English:
[0002] The work described herein was supported, in part, by a grant no. 32412 from the National Institutes of Health. The United States government may have certain rights in the invention.
[0003] The dramatic increase in the incidence of obesity in most parts of the world has contributed to an increased incidence of insulin resistance, type 2 diabetes, and cardiovascular disease. These obesity-associated diseases have become serious threats to human health.
[0006] Many of the conditions that have been shown to trigger ER stress have also been found to occur in obesity, and associated diseases such as type 2 diabetes, hyperglycemia, and insulin resistance. For example, obesity increases the demand on the synthetic machinery of the cell in many secretory organ systems and is also associated with abnormalities in intracellular energy fluxes and nutrient availability. The present invention stems from the recognition that many of these diseases associated with obesity cause ER stress, particularly in peripheral tissues, and that ER stress is involved in triggering insulin resistance and type 2 diabetes, two sequelae of obesity. Therefore, agents that reduce ER stress are useful in treating obesity, peripheral insulin resistance, hyperglycemia, and type 2 diabetes. The agents useful in the treatment of these diseases include small molecules, proteins, nucleic acids, and any other chemical compounds known to reduce or prevent ER stress. These agents make act in any manner that reduces or prevents ER stress such as reducing the production of mutant or misfolded proteins, increasing the expression of ER chaperones, increasing the stability of proteins, boosting the processing capacity of the ER, etc. Particularly useful agents include chemical chaperones such as 4-phenyl butyrate (PBA), tauroursodeoxycholic acid (TUDCA), trimethylamine N-oxide (TMAO), glycerol, D2O, dimethylsufloxide, glycine betaine, methyl amines, and glycerophosphocholine. In particular, both PBA and TUDCA have been shown to regulate ER stress in animals as measured by the reduced phosphorylation of PERK, reduced activation of JNK, and reduced phosphorylation of IRE-I α, as determined by western blot after treatment of the animal with the compound. In addition, both PBA and TUDCA regulate insulin receptor signaling in animals, as measured by increased tyrosine phosphorylation of insulin receptor, insulin substrate 1 -IRS-I and IRS-I, and increased serine phosphorylation of Akt. TMAO has been shown to act as an anti¬ diabetic agent in vivo, lowering glucose and insulin levels when administered to animals with insulin resistance and type 2 diabetes. The agent or a pharmaceutical composition of the agent is administered to a subject {e.g., human, dog, cat, mammal, animal) in doses effective to reduce ER stress, and thereby reduce the signs, symptoms, and consequences of obesity, insulin resistance, and type 2 diabetes. The invention also provides methods of treating and/or preventing obesity, insulin resistance, type 2 diabetes, and hyperglycemia by administering agents that reduce ER stress. The agents may be administered in any manner known in the drug delivery art although preferably the agent is delivered orally or parenterally. Dose ranges for these agents depend on the agent being delivered as well as other factors but will typically be from 10 mg/kg/day to 10 g/kg/day. [0007] In certain embodiments, the agent used to reduce ER stress is 4-phenyl butyric acid (PBA).
[0008] In another embodiment, tauroursodeoxycholic acid (TUDCA), a bile acid, is the agent used to reduce ER stress.
[0012] In another aspect, the invention provides a method of diagnosing insulin resistance, hyperglycemia, or type 2 diabetes by measuring the level of expression of ER stress markers. Markers which may be analyzed in the inventive diagnostic method include spliced forms of XBP-I, phosphorylation status of PERK, phosphorylation of eIF2α, mRNA levels of GRP78/BIP, protein levels of GRP78/BIP, and JNK activity. Any other cellular marker known to be indicative of ER stress may also be used. The levels of these markers may be measured by any method known in the art including western blot, northern blot, immunoassay, or enzyme assay. An increase in the level of an ER stress markers indicates that the subject it at risk for insulin resistance, hyperglycemia, or type 2 diabetes.
[0013] "Animal": The term animal, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In certain embodiments, the animal is a human.
