miR-1 Exacerbates Cardiac Ischemia-Reperfusion Injury in Mouse Models

Recent studies have revealed the critical role of microRNAs (miRNAs) in regulating cardiac injury. Among them, the cardiac enriched microRNA-1(miR-1) has been extensively investigated and proven to be detrimental to cardiac myocytes. However, solid in vivo evidence for the role of miR-1 in cardiac injury is still missing and the potential therapeutic advantages of systemic knockdown of miR-1 expression remained unexplored. In this study, miR-1 transgenic (miR-1 Tg) mice and locked nucleic acid modified oligonucleotide against miR-1 (LNA-antimiR-1) were used to explore the effects of miR-1 on cardiac ischemia/reperfusion injury (30 min ischemia followed by 24 h reperfusion). The cardiac miR-1 level was significantly increased in miR-1 Tg mice, and suppressed in LNA-antimiR-1 treated mice. When subjected to ischemia/reperfusion injury, miR-1 overexpression exacerbated cardiac injury, manifested by increased LDH, CK levels, caspase-3 expression, apoptosis and cardiac infarct area. On the contrary, LNA-antimiR-1 treatment significantly attenuated cardiac ischemia/reperfusion injury. The expression of PKCε and HSP60 was significantly repressed by miR-1 and enhanced by miR-1 knockdown, which may be a molecular mechanism for the role miR-1 in cardiac injury. Moreover, luciferase assay confirmed the direct regulation of miR-1 on protein kinase C epsilon (PKCε) and heat shock protein 60 (HSP60). In summary, this study demonstrated that miR-1 is a causal factor for cardiac injury and systemic LNA-antimiR-1 therapy is effective in ameliorating the problem.     

Phlorizin recognition in a C-terminal fragment of SGLT1 studied by tryptophan scanning and affinity labeling

SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2'-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by site-directed single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564-638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission maxima were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence ( approximately 14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (Kd approximately 54 microm). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (KSV = 11 m-1 in the absence of phlorizin and 55 m-1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two alpha-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4- and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein.     

线粒体分裂抑制剂Mdivi-1对大鼠心肌微血管内皮细胞缺血再灌注损伤的保护作用

目的:探讨Mdivi-1对缺血再灌注损伤后大鼠心肌微血管内皮细胞(cardiac microvascular endothelial cells,CMECs)的保护作用.方法:分离培养大鼠CMECs,建立缺血再灌注(SI/R)模型,随机分为对照组、SI/R组、和SI/R+Mdivi-1组.采用MTT法检测细胞增殖能力;Transwell法检测细胞迁移能力;Annexin V-FITC/PI双标记流式细胞术检测CMECs凋亡率;western blot法检测各组细胞Drp1,Fis1水平;活性氧检测试剂盒测细胞ROS水平.结果:与对照组比较,SI/R组和SI/R+Mdivi-1组细胞增殖和迁移能力明显降低,凋亡明显增加,Drp1,Fis1表达增高,ROS水平明显升高,结果具有统计学意义(P＜0.05);与SI/R组比较,SI/R+Mdivi-1组细胞增殖和迁移能力明显增加,凋亡明显降低,ROS水平明显降低,结果具有统计学意义(P＜0.05),而Drp1,Fis1表达则无明显改变.结论:Mdivi-1可减轻CMECs的缺血再灌注损伤,这种保护作用可能是通过抗氧化应激来实现的.     

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s?1) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.     

Two-Step Mechanism of Phlorizin Binding to the SGLT1 Protein in the Kidney

The relationships between phlorizin binding and Na⁺-glucose cotransport were addressed in rabbit renal brush-border membrane vesicles. At pH 6.0 and 8.6, high affinity phlorizin binding followed single exponential kinetics. With regard to phlorizin concentrations, the binding data conformed to simple Scatchard kinetics with lower apparent affinities of onset binding (K _di = 12–30 μm) compared to steady-state binding (K _de = 2–5 μm), and the first-order rate constants demonstrated a Michaelis-Menten type of dependence with K _m values identical to K _di . Phlorizin dissociation from its receptor sites also followed single exponential kinetics with time constants insensitive to saturating concentrations of unlabeled phlorizin or d-glucose, but directly proportional to Na⁺ concentrations. These results prove compatible with homogeneous binding to SGLT1 whereby fast Na⁺ and phlorizin addition on the protein is followed by a slow conformation change preceding further Na⁺ attachment, thus occluding part of the phlorizin-bound receptor complexes. This two-step mechanism of inhibitor binding invalidates the recruitment concept as a possible explanation of the fast-acting slow-binding paradigm of phlorizin, which can otherwise be resolved as follows: the rapid formation of an initial collision complex explains the fast-acting behavior of phlorizin with regard to its inhibition of glucose transport; however, because this complex also rapidly dissociates in a rapid filtration assay, the slow kinetics of phlorizin binding are only apparent and reflect its slow isomerization into more stable forms. Received: 22 June 2000/Revised: 1 November 2000     

