporesponsiveness, but fail to induce donor-reactive T-cell deletion; whereas establishment of stable hematopoietic chimerism promotes specific tolerance mediated by deletion of donor-reactive cells in the periphery.     

SH2B1 Enhances Insulin Sensitivity by Both Stimulating the Insulin Receptor and Inhibiting Tyrosine Dephosphorylation of Insulin Receptor Substrate Proteins

OBJECTIVE

SH2B1 is a SH2 domain-containing adaptor protein expressed in both the central nervous system and peripheral tissues. Neuronal SH2B1 controls body weight; however, the functions of peripheral SH2B1 remain unknown. Here, we studied peripheral SH2B1 regulation of insulin sensitivity and glucose metabolism.

RESEARCH DESIGN AND METHODS

We generated TgKO mice expressing SH2B1 in the brain but not peripheral tissues. Various metabolic parameters and insulin signaling were examined in TgKO mice fed a high-fat diet (HFD). The effect of SH2B1 on the insulin receptor catalytic activity and insulin receptor substrate (IRS)-1/IRS-2 dephosphorylation was examined using in vitro kinase assays and in vitro dephosphorylation assays, respectively. SH2B1 was coexpressed with PTP1B, and insulin receptor–mediated phosphorylation of IRS-1 was examined.

RESULTS

Deletion of peripheral SH2B1 markedly exacerbated HFD-induced hyperglycemia, hyperinsulinemia, and glucose intolerance in TgKO mice. Insulin signaling was dramatically impaired in muscle, liver, and adipose tissue in TgKO mice. Deletion of SH2B1 impaired insulin signaling in primary hepatocytes, whereas SH2B1 overexpression stimulated insulin receptor autophosphorylation and tyrosine phosphorylation of IRSs. Purified SH2B1 stimulated insulin receptor catalytic activity in vitro. The SH2 domain of SH2B1 was both required and sufficient to promote insulin receptor activation. Insulin stimulated the binding of SH2B1 to IRS-1 or IRS-2. This physical interaction inhibited tyrosine dephosphorylation of IRS-1 or IRS-2 and increased the ability of IRS proteins to activate the phosphatidylinositol 3-kinase pathway.