[0015] "Effective amount": In general, the "effective amount" of an active agent, such as an ER stress reducer or a pharmaceutical composition thereof, refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent that reduces or prevents ER stress may vary depending on such factors as the desired biological endpoint, the agent being delivered, the disease being treated, the subject being treated, etc. For example, the effective amount of agent used to treat hyperglycemia or type 2 diabetes is the amount that results in a reduction in blood glucose levels by at least about 10%, 20%, 30%, 40%, or 50%. In other embodiments, the effective amount of the ER stress modulator reduces the levels of at least one ER stress marker (e.g., spliced forms of XBP-I, phosphorylation status of PERK, phosphorylation of eIF2α, mRΝA levels of GRP78/BIP, protein levels of GRP78/BIP, and JΝK activity). In certain embodiments, the levels of at least two, three, four, or more ER stress markers are reduced. The ER stress marker may be reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70,%, 80%, 90%, 95%, 98%, 99%, or 100%. [0016] "Peptide" or "protein": According to the present invention, a
[0024] Figure 6 shows that the treatment of ob/ob mice with tauroursodexoycholic acid (TUDCA) increases insulin sensitivity and reverses diabetes. 9-10 weeks old ob/ob and their age and sex matched lean controls were used to analyze the effects of TUDCA on glucose metabolism and diabetes. After acclimation with PBS injection (once a day, 200 μl i.p. injection) for 4 days, mice were injected intraperitoneally with TUDCA (500 mg/kg once a day in 200 μl of PBS) (n=6 for lean and n=7 for ob/ob) and PBS (200 μl) (n=6 lean and n=7 for ob/ob). Blood glucose levels on the 4th, 7th, and 10th days after first injection (a). ITT on 10 days after the first injection (b).
[0026] Figure 8 shows an effective combination of sodium 4-phenylbutyrate and metformin in the treatment of diabetes. Experiments are performed using the ob/ob genetic model of obesity and insulin resistance (C57B6. V-Z,epøέ/OlaHsd mice were purchased from Harlan Teklad, Madison, WI). At seven weeks of age, treatments were started after acclimatization for 5 days by administration of PBS. After this period, PBS, sodium 4-phenylbutyrate (200 mg/kg/day), metformin (200 mg/kg/day), and sodium 4-phenylbutyrate plus metformin (200 mg/kg/day each) were administered into four separate experimental groups of mice (with at least six mice in each group) by oral gavage daily. At these doses, only the combined sodium 4- phenylbutyrate and metformin proved to be effective as a blood glucose lowering regimen and single agents did not have any effect on blood glucose. Blood glucose levels were measured at the fed state at 8:00 AM.
[0027] Figure 9 shows regulation of GRP78 expression by glucose in vitro and hyperglycemia in vivo. (A) Fao cells were treated with various doses of glucose (0, 5, 10, 25, and 75 mM) for 24 hours. The mRNA level of GRP78 was examined by Northern blot using the total RNAs isolated from these cells. Ethidium bromide staining is shown as a control for loading and integrity of RNA. (B) Streptozotocin (STZ, 200 mg/kg) was injected intaperitoneally into male mice. Three days after injection, blood glucose levels were measured to confirm STZ-induced hyperglycemia. Livers were collected 10 days after injection and GRP78 expression was examined by Northern blot analysis using the liver total RNA. [0028] Figure 10 shows ER stress indicators in adipose tissues of obese mice.
[0034] Figure 16 shows ER stress and insulin receptor signaling in XBP-1+/" mice. PERK phosphorylation (p-PERK) (A), JNK activity (p-c-Jun) (B), and IRS-I Ser307 (IRS-lpSer307) (C) were examined in the livers of XBP-1+/- and XBP-1+/+ mice after 16 weeks on high fat diet. After infusion of insulin (lU/kg) through portal vein, insulin receptor (IR) tyrosine phosphorylation (pY) (D), IRS-I tyrosine phosphorylation (E), IRS-2 tyrosine phosphorylation (F), and Akt Ser473 phosphorylation (G) were examined in livers of XBP-I +/" and XBP- 1+/+ mice after 16 weeks on high fat diet.
[0039] Figure 21 shows increased JNK activity in the liver tissue of obese mice followed by normalization of JNK activity after treatment with PBA.
[0042] The administration of an effective dose of an ER stress modulator, or a combination therapty including an ER stress modulator, to a subject to treat or prevent obesity, insulin resistance, type 2 diabetes, hyperglycemia, or other related disease may cure the disease being treated, alleviate or reduce at least one sign or symptoms of the disease being treated, reduce the short term consequences of the disease, reduce the long term consequences of the disease, or provide some other transient beneficial effect to the subject. In certain embodiments, the inventive treatment increases insulin sensitivity. In other embodiments, the inventive treatment decreases blood glucose levels. In other embodiments, the inventive treatment prevents the long term consequences of diabetes including atherosclerosis, diabetic retinopathy, peripheral neuropathy, etc. In certain embodiments, the inventive treatment reduces levels of ER stress markers {e.g., spliced froms of XBP-I, phosphorylation status of PERK, phosphorylation of eIF2α, mRNA levels of GRP78/BIP, protein levels of GRP78/BIP, JNK activity) in cells {e.g., adipocytes, hepatocytes). In certain embodiments, the inventive treatment increases insulin action. In other embodiments, the inventive treatment increases insulin receptor signalling (e.g., phosphorylation of insulin receptor, IRS-I, IRS-2, akt). In certain embodiments, the inventive treatment suppresses appetite. In other embodiments, the inventive treatment prevents weight gain or promotes weight loss. In certain embodiments, the inventive treatment prevents the development of type 2 diabetes. In certain embodiments, the inventive treatment prevents the development of obesity. In certain embodiments, the inventive treatment prevents the development of hyperglycemia.