雌激素受体在脑内的表达与缺血再灌注损伤

雌激素作为一种内源性激素参与多器官生理过程的调节,通过与雌激素受体结合或介导不同雌激素受体间的协作而发挥效应,其中在中枢神经系统疾病的发展中具有保护作用已受到人们的广泛关注。脑卒中作为一种高发疾病,具有明显的性别差异性,因此对脑内雌激素的研究为脑卒中的预防及治疗提供了新的方向。我们针对雌激素受体不同亚型在脑内的分布及其在缺血再灌注损伤中的作用研究进展做简要综述。     

T淋巴细胞在小鼠肾缺血再灌注损伤中的作用

目的研究T淋巴细胞在肾缺血再灌注损伤(IRI)导致的急性肾损害中的作用。方法BALB/c小鼠和BALB/c裸小鼠各24只,分别随机分为A1-4组和B1-4组,每组6只。双肾蒂阻断45 min后恢复血流建立肾IRI模型,假手术对照组I、RI后24、48和72 h时检测Scr、尿蛋白定量及肾病理学,A组检测脾T细胞亚群;对比BALB/c小鼠和BALB/c裸小鼠的肾功能下降、组织学损害程度以及脾T淋巴细胞亚群变化。结果A2-4组和B2-4组均有Scr和尿蛋白定量明显升高(P<0.05),且A组损害程度明显重于B组(P<0.05);A2-4组出现典型的IRI组织损害表现(P<0.05),B2-4组无明显IRI组织损害(P>0.05);A2-3组脾CD3 T细胞百分比较A1组升高(P<0.05),而CD4 /CD8 比值无明显变化(P>0.05)。结论T淋巴细胞是小鼠肾IRI导致急性肾损害的重要病理生理学因素。     

血清NGAL 在大鼠肾脏缺血再灌注损伤不同时段 的表达及其意义

目的:观察大鼠肾脏缺血再灌注损伤不同时段血中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的表达并探讨其在急性肾脏缺血再灌注损伤中的意义.方法:建立大鼠肾脏缺血再灌注损伤模型,将50只大鼠随机分为假手术组(S组)和模型组(M组),每组分为5个亚组,包括2h、6h、12h、24h、48h,每亚组大鼠5只.观察各组血NGAL,β2-微球蛋白及血尿素氮,肌酐的变化.结果:M组血NGAL于再灌注损伤后早期(2h)即开始升高,于24h达高峰,至48h仍高于正常(P＜0.05);β2-微球蛋白于12h升高至48h达高峰(P＜0.01);尿素氮于6h升高于48h达高峰(P＜0.01);而血肌酐则于48h才显著升高(P＜0.05).病理显示:M组2h时可见受损肾小管上皮细胞肿胀,管腔扩张、刷状缘消失,至6h时少量上皮细胞脱落、变性甚至坏死,管腔内可见坏死脱落的细胞碎屑,蛋白管型出现,12h时可见间质水肿压迫至管腔明显狭窄,于24h、48h可见蛋白管型显著增多.结论:血NGAL可作为肾脏缺血再灌注损伤早期敏感的生物标志物.     

Protective Effect of Ginsenoside Rb1 against Intestinal Ischemia-Reperfusion Induced Acute Renal Injury in Mice

Ginsenoside Rb1 (RB1), the most clinically effective constituent of ginseng, possesses a variety of biological activities. The objectives of this study were to investigate the protective effects of RB1 and its underlying mechanism on renal injury induced by intestinal ischemia-reperfusion (IIR) in mice. RB1 was administered prior to inducing IIR achieved by occluding the superior mesenteric artery for 45 min followed by 120 min of reperfusion. All-trans-retinoic acid (ATRA) was used as an inhibitor of NF-E2-related factor-2 (Nrf2) signaling. Adult male C57BL/6J mice were randomly divided into six groups: (1) sham group, (2) IIR group, (3) RB1 group, (4) sham + ATRA group, (5) IIR + ATRA group, and (6) RB1 + ATRA group. Intestinal histology and pathological injury score were observed. Intestinal mucosal injury was also evaluated by measuring serum diamine oxidase (DAO). Renal injury induced by IIR was characterized by increased levels of histological severity score, blood urea nitrogen (BUN), serum creatinine (Scr) and neutrophil gelatinase-associated lipocalin (NGAL), which was accompanied with elevated renal TUNEL-positive cells and the Bcl-2/Bax expression ratio. RB1 significantly reduced renal injury and apoptosis as compared with IIR group, which was reversed by ATRA treatment. Immunohistochemistry and Western blot analysis demonstrated that RB1 significantly upregulated the protein expression of heme oxygenase-1 (HO-1) and Nrf2, which were attenuated by ATRA treatment. Taken together, these results suggest that the protective effects of RB1 pretreatment against renal injury induced by IIR are associated with activation of the Nrf2/ anti-oxidant response element (ARE) pathway.     