CONCLUSIONS

SH2B1 is an endogenous insulin sensitizer. It directly binds to insulin receptors, IRS-1 and IRS-2, and enhances insulin sensitivity by promoting insulin receptor catalytic activity and by inhibiting tyrosine dephosphorylation of IRS proteins.Insulin decreases blood glucose both by promoting glucose uptake into skeletal muscle and adipose tissue and by suppressing hepatic glucose production. In type 2 diabetes, the ability of insulin to reduce blood glucose is impaired (insulin resistance) because of a combination of genetic and environmental factors, resulting in hyperglycemia. Insulin resistance is not only the hallmark but also a determinant of type 2 diabetes.Insulin binds to and activates the insulin receptor. Insulin receptor tyrosyl phosphorylates insulin receptor substrates (IRS-1, -2, -3, and -4). IRS proteins, particularly IRS-1 and IRS-2, initiate and coordinate multiple downstream pathways, including the phosphatidylinositol 3-kinase/Akt pathway (1). Genetic deletion of IRS-1, IRS-2, or Akt2 causes insulin resistance in mice, indicating that the IRS protein/phosphatidylinositol 3-kinase/Akt2 pathway is required for regulation of glucose homeostasis by insulin (2–5). Insulin receptor and IRS proteins are negatively regulated by various intracellular molecules, including PTP1B, Grb10, Grb14, SOCS1, SOCS3, JNK, PKCθ, S6K, and IKKβ (6–23). The relative contribution of these negative regulators to the progression of insulin resistance has been extensively studied (6–24). However, insulin signaling is likely to also be modulated by positive regulators. In this study, we demonstrate that SH2B1 is a novel endogenous insulin sensitizer.SH2B1 is a member of the SH2B family of adapter proteins that also includes SH2B2 (APS) and SH2B3 (Lnk). SH2B1 and SH2B2 are expressed in multiple tissues, including insulin target tissues (e.g., skeletal muscle, adipose tissue, liver, and the brain); by contrast, SH2B3 expression is restricted to hematopoietic tissue (25,26). Structurally, SH2B family members have an NH₂-terminal dimerization domain, a central pleckstrin homology domain, and a COOH-terminal Src homology 2 (SH2) domain. The dimerization domain mediates homodimerization or heterodimerization between different SH2B proteins (27). SH2B1 and SH2B2 bind via their SH2 domains to a variety of tyrosine phosphorylated proteins, including JAK2 and insulin receptor, in cultured cells (28). Genetic deletion of SH2B1 results in marked leptin resistance, obesity, insulin resistance, and type 2 diabetes in mice, demonstrating that SH2B1 is required for the maintenance of normal body weight, insulin sensitivity, and glucose metabolism (29–32). Surprisingly, SH2B2-null mice have normal body weight and slightly improved insulin sensitivity (32,33), suggesting that SH2B1 and SH2B2 have distinct functions in vivo. However, it remains unclear whether SH2B1 cell autonomously regulates insulin sensitivity in peripheral insulin target tissues because systemic deletion of SH2B1 causes obesity, which may cause insulin resistance in SH2B1-null mice.We generated a mouse model in which recombinant SH2B1 is specifically expressed in the brain of SH2B1-null mice (TgKO) using transgenic approaches (31). Neuron-specific restoration of SH2B1 corrects both leptin resistance and obesity, suggesting that neuronal SH2B1 regulates energy balance and body weight by enhancing leptin sensitivity (31). Consistent with these conclusions, polymorphisms in the SH2B1 loci are linked to leptin resistance and obesity in humans (34–36). In this work, we demonstrate that deletion of SH2B1 in peripheral tissues impairs insulin sensitivity independent of obesity in TgKO mice. Moreover, we demonstrate that SH2B1 directly promotes insulin responses by stimulating insulin receptor catalytic activity and by protecting IRS proteins from tyrosine dephosphorylation.     

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) R47H Variant Causes Distinct Age- and Sex-Dependent Musculoskeletal Alterations in Mice

Previous studies proposed the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone, as a link between brain and bone disease. The TREM2 R47H variant is a known risk factor for Alzheimer's disease (AD), the most common form of dementia. To investigate whether altered TREM2 signaling could contribute to bone and skeletal muscle loss, independently of central nervous system defects, we used mice globally hemizygous for the TREM2 R47H variant (TREM2^R47H/+), which do not exhibit AD pathology, and wild-type (WT) littermate control mice. Dxa/Piximus showed bone loss in female TREM2^R47H/+ animals between 4 and 13 months of age and reduced cancellous and cortical bone (measured by micro-computed tomography [μCT]) at 13 months, which stalled out by 20 months of age. In addition, they exhibited decreased femoral biomechanical properties measured by three-point bending at 13 months of age, but not at 4 or 20 months. Male TREM2^R47H/+ animals had decreased trabecular bone geometry but increased ultimate strain and failure force at 20 months of age versus WT. Only male TREM2^R47H/+ osteoclasts differentiated more ex vivo after 7 days with receptor activator of nuclear factor κB ligand (RANKL)/macrophage colony-stimulating factor (M-CSF) compared to WT littermates. Yet, estrogen receptor alpha expression was higher in female and male TREM2^R47H/+ osteoclasts compared to WT mice. However, female TREM2^R47H/+ osteoclasts expressed less complement 3 (C3), an estrogen responsive element, and increased protein kinase B (Akt) activity, suggesting altered estrogen signaling in TREM2^R47H/+ cells. Despite lower bone volume/strength in TREM2^R47H/+ mice, skeletal muscle function measured by plantar flexion and muscle contractility was increased in 13-month-old female mutant mice. Overall, these data demonstrate that an AD-associated TREM2 variant can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of central neuropathology, potentially mediated through changes in osteoclastic intracellular signaling. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