[0077] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims. Examples
[0078] To examine whether ER stress is increased in obesity, we investigated the expression patterns of several molecular indicators of ER stress in dietary (high fat diet-induced) and genetic (ob/ob) models of murine obesity. The pancreatic ER kinase or PKR like kinase (PERK) is an ER transmembrane protein kinase that phosphorylates the α subunit of translation initiation factor 2 (eIF2α) in response to ER stress (Shi et al, MoI. Cell Biol. 18, 7499 (1998); Harding et al., Nature 397, 271 (Jan 21, 1999); each of which is incorporated herein by reference). The phosphorylation status of PERK and eIF2α is therefore a key indicator of the presence of ER stress. We determined the phosphorylation status of PERK (Thr980) and eIF2α (Ser51) using phospho-specific antibodies. These experiments demonstrated increased PERK and eIF2α phosphorylation in liver extracts of obese mice compared with lean controls (Figures IA and IB). ER stress also leads to JNK activation. Consistent with earlier observations (J. Hirosumi et al., Nature 420, 333 (2002); incorporated herein by reference), total JNK activity, indicated by c-Jun phosphorylation, was also dramatically elevated in the obese mice (Figures IA and IB).
[0080] We also tested adipose and muscle tissues, important sites for metabolic homeostasis, for indications of ER stress in obesity. Similar to liver, PERK phosphorylation, JNK activity, and GRP78 expression were all significantly increased in adipose tissue of obese animals compared with lean controls (Figure 1 OA-C). However, no indication for ER stress was evident in the muscle tissue of obese animals (data not shown). Taken together, these results indicate that obesity is associated with induction of ER stress predominantly in liver and adipose tissues.
[0082] We next examined the role of JNK in IRS-I serine phosphorylation and inhibition of insulin-stimulated IRS-I tyrosine phosphorylation by ER stress (Figures 11C and 1 ID). Inhibition of JNK activity with the synthetic inhibitor, SP600125, reversed the ER stress-induced serine phosphorylation of IRS-I (Figures 11C and 1 ID). Pretreatment of Fao cells with a highly specific inhibitory peptide derived from the JNK binding protein, JIP (Barr et al, J. Biol. Chem. 277:10987 (2002); incorporated herein by reference), also completely preserved insulin receptor signaling in cells exposed to tunicamycin (Figures 1 IE and 1 IF). Similar results were obtained with the synthetic JNK inhibitor, SP600125 (data not shown). These results indicate that ER stress promotes a JNK-dependent serine phosphorylation of IRS-I, which in turn inhibits insulin receptor signaling.
[0083] In the presence of ER stress, increased phosphorylation of inositol requiring kinase- lα (IRE-lα) leads to recruitment of TNF-α receptor-associated factor 2 (TRAF2) protein and activates JNK (Urano et al, Science 287:664 (2000); incorporated herein by reference). To address whether ER stress-induced insulin resistance is dependent on intact IRE-lα, we measured JNK activation, IRS-I serine phosphorylation, and insulin receptor signaling upon exposure of IRE-lα"'" and wild type (WT) fibroblasts to tunicamycin. In the WT but not IRE-lα"7" cells, induction of ER stress by tunicamycin resulted in strong activation of JNK (Figure 1 IG). Tunicamycin also stimulated phosphorylation of IRS-I at Ser307 residue in WT (Figure 1 IG) but not IRE-lα"Λ fibroblasts (Figure 1 IE). Importantly, tunicamycin inhibited insulin-stimulated tyrosine phosphorylation of IRS-I in the WT cells, whereas no such effect was detected in the IRE-lα"7" cells (Figure 1 IH). The level of insulin-induced tyrosine phosphorylation of IRS-I was dramatically higher in IRE-lα" ;" cells despite lower total IRS-I protein levels (Figure 1 IH). These results demonstrate that ER stress-induced inhibition of insulin action is mediated by an IRE- lα- JNK-dependent protein kinase cascade.
