Retinol binding protein 4 primes the NLRP3 inflammasome by signaling through Toll-like receptors 2 and 4

Adipose tissue (AT) inflammation contributes to systemic insulin resistance. In obesity and type 2 diabetes (T2D), retinol binding protein 4 (RBP4), the major retinol carrier in serum, is elevated in AT and has proinflammatory effects which are mediated partially through Toll-like receptor 4 (TLR4). We now show that RBP4 primes the NLRP3 inflammasome for interleukin-1β (IL1β) release, in a glucose-dependent manner, through the TLR4/MD2 receptor complex and TLR2. This impairs insulin signaling in adipocytes. IL1β is elevated in perigonadal white AT (PGWAT) of chow-fed RBP4-overexpressing mice and in serum and PGWAT of high-fat diet-fed RBP4-overexpressing mice vs. wild-type mice. Holo- or apo-RBP4 injection in wild-type mice causes insulin resistance and elevates PGWAT inflammatory markers, including IL1β. TLR4 inhibition in RBP4-overexpressing mice reduces PGWAT inflammation, including IL1β levels and improves insulin sensitivity. Thus, the proinflammatory effects of RBP4 require NLRP3-inflammasome priming. These studies may provide approaches to reduce AT inflammation and insulin resistance in obesity and diabetes.

The incidence of type 2 diabetes (T2D) continues to increase worldwide and constitutes a major global health threat (1). Insulin resistance in muscle, adipose tissue (AT), and liver is a hallmark of T2D, which is associated with low-grade chronic inflammation, characterized by increased serum cytokine levels and a proinflammatory immune profile in AT (2–4). Pattern recognition receptors including Toll-like receptors (TLRs) sense pathogenic microbial components (pathogen-associated molecular patterns, PAMPs) and also host-derived damage-associated molecular patterns (DAMPs), which results in nonpathogen-initiated inflammation often referred to as “sterile inflammation” (5). In obesity, TLR2 and TLR4 are implicated in inflammation-induced insulin resistance in AT, and genetic deletion of TLR2 or TLR4 in mice results in improved insulin sensitivity (6, 7). In obese, insulin-resistant humans, TLR2 and TLR4 expression and activation are increased in AT compared to healthy controls (8) and some evidence suggests that people taking an antiinflammatory agent (abatacept, also known as CTLA4-Ig) which blocks antigen presentation, may have improved insulin sensitivity (9, 10). TLR2 and TLR4 signal through the adaptor proteins myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adaptor-inducing IFN (TRIF) to activate proinflammatory signaling cascades including nuclear factor kappa B (NFκB) and c-Jun N-terminal kinase (JNK) (5, 11). This results in AT immune cell migration, activation, and cytokine secretion which augments insulin resistance in adipocytes (12). Thus, TLR2 and TLR4 contribute to AT inflammation and insulin resistance and their targeting may provide treatment strategies for T2D.Retinol binding protein 4 (RBP4), the major serum retinol transporter, is secreted by the liver and AT (13, 14). Serum RBP4 levels are elevated in obese, insulin-resistant mice and humans (15, 16). Genetic and pharmacological elevation of RBP4 induces insulin resistance in wild-type (WT) mice (16, 17) and reducing RBP4 levels improves insulin sensitivity (16, 18). In humans, RBP4 levels are elevated with prediabetes and correlate positively with many metabolic syndrome-related components, including increased waist/hip ratio, intraabdominal fat mass, dyslipidemia, hypertension, and cardiovascular disease in large epidemiological studies (15, 19–21). A gain-of-function polymorphism in the RBP4 promoter which increases adipose RBP4 expression is associated with an 80% increased risk of T2D in humans (19, 22).Inflammation is critical for RBP4-induced insulin resistance which is partially mediated through TLR4 (17, 18, 23). RBP4 induces insulin resistance in adipocytes indirectly by increasing proinflammatory cytokine secretion from macrophages (23). Insulin resistance and AT inflammation have been observed in two mouse models of RBP4 overexpression. Mice overexpressing RBP4 driven by a muscle-specific promoter (RBP4-Ox) have elevated serum and perigonadal white adipose tissue (PGWAT) RBP4 protein levels and PGWAT inflammation (17). Elevated AT RBP4 in these mice activates antigen presentation through the JNK pathway which results in proinflammatory CD4 T cell proliferation and Th1 polarization (17). Mice overexpressing RBP4 selectively in adipocytes also have AT inflammation and glucose intolerance even on a chow diet (14). Transfer of RBP4-activated dendritic cells into normal mice is sufficient to cause AT inflammation and insulin resistance (17). Interestingly, overexpression of RBP4 selectively in hepatocytes has been reported not to cause elevated circulating RBP4 levels and is not associated with insulin resistance (24) even though hepatocytes are thought to be the major site for RBP4 secretion (25). Adipocytes can contribute to circulating RBP4 levels especially in obesity (14). Taken together, these data suggest that RBP4 in AT may be an obesity and insulin-resistance-related damage-associated molecule pattern.Activation of PAMP and DAMP receptors in immune cells leads to the assembly of inflammasomes, which are multiprotein complexes that cleave and activate interleukin-1 beta (IL1β) and interleukin-18 (IL18) (26). The nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) inflammasome plays a role in the pathogenesis of obesity, type 1 diabetes, type 2 diabetes, and metabolic syndrome (26–28). NLRP3 knockout (KO) protects against insulin resistance in mice (27). NLRP3 activation requires a two-step process. The first “priming” occurs in response to TLR-mediated activation of the NFκB pathway resulting in expression of pro-IL1β (26). The second “activating” step directly induces inflammasome assembly which recruits and cleaves procaspase-1 to its active form caspase-1 (26). Caspase-1 mediates the cleavage of pro-IL1β resulting in IL1β release (26). The NLRP3 inflammasome can be activated by metabolites which are elevated in obesity and insulin resistance, such as palmitate (29). While an ever-expanding list of several metabolites is known to provide the second signal in inflammasome activation, there is a paucity of data on the endogenous proteins and metabolites that provide the first priming signal in obesity and T2D. Here we show that RBP4 is an endogenous NLRP3-inflammasome priming agent and we investigate its upstream signaling pathways.Our data show that elevating RBP4 levels by RBP4 injection in WT mice or genetically induced RBP4 overexpression markedly elevates adipose IL1β expression, which leads to PGWAT inflammation and insulin resistance. IL1β is elevated in PGWAT of chow-fed RBP4-Ox mice and in serum and PGWAT of HFD-fed RBP4-Ox mice compared to WT mice. The RBP4-mediated proinflammatory effects are mediated specifically through TLR2 and a TLR4/MD2 receptor complex, which does not require the other adaptor proteins LPS-binding protein and CD14. The activation of macrophages by RBP4 through TLR2 and TLR4/MD2 requires signaling through the downstream pathways MyD88 and TRIF. TLR4 inhibition in RBP4-Ox reduces IL1β levels in PGWAT and improves insulin sensitivity. The RBP4-mediated increase in IL1β release from macrophages is glucose dependent. Thus, targeting the NLRP3 inflammasome or the upstream activating receptors or pathways may provide therapeutic avenues to ameliorate RBP4-mediated insulin resistance and T2D.     