生长激素促分泌素受体基因敲除小鼠胚胎干细胞的建立

目的建立生长激素促分泌素受体（ghrelin receptor，GHS-R）基因敲除小鼠胚胎干细胞（ES细胞）杂合子模型，为研究GHS—R基因的功能奠定基础。方法用TK-neo置换原X-pPNT载体的PGK—neo构建目标载体。以小鼠基因组DNA为模板，用PCR方法扩增2条同源臂，将其按照一定方向装入含有TK—neo的X—pPNT载体，并测序鉴定。载体线性化及纯化后电穿孔转染小鼠ES细胞，用G418和更昔洛韦（Gancyclovir）对电穿孔转染后的ES细胞进行正、负筛选培养，得到双药抗性ES细胞，克隆后抽提基因组DNA，分别用PCR方法鉴定2条同源臂，并测序确定成功同源重组的ES细胞克隆。结果改建X—pPNT载体成功，PCR获得2条同源臂片段，测序正确，并按一定方向装入打靶载体，ES细胞转染后经双药筛选得到328个阳性ES细胞克隆，PCR及测序鉴定证实3个克隆发生同源重组。结论本研究成功获得了GHS-R（-／＋）杂合子小鼠ES细胞克隆，为进一步通过显微注射及杂交育种获得GHS—R基因敲除小鼠打下了基础。     

Obesity-Induced Infertility and Hyperandrogenism Are Corrected by Deletion of the Insulin Receptor in the Ovarian Theca Cell

Women with polycystic ovary syndrome (PCOS) exhibit elevated androgen levels, oligoanovulation, infertility, and insulin resistance in metabolic tissues. The aims of these studies were to determine the role of insulin signaling in the development and function of ovarian theca cells and the pathophysiologic effects of hyperinsulinism on ovarian function in obesity. We disrupted the insulin receptor (IR) gene specifically in the theca-interstitial (TI) cells of the ovaries (Cyp17IRKO). No changes in reproductive development or function were observed in lean Cyp17IRKO female mice, suggesting that insulin signaling in TI cell is not essential for reproduction. However, when females were fed a high-fat diet, diet-induced obesity (DIO) wild-type (DIO-WT) mice were infertile and experienced increased circulating testosterone levels, whereas DIO-Cyp17IRKO mice exhibited improved fertility and testosterone levels comparable to those found in lean mice. The levels of phosphorylated IRS1 and CYP17 protein were higher in the ovary of DIO-WT compared with DIO-Cyp17IRKO or lean mice. Ex vivo studies using a whole ovary culture model demonstrated that insulin acts independently or additively with human chorionic gonadotropin to enhance androstenedione secretion. These studies reveal the causal pathway linking hyperinsulinism with ovarian hyperandrogenism and the infertility of obesity.     

γ-Aminobutyric Acid Type A Receptor β2 Subunit Mediates the Hypothermic Effect of Etomidate in Mice

Background: The authors have previously described that the [gamma]-aminobutyric acid type A (GABAA) receptor [beta]2N265S mutation results in a knock-in mouse with reduced sensitivity to etomidate. After recovery from etomidate anesthesia, these mice have improved motor performance and less slow wave sleep. Because most clinically used anesthetics produce hypothermia, the effect of this mutation on core body temperature was investigated.

Methods: The effect of etomidate and propofol on core body temperature were measured using radiotelemetry in freely moving GABAA receptor [beta]2N265S mutant mice and wild-type controls.

Results: [beta]2N265S mutant mice have a reduced hypothermic response to anesthetic doses of etomidate compared with wild-type controls and after a transient loss of righting reflex regain normothermia more rapidly compared with wild-type controls. Subanesthetic doses of etomidate produce hypothermia, which was not observed in the mutant mice. Vehicle administration resulted in a stress-induced hyperthermic response in both genotypes. Propofol produced a hypothermic response that was similar in both genotypes.