[0086] Complete XBP-I deficiency results in embryonic lethality (Reimold et al, Genes Dev. 14:152, 2000; incorporated herein by reference). To investigate the role of XBP-I in ER stress, insulin sensitivity and systemic glucose metabolism in vivo, we studied Balb/C-XBP-1+/" mice with a null mutation in one XBP-I allele. We studied mice on the Balb/C genetic background, since this strain exhibits strong resistance to obesity-induced alterations in systemic glucose metabolism. Based on our results with cellular systems, we hypothesized that XBP-I deficiency would predispose mice to the development of insulin resistance and type 2 diabetes. [0087] We placed XBP-1+/" mice and their WT littermates on a high fat diet
[0090] Our experiments with cultured cells demonstrated an increase in ER stress and a decrease in insulin signaling capacity in XBP-I -deficient cells and reversal of these phenotypes upon expression of high levels of XBP-Is. If this mechanism is the basis of the insulin resistance seen in XBP-I+7" mice, these animals should exhibit high levels of ER stress coupled with impaired insulin receptor signaling. To test this, we first evaluated ER stress by examining PERK phosphorylation and JNK activity in the livers of obese XBP-I+7" and WT mice. These experiments revealed an increase in PERK levels and seemingly an increase in liver PERK phosphorylation in obese XBP-I+ " mice compared with WT controls on HFD (Figure 18A). There was also a significant increase in JNK activity in XBP-I+7" mice compared with WT controls (Figure 18B). Consistent with these results, serine 307 phosphorylation of IRS-I was also increased in XBP-I+7" mice compared with WT controls on HFD (Figure 18C). Finally, we studied in vivo insulin-stimulated insulin receptor-signaling capacity in these mice. There was no detectable difference in any of the insulin receptor signaling components in liver and adipose tissues between genotypes on regular diet (Figure 19). However, following exposure to HFD, major components of insulin receptor signaling in the liver, including insulin- stimulated IR, IRS-I and IRS-2 tyrosine- and Akt serine-phosphorylation were all decreased in XBP-1+/" mice compared with WT controls (Figure 18D-G). A similar suppression of insulin receptor signaling was also evident in the adipose tissues of XBP-1+/' mice compared with XBP-1+/+ mice on HFD (Figure 20). The suppression of IR tyrosine phosphorylation in XBP- 1+/" mice differs from the observations made in XBP- Y1' cells where ER stress inhibited insulin action at a post-receptor level. It is likely that this reflects the effects of chronic hyperinsulinemia in vivo on insulin receptors. Hence, our data demonstrate the link between ER stress and insulin action in vivo but are not conclusive in determining the exact locus in insulin receptor signaling pathway that is targeted through this mechanism.
[0093] The critical role of ER stress responses in insulin action may represent an evolutionarily conserved mechanism by which stress signals are integrated with metabolic regulatory pathways. Such integration through ER stress would have been advantageous since proper regulation of energy fluxes and suppression of major anabolic pathways such as insulin action might be favorable during acute stress, pathogen invasion and immune responses. However, in the presence of a chronic ER stress such as in obesity, this close link between ER stress and metabolic regulation would lead to development of insulin resistance and eventually, type 2 diabetes. Finally, if the integration of stress signals and metabolic homoeostasis through ER stress has a potential positive impact on survival, a highly responsive system would be subject to selection. This selection might be a potential underlying mechanism for the dramatically high prevalence of metabolic diseases in modern times as exposure to excess caloric load would create continuous stress for ER. In terms of therapeutics, our findings suggest that manipulation of the ER stress response offers new opportunities for preventing and treating type 2 diabetes.