Sirtuin 1 (SIRT1) Activation Mediates Sildenafil Induced Delayed Cardioprotection against Ischemia-Reperfusion Injury in Mice

Background

It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury.

Objective/Hypothesis

We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1.

Methods

Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis.

Results

Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001) as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM) or RSV (1 µM) for one hour in vitro also upregulated SIRT1 activity (P<0.05). We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV) 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05). Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p.) given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001).

Conclusion

Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.     

NDRG2在大鼠肺缺血再灌注损伤中的表达

目的:通过建立大鼠肺缺血再灌注损伤(Lung ischemia-reperfusion injury,LIRI)模型,观察肺缺血再灌注损伤后,肺组织中N-myc下游调节基因2(N-myc downstream regulated gene,NDRG2)表达水平的变化.方法:将70只健康成年雄性SD大鼠随机分成对照组(C)、缺血组(I)、缺血再灌注组(I/R)(后两组各含3个亚组),每组10只.麻醉固定大鼠,颈部切口行气管插管.右侧开胸,肺缺血组依次分别选择游离夹闭右肺门(即右主支气管,右肺动、静脉)缺血30 min、60 min、120 min后,麻醉处死大鼠获取肺组织.肺缺血再灌注组同样选择游离夹闭右肺门,于夹闭右肺门60 min后松开,分别取再灌注30 min、60 min、120m in后麻醉处死大鼠获取肺组织样本.采用免疫组化对肺组织NDRG2进行蛋白定位检测、RT-PCR对肺组织NDRG2 mRNA含量进行检测、Western-blot对肺组织NDRG2蛋白含量进行检测.结果:肺缺血组与对照组比较,肺组织NDRG2的表达无明显变化(P＞0.05);肺缺血再灌注组与对照组比较,NDRG2蛋白含量和mRNA表达量逐渐下降,在60 min时达最低,之后又有所回升,但仍低于对照组(P＜0.05).结论:肺缺血再灌注损伤可下调肺组织中NDR G2的表达含量,NDRG2可能是肺缺血再灌注损伤的靶向调控位点.     

Glycoside Binding and Translocation in Na+-Dependent Glucose Cotransporters: Comparison of SGLT1 and SGLT3

Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na⁺/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K_0.5 0.06 mm) and 2-naphthylglucose with lower affinity (K_0.5 0.5 mm) than α-methyl-d-glucopyranoside (αMDG, 0.2 mm). Both were poorly transported (maximal velocities, I _max , 14% and 8% of αMDG). Other compounds were inhibitors (K _i s 1–13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than αMDG (K_0.5s 0.9, 0.2 and 2.5 mm and relative I _max s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I _max s (130 and 120%) and comparable K_0.5s (8 and 1 mm). Increased affinity of indican relative to αMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value. Received: 18 February 2000/Revised: 13 April 2000     

Identification of a Region Critically Involved in the Interaction of Phlorizin with the Rabbit Sodium-D-Glucose Cotransporter SGLT1

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604–610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of α-methyl-d-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K _i for phlorizin increased at least by a factor of 5 compared to the wild-type K _i of 4.6 ± 0.7 μmol/l; most striking changes were observed for Y604K (K _i = 75.3 ± 19.0 μmol/l) and C608K (K _i = 83.6 ± 13.9 μmol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (K _m = 0.8 ± 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside. Received: 29 March 2001/Revised: 15 June 2001     

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality^1-4 and which is most frequently caused by whole-body hypoperfusion.^5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with difficulty.^7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.^11,12 However, in recent years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.¹³ This model reliably reproduces physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience with use of this model to assess a number of organ-specific outcomes in AKI.^14,15     

Chronic Testosterone Replacement Exerts Cardioprotection against Cardiac Ischemia-Reperfusion Injury by Attenuating Mitochondrial Dysfunction in Testosterone-Deprived Rats

Background

Although testosterone deficiency is associated with increased risks of heart disease, the benefits of testosterone therapy are controversial. Moreover, current understanding on the cardiac effect of testosterone during cardiac ischemia-reperfusion (I/R) periods is unclear. We tested the hypothesis that testosterone replacement attenuates the impairment of left ventricular (LV) function and heart rate variability (HRV), and reduces the infarct size and arrhythmias caused by I/R injury in orchiectomized (ORX) rats.