Changes of hexachlorobutadiene nephrotoxicity after piperonyl butoxide treatment

The effects of piperonyl butoxide on hexachlorobutadiene (HCBD) nephrotoxicity were measured. The time course and severity of toxicity were affected. Five hours after either piperonyl butoxide or HCBD glomerular filtration rate (GFR) was decreased; at 24 h GFR had recovered for the piperonyl butoxide group but continued to fall in the HCBD group. The group treated with piperonyl butoxide and HCBD had the same GFR as the group treated with just HCBD. At 24 h after HCBD the piperonyl butoxide pretreated group was not different from the oil pretreated controls. At 48 h after HCBD, reabsorbtion of water and glucose was more severely impaired in the group pretreated with piperonyl butoxide. These results support the hypothesis that HCBD metabolites are involved in renal tubular dysfunction.     

RNA结合蛋白与肿瘤生物治疗：新机遇与新策略

RNA结合蛋白（RBP）由于其独特的生物学功能,目前已经成为肿瘤生物治疗相关靶点筛选的宠儿,很可能为肿瘤生物治疗带来新的机遇。RBP能调控肿瘤细胞及肿瘤微环境免疫细胞和间质细胞的DNA-RNA-蛋白质相互作用网络,进而广泛影响肿瘤发生发展、抗肿瘤免疫应答及肿瘤免疫逃逸过程,目前RBP相关肿瘤生物治疗的研发,主要聚焦在治疗性疫苗、免疫细胞治疗、表观调控治疗等方面,部分研发成果已处于临床试验阶段。随着新理论、新技术的发展以及研究模式的创新,靶向RBP的治疗逐渐摆脱了既往靶向难、疗效欠佳的困局,迎来了新的机遇,通过改良精准靶向和优化组合用药等新策略,为肿瘤生物治疗注入了新的活力,对精准个体化医疗的发展具有重要意义。     