[00100] Insulin Infusion and Tissue Protein Extraction: Insulin was injected through the portal vein as previously described (Uysal et al, Nature 389:610 (1997); Hirosumi et al., Nature 420:333 (2002); each of which is incorporated herein by reference). Three minutes after insulin infusion, liver was removed and frozen in liquid nitrogen and kept at -800C until processing. For protein extraction, liver tissue (~0.3g) was placed in 10 ml of lysis buffer containing 25 mM Tris-HCl (pH7.4), 10 mM Na3VO4, 100 mM NaF, 50 mM Na4P2O7, 10 mM EGTA, 10 mM EDTA, 1% NP- 40, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 10 nM okadaic acid, and 2 mM PMSF. After homogenization on ice, the tissue lysate was centrifuged at 4,000 rpm for 15 minutes at 40C followed by 55,000 rpm for 1 hour at 4°C. One milligram of total tissue protein was used for immunoprecipitation and subsequent immunoblotting, whereas 100-150 μg total tissue protein was used for direct immunoblotting (Hirosumi et al, Nature 420:333 (2002); incorporated herein by reference). Other Embodiments

中文:
[0002]本发明所述的工作部分受到国立卫生研究所(the NationalInstitutes of Health)资助的第32412号项目的支持。美国政府可拥有本发明的某些权利。
[0003]世界大部分地区肥胖症的发病率的戏剧性增加促成胰岛素抗性、2型糖尿病和心血管疾病发病率的增加。这些与肥胖症有关的疾病已对人体健康构成严重威胁。
[0006]现已发现很多呈现出触发ER应力的病症都出现于肥胖症以及诸如2型糖尿病、高血糖症和胰岛素抗性的相关疾病中。例如,肥胖症增加很多分泌器官系统内细胞的合成结构的要求,并且也与细胞内能量流和营养可用性的异常有关。本发明认为:很多这些与肥胖症有关的疾病引起ER应力,尤其在外周组织中,并且ER应力与触发胰岛素抗性和2型糖尿病(肥胖症的两种后遗症)有关。因此,能降低ER应力的药物可用于治疗肥胖症、外周胰岛素抗性、高血糖症和2型糖尿病。用于治疗这些疾病的药物包括小分子、蛋白质、核酸以及已知能降低或阻止ER应力的任何其它化学化合物。这些药物以任何能降低或阻止ER应力的方式发挥作用,例如降低或产生突变体或错折叠蛋白质、增加ER陪伴分子的表达、增加蛋白质的稳定性、推进ER的处理能力等。尤其有用的药物包括化学陪伴分子,如4-苯基丁酸(PBA)、牛熊去氧胆酸(tauroursodeoxycholic acid)(TUDCA)、三甲胺N-氧化物(TMAO)、甘油、D2O、二甲亚砜、甘氨酸三甲铵乙内酯(glycine betaine)、甲胺和甘油磷酸胆碱。特别是,已显示PBA和TUDCA能在动物中调节ER应力,其通过PERK磷酸化降低来测定,并显示它们能降低JNK的激活以及降低IRE-Iα的磷酸化,其通过在用本发明化合物治疗动物后进行的蛋白质印迹测定。另外,PBA和TUDCA能在动物中调节胰岛素受体信号,其可通过增加的胰岛素受体、胰岛素底物1-IRS-1和IRS-1酪氨酸磷酸化以及增加的Akt的丝氨酸磷酸化来测定。当将TMAO给予患有胰岛素抗性和2型糖尿病的动物时,TMAO在体内已呈现出抗糖尿病药物作用,降低葡萄糖和胰岛素水平。将所述药物或该药物的药用组合物以降低ER应力的有效剂量给予患者(例如人、狗、猫、哺乳动物、动物),从而降低肥胖症、胰岛素抗性和2型糖尿病的体征、症状和后遗症。本发明还提供通过给予降低ER应力的药物来治疗和/或预防肥胖症、胰岛素抗性、2型糖尿病和高血糖症的方法。虽然优选将所述药物口服或非肠道给药,但可将以药物传递领域已知的任何方式给予所述药物。这些药物的剂量范围依据所要传递的药物以及其它因素而变化,但一般为10mg/kg/日-10g/kg/日。