Methodology

ORX or sham-operated male Wistar rats (n = 24) were randomly divided and received either testosterone (2 mg/kg, subcutaneously administered) or the vehicle for 8 weeks. The ejection fraction (EF) and HRV were determined at baseline and the 4^th and 8^th week. I/R was performed by left anterior descending coronary artery ligation for 30 minutes, followed by a 120-minute reperfusion. LV pressure, arrhythmia scores, infarct size and cardiac mitochondrial function were determined.

Results

Prior to I/R, EF and HRV were impaired in the ORX group, but were restored in the testosterone-treated group. During I/R, arrhythmia scores and the infarct size were greater, and cardiac mitochondrial function was impaired, whereas the time to 1^st VT/VF onset and the LV end-systolic pressure were decreased in the ORX group when compared to the sham group. Testosterone replacement attenuated the impairment of these parameters in ORX rats during I/R injury, but did not show any benefit or adverse effect in non-ORX rats.

Conclusions

Testosterone replacement restores cardiac function and autonomic regulation, and exerts cardioprotective effects during the I/R period via mitochondrial protection in ORX rats.     

Toll-Like Receptor 2 Mediates Ischemia-Reperfusion Injury of the Small Intestine in Adult Mice

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.     

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.     

Sympathetic Regulation of Glucose Uptake by the α1‐Adrenoceptor in Human Obesity

Objective: To investigate the involvement of α₁‐adrenoceptors in the sympathetic regulation of glucose uptake in human adipocytes. Research Methods and Procedures: Twenty‐four severely obese subjects participated in this study. The microdialysis technique was used to determine interstitial glucose concentration after stimulation of abdominal subcutaneous adipose tissue with the α₁‐agonist norfenefrine, the α_{1, 2}β‐agonist norepinephrine, and both agents in combination with the α₁‐antagonist urapidil. The effect of β‐adrenoceptor stimulation was assessed by orciprenaline. Changes in local blood flow were determined using the ethanol escape technique. Results: Both norfenefrine and norepinephrine induced a concentration‐dependent decrease of interstitial glucose concentration, with a greater decrease observed with norepinephrine. Preperfusion of adipose tissue with urapidil inhibited glucose decrease. The inhibition was overcome with high concentrations of norfenefrine and norepinephrine, respectively. Both adrenergic agents induced tachyphylaxia. Urapidil enhanced extracellular glucose level at high concentration. Blood flow decreased in the presence of norfenefrine and norepinephrine but increased in response to urapidil. The accelerated blood flow due to urapidil was counteracted by norepinephrine and norfenefrine. Orciprenaline decreased interstitial glucose concentration and increased nutritive blood flow. The observed changes in blood flow induced by adrenergic agents were not related to glucose uptake. Discussion: The stimulatory effect of the sympathetic nerves on glucose uptake in subcutaneous adipose tissue appears to be mediated by the α₁‐adrenoceptor. Norepinephrine enhances glucose entry into adipocytes independently of insulin action. In obese subjects with insulin resistance, the α₁‐adrenergic receptor may provide an important alternative pathway for glucose uptake.     

SIRT6 Deficiency Results in Severe Hypoglycemia by Enhancing Both Basal and Insulin-stimulated Glucose Uptake in Mice

Glucose homeostasis in mammals is mainly regulated by insulin signaling. It was previously shown that SIRT6 mutant mice die before 4 weeks of age, displaying profound abnormalities, including low insulin, hypoglycemia, and premature aging. To investigate mechanisms underlying the pleiotropic phenotypes associated with SIRT6 deficiency, we generated mice carrying targeted disruption of SIRT6. We found that 60% of SIRT6^−/− animals had very low levels of blood glucose and died shortly after weaning. The remaining animals, which have relatively higher concentrations of glucose, survived the early post-weaning lethality, but most died within one year of age. Significantly, feeding the mice with glucose-containing water increased blood glucose and rescued 83% of mutant mice, suggesting that the hypoglycemia is a major cause for the lethality. We showed that SIRT6 deficiency results in more abundant membrane association of glucose transporters 1 and 4, which enhances glucose uptake. We further demonstrated that SIRT6 negatively regulates AKT phosphorylation at Ser-473 and Thr-308 through inhibition of multiple upstream molecules, including insulin receptor, IRS1, and IRS2. The absence of SIRT6, consequently, enhances insulin signaling and activation of AKT, leading to hypoglycemia. These data uncover an essential role of SIRT6 in modulating glucose metabolism through mediating insulin sensitivity.