山茱萸对苯扎氯铵诱导的干眼症小鼠视黄醇转运调控作用研究

目的探讨山茱萸对视黄醇转运的调控作用,阐述其治疗干眼症的作用机制,丰富"酸补肝明目"的分子基础。方法 60只BALB/c小鼠随机分为6组,每组10只,分别为对照组、模型组、阳性对照(石斛夜光丸2.9 g/kg)组及山茱萸低、中、高剂量(12、24、48g/kg)组。采用苯扎氯铵滴眼法诱导干眼症模型小鼠,ig给予不同剂量的山茱萸水煎液,通过检测各组小鼠泪液分泌量、泪膜破裂时间,评价山茱萸水煎剂对干眼症模型小鼠的干预作用。ELISA法检测血清前蛋白(PA)、视黄醇结合蛋白(RBP)水平、肝脏中视黄醇水平;免疫组织化学法(IHC)及Western blotting法检测泪腺组织中胞内视黄醇结合蛋白(CRBP)及RBP受体(STRA6)的表达水平。结果与对照组比较,模型组小鼠泪液分泌量显著减少,泪膜破裂时间显著缩短(P0.01),泪腺组织中CRBP及STRA6蛋白表达水平显著降低(P0.01)。与模型组比较,各给药组小鼠血清PA、RBP及肝脏组织中视黄醇水平显著升高(P0.01),泪腺组织中CRBP及STRA6蛋白表达水平显著升高(P0.05、0.01)。结论干眼症发生时,机体伴随视黄醇缺乏和转运障碍,而酸味药山茱萸水煎液可以促进视黄醇吸收,改善机体视黄醇缺乏状态,进而调控其转运过程,这可能是"酸入肝,明目"的分子机制之一。     

High circulating levels of RBP4 and mRNA levels of aP2, PGC-1alpha and UCP-2 predict improvement in insulin sensitivity following pioglitazone treatment of drug-naïve type 2 diabetic subjects

Context. High levels of circulating retinol‐binding protein 4 ( RBP4) and baseline expression of adipogenic genes correlate with subsequent improvement in insulin sensitivity following Thiazolidinedione (TZD) treatment. Objective. The aim was to identify baseline characteristics and early changes related to TZD treatment that could predict a good treatment response. Design. Subjects were examined with oral glucose tolerance test, intravenous glucose tolerance test, hyperinsulinaemic euglycaemic clamp, body composition and standard blood sampling at baseline and after 4 and 12 weeks treatment. Subcutaneous adipose tissue biopsies were taken from the abdominal region at baseline, after 3 days and 4 weeks treatment to examine the gene expression profile. Setting. Research laboratory in a University hospital. Participants. Ten newly diagnosed and previously untreated type 2 diabetic subjects were treated with pioglitazone for 3 months. Main outcome measures. Baseline characteristics and early changes related to TZD treatment that could predict the response after 3 months. Results. Pioglitazone improved insulin sensitivity after 4 weeks combined with lower glucose and insulin levels without any change in BMI. It was accompanied by lower circulating resistin and plasminogen activator inhibitor‐1 levels rapidly increased levels of circulating total and high molecular weight adiponectin as well as adiponectin and adipocyte fatty acid‐binding protein (aP2) mRNA expression in the adipose tissue. High levels of circulating RBP4 at baseline and adipose tissue expression of aP2, proliferator‐activated receptor‐gamma coactivator 1 alpha (PGC‐1α) and uncoupling protein 2 (UCP‐2) predicted a good treatment response measured as improvement in insulin‐stimulated whole‐body glucose uptake after 3 months. Conclusions. Circulating levels of RBP4 as an index of insulin sensitivity and mRNA levels of adipogenic genes correlate with the subsequent improvement in insulin sensitivity following TZD treatment.     

养阴降糖片联合格列美脲治疗2型糖尿病的临床研究

目的 探讨养阴降糖片联合格列美脲片治疗2型糖尿病的临床疗效。方法 选取2021年3月—2022年3月在山西运城同德医院治疗的106例2型糖尿病患者,根据住院号的奇偶性分为对照组和治疗组,每组各53例。对照组早餐时口服格列美脲片,初始2 mg/次,1次/d,对降糖药敏感者1 mg/次,1次/d,维持剂量1～4 mg/次,1次/d,最大维持量不超过6 mg/次,1次/d;在此基础上治疗组口服养阴降糖片,4片/次,3次/d。两组患者均连续治疗4周进行效果对比。观察两组患者临床疗效,比较治疗前后两组患者空腹血糖（FPG）、餐后2 h血糖（2 h PG）、糖化血红蛋白（HbAlc）、血胰岛素（FINS）和胰岛素抵抗指数（HOMA-IR）水平,HFS-II评分、DDS评分、中医证候积分、DSC评分和CDES-SF评分,血清视黄醇结合蛋白4(RBP4)、摄食抑制因子-1(Nesfatin-1)、抑瘤素M(OSM)、鸢尾素和血清网膜素-1(Omentin-1)水平。结果 治疗后,对照组临床有效率为81.13%,明显低于治疗组98.11%(P<0.05)。治疗后,两组FPG、2 h PG、HbAl...     

检测尿视黄醇结合蛋白对窒息新生儿肾功能损害的诊断价值

目的 探讨尿视黄醇结合蛋白（Ｒｅｔｉｎｏｌ ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ，ＲＢＰ）在窒息新生儿肾功能损害早期诊断的临床价值。方法 应用双抗体夹心酶联免疫吸附法（ＥＬＩＳＡ）对１５例窒息新生儿及２１例正常新生儿分别于生后１、３、７ｄ进行ＲＢＰ检测。结果 窒息组第１、３、７天尿ＲＢＰ水平明显高于对照组（Ｐ〈０．０１）。结论 尿ＲＢＰ可用于窒息新生儿肾功能损害的早期诊断。