[0008]在另一实施方案中,牛熊去氧胆酸(TUDCA),一种胆汁酸,是用于降低ER应力的药物。
[0012]在另一方面,本发明提供一种通过测定ER应力标记的表达水平而诊断胰岛素抗性、高血糖症或2型糖尿病的方法。可在本发明诊断方法中分析的标记包括剪接形式的XBP-1、磷酸化状态的PERK、磷酸化的eIF2α、mRNA水平的GRP78/BIP、蛋白水平的GRP78/BIP和JNK活性。也可使用已知能指示ER应力的任何其它细胞标记。这些标记的水平可通过本领域已知的任何方法测定,包括蛋白质印迹法、RNA印迹法、免疫测定法或酶测定法。ER应力标记水平的增加表明受试者面临胰岛素抗性、高血糖症或2型糖尿病的风险。
[0013]“动物”:此处所用的术语动物指人或非人动物,包括例如哺乳动物、鸟类、爬行类动物、两栖类动物和鱼类。非人动物优选为哺乳动物(例如啮齿类动物、小鼠、大鼠、兔、猴、狗、猫、灵长类动物或猪)。在某些实施方案中,所述动物为人。
[0015]“有效量”:通常活性药物(如ER应力降低剂或其药用组合物)的“有效量”指引发所要求的生物学响应所必需的量。本领域普通技术人员清楚,能降低或阻止ER应力的药物的有效量可依据诸如所要求的生物学结果、正在传递的药物、正在治疗的疾病、所治疗的患者等这些因素而变化。例如,用于治疗高血糖症或2型糖尿病的药物的有效量是能导致血糖水平降低至少约10%、20%、30%、40%或50%的量。在其它实施方案中,ER应力调节剂的有效量降低至少一种ER应力标记(例如剪接形式的XBP-1、磷酸化状态的PERK、磷酸化的eIF2α、mRNA水平的GRP78/BIP、蛋白水平的GRP78/BIP和JNK活性)的水平。在某些实施方案中,可降低至少2、3、4或更多种ER应力标记的水平。可将ER应力标记降低大约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、98%、99%或100%。
[0024]图6显示用牛熊去氧胆酸(TUDCA)治疗ob/ob小鼠能增强胰岛素敏感性病并能逆转糖尿病。使用9-10周龄ob/ob以及与其年龄和性别匹配的瘦对照组小鼠来分析TUDCA对葡萄糖代谢和糖尿病的影响。用PBS注射(每日一次,200μl腹腔注射)适应4日后,给小鼠腹膜内注射TUDCA(一日一次,500mg/kg在200μl PBS中)(瘦组n=6,ob/ob组n=7)和PBS(200μl)(瘦组n=6,ob/ob组n=7)。第一次注射后第4、7和10日的血糖水平(a)。第一次注射后第10日的ITT(b)。
[0026]图8显示4-苯基丁酸盐和二甲双胍在治疗糖尿病中的有效组合。实验采用肥胖症和胰岛素抗性的ob/ob遗传模型(C57B6.V-Lepob/OlaHsd小鼠,购自Harlan Teklad,Madison,WI)进行。在七周龄时,给予PBS适应5日后开始治疗。该期间过后,每日通过管饲法,分别给予四个独立实验组小鼠(每组至少6只小鼠)PBS、4-苯基丁酸钠(200mg/kg/日)、二甲双胍(200mg/kg/日)和4-苯基丁酸钠+二甲双胍(各200mg/kg/日)。在这些剂量中,仅有4-苯基丁酸钠和二甲双胍组合显示能作为一种有效的降低血糖用药方案,而单一药物对血糖不具任何作用。在8:00AM,在喂食时测定血糖水平。
[0027]图9显示体外葡萄糖和体内高血糖对GRP78表达的调节作用。(A)将Fao细胞用各种剂量的葡萄糖(0、5、10、25和75mM)处理24小时。通过RNA印迹法,采用从这些细胞中分离的总RNAs,测定GRP78的mRNA水平。所显示的溴化乙锭染色法为一种装填和整合RNA的对照。(B)向雄性小鼠腹膜内注射链唑霉素(streptozotocin)(STZ,200mg/kg)。注射3日后,测定血糖水平以确定STZ-诱发高血糖症。注射10日后,收集肝,通过RNA印迹分析,采用肝总RNA测定GRP78表达。
[0034]图16显示XBP-1+/-小鼠中ER应力和胰岛素受体信号。在高脂肪饮食16周后,检测XBP-1+/-和XBP-1+/+小鼠的肝中PERK磷酸化(p-PERK)(A)、JNK活性(p-c-Jun)(B)和IRS-1 Ser307(IRS-1pSer307)(C)。门静脉输注胰岛素(1U/kg)后,在高脂肪饮食16周的XBP-1+/-和XBP-1+/+小鼠的肝中,检测胰岛素受体(IR)酪氨酸磷酸化(pY)(D)、IRS-1酪氨酸磷酸化(E)、IRS-2酪氨酸磷酸化(F)和Akt Ser473磷酸化(G)。
[0039]图21显示肥胖小鼠肝组织中JNK活性的增加,以及随后的用PBA处理后JNK活性的归一化(normalization)。
[0042]给予患者有效治疗或预防肥胖症、胰岛素抗性、2型糖尿病、高血糖症或其它相关疾病的量的ER应力调节剂或包括ER应力调节剂的联合药物可以治愈正在治疗的疾病、缓解或降低至少一种正在治疗的疾病的体征和症状、降低疾病的短期后遗症、降低疾病的长期后遗症,或者提供对患者的某些其它的暂时的有益作用。在某些实施方案中,本发明疗法增加胰岛素敏感性。在其它实施方案中,本发明疗法降低血糖水平。在其它实施方案中,本发明疗法预防糖尿病的长期后遗症,包括动脉粥样硬化、糖尿病性视网膜病、外周性神经病等。在某些实施方案中,本发明疗法降低细胞(例如脂肪细胞、肝细胞)中ER应力标记的水平(例如剪接形式的XBP-1、磷酸化状态的PERK、磷酸化的eIF2α、mRNA水平的GRP78/BIP、蛋白水平的GRP78/BIP和JNK活性)。在某些实施方案中,本发明疗法增加胰岛素作用。在其它实施方案中,本发明疗法增加胰岛素受体信号(例如胰岛素受体、IRS-1、IRS-2、akt的磷酸化)。在某些实施方案中,本发明疗法抑制食欲。在其它实施方案中,本发明疗法抑制体重增加或促进体重降低。在某些实施方案中,本发明疗法预防2型糖尿病的发展。在某些实施方案中,本发明的疗法预防肥胖症的发展。在某些实施方案中,本发明治疗方法预防高血糖症的发展。
[0077]本发明的这些和其它方面通过下列实施例被进一步解释说明,所述实施例用于说明本发明的某些具体的实施方案,但并不构成对本发明范围的限定,本发明范围通过权利要求书界定。
[0078]为检测ER应力在肥胖症中是否增加,我们在小鼠肥胖的饮食性(高脂肪饮食诱发)和遗传性(ob/ob)模型中研究了ER应力的几种分子指示器的表达模式。胰ER激酶或PKR样激酶(PERK)是ER跨膜蛋白激酶,其在响应于ER应力下使翻译起始因子2的α亚单位(eIF2α)磷酸化(Shi等,MoI.Cell Biol.18,7499(1998);Harding等,Nature 397,271(Jan 21,1999);其各自通过引用结合到本文中)。因此,PERK和eIF2α的磷酸化状态是ER应力存在的关键指示器。我们使用磷酸特异性抗体测定PERK(Thr980)和eIF2α(Ser51)的磷酸化状态。这些实验表明:与瘦的小鼠对照组比较,肥胖小鼠肝提取物中PERK和eIF2α磷酸化增加(图1A和1B)。ER应力还导致JNK活性。与早期发现(J.Hirosumi等,Nature 420,333(2002);通过引用结合到本文中)一致的是,通过c-Jun磷酸化指示的总JNK活性也在肥胖小鼠中极大地增高(图1A和1B)。
[0080]我们还对脂肪和肌肉组织,即对代谢体内平衡、肥胖症中ER应力的指征重要的部位进行了试验。与肝类似,与瘦的对照组比较,在肥胖动物的脂肪组织中PERK磷酸化、JNK活性和GRP78表达都明显增加(图10A-C)。但是,在肥胖动物的肌肉组织中没有出现明显的ER应力指征(数据未显示)。综上所述,这些结果表明肥胖症与主要在肝和脂肪组织中的ER应力的诱导有关。
[0082]然后,我们检测了JNK对IRS-1丝氨酸磷酸化的作用以及ER应力对胰岛素刺激的IRS-1酪氨酸磷酸化的抑制作用(图11C和11D)。用合成抑制剂SP600125抑制JNK活性逆转ER应力诱导的IRS-1的丝氨酸磷酸化(图11C和11D)。用源于JNK结合蛋白JIP(Barr等,J.Biol.Chem.277:10987(2002);通过引用结合到本文中)的高特异性抑制肽预处理Fao细胞也完全保护暴露于衣霉素的细胞中的胰岛素受体信号(图11E和11F)。用合成JNK抑制剂SP600125也得到类似的结果(数据未显示)。这些结果表明ER应力促进IRS-1的JNK-依赖性丝氨酸磷酸化,然后依次抑制胰岛素受体信号。
[0083]在ER应力存在下,需要激酶-1α(IRE-1α)的肌醇的磷酸化的增高导致TNF-α受体-相关因子2(TRAF2)蛋白的复原并激活JNK(Urano等,Science 287:664(2000);通过引用结合到本文中)。为确定ER应力诱发的胰岛素抗性是否依赖完整的IRE-1α,我们在暴露于衣霉素的IRE-1α-/-和野型(WT)成纤维细胞中测定JNK活性、IRS-1丝氨酸磷酸化以及胰岛素受体信号。在WT但非IRE-1α-/-细胞中,衣霉素诱导的ER应力导致强烈的JNK激活(图11G)。衣霉素还刺激WT中(图11G)但非IRE-1α-/-成纤维细胞(图11E)中的Ser307残基上的磷酸化。重要的是,衣霉素在WT细胞中抑制IRS-1的胰岛素刺激的酪氨酸磷酸化,然而在IRE-1α-/-细胞中未检测到这种结果(图11H)。尽管总IRS-1蛋白水平较低,但IRS-1的胰岛素刺激的酪氨酸磷酸化的水平在IRE-1α-/-细胞中特别高(图11H)。这些结果表明ER应力-诱导的胰岛素作用的抑制作用由IRE-1α-JNK-依赖性蛋白激酶级联所介导。
[0086]完全XBP-1缺陷导致胚胎死亡(Reimold等,Genes Dev.14:152,2000;通过引用结合到本文中)。为研究XBP-1对体内ER应力、胰岛素敏感性和系统葡萄糖代谢的作用,我们研究在一XBP-1等位基因上具有无效突变的Balb/C-XBP-1+/-小鼠。我们在Balb/C遗传背景下研究小鼠,因为这种品系小鼠对系统葡萄糖代谢中肥胖诱导的改变具有强烈的抗性。基于我们对细胞系统的效果,我们假定XBP-1缺陷易于使小鼠产生胰岛素抗性和2型糖尿病。
[0090]我们的培养细胞试验证实在XBP-1缺陷细胞中ER应力增加及胰岛素信号能力降低,并且在高水平XBP-1s表达下这些显型逆转。如果该机理是XBP-1+/-小鼠中所观测到的胰岛素抗性的基础,那么这些动物呈现与受损的胰岛素受体信号偶合的高水平ER应力。为进行这种检测,我们首先在肥胖的XBP-1+/-和WT小鼠的肝中通过测定PERK磷酸化和JNK活性而评估ER应力。这些试验显示:与HFD下的WT对照组比较,肥胖的XBP-1+/-小鼠中PERK水平增加,并且肝PERK磷酸化也似乎增加(图18A)。与WT对照组比较,XBP-1+/-小鼠中JNK活性也明显增加(图18B)。与这些结果一致的是,与HFD下的WT对照组比较,肥胖的XBP-1+/-小鼠中IRS-1的丝氨酸307磷酸化也增加(图18C)。最后,我们研究这些小鼠中体内胰岛素刺激的胰岛素受体信号能力。在正常饮食的显型之间,肝和脂肪组织中任何胰岛素受体信号组分都无能检测到的差别(图19)。但是,暴露于HFD后,与WT对照组比较,XBP-1+/-小鼠的肝中的胰岛素受体信号的主要成分,包括胰岛素-刺激的IR、IRS-1和IRS-2酪氨酸-和Akt丝氨酸-磷酸化都降低(图18D-G)。与HFD下的XBP-1+/+小鼠相比,在XBP-1+/-小鼠的脂肪组织中也出现类似的胰岛素受体信号的抑制(图20)。XBP-1+/-小鼠中IR酪氨酸磷酸化的抑制与在XBP-1-/-细胞中所作的观察不同,其中ER应力在受体后水平上抑制胰岛素作用。这很可能反映体内慢性高血糖症对胰岛素受体的作用。因此,我们的数据表明体内ER应力和胰岛素作用之间又关,但在确定胰岛素受体信号途径中的准确位置上(通过该机理靶向作用点)尚不明确。
[0093]胰岛素作用中ER应力响应的关键作用可能代表一种进化性的保守机理,其将应力信号与代谢性调整途经整合在一起。通过ER应力的此种整合作用具有多种优点,原因是在急性应力、病原体侵入和免疫反应过程中,能量流的适当调整和主要合成代谢途径的抑制(如胰岛素作用)可能是有用的。但是,在慢性ER应力(如肥胖症)存在下,ER应力和代谢性调节之间这种紧密的关联将导致产生胰岛素抗性,并最终产生2型糖尿病。最后,如果通过ER应力的应力信号和代谢体内平衡的整合对存活率具有潜在的积极作用,那么可对高响应性系统进行选择。这种选择可能是一种当今时代代谢疾病戏剧性高度流行的可能的潜在机制,原因是接触过度热量负载将产生持续的ER应力。从治疗方面来看,我们的发现提出对ER应力响应的控制为预防和治疗2型糖尿病提供了新的机遇。
[00100]胰岛素输注和组织蛋白的提取:如前所述,经门静脉注射胰岛素(Uysal等,Nature 389:610(1997);Hirosumi et al.,Nature420:333(2002);各自通过引用结合到本文中)。胰岛素输注3分钟后,移出肝,在液氮中冷冻,在-80°C下保持直至处理。为进行蛋白提取,将肝组织(~0.3g)置于包含25mM Tris-HCl(pH7.4)、10mM Na3VO4、100mM NaF、50mM Na4P2O7、10mM EGTA、10mM EDTA、1%NP-40、5μg/ml亮抑酶肽、5μg/ml抑肽酶、10nM冈田酸和2mM PMSF的10ml溶胞缓冲液中。在冰上匀浆化后,在4°C下,以4,000rpm速度,将组织溶胞产物离心15分钟,接着在4°C下,以55,000rpm速度离心1小时。将其中总组织蛋白的1毫克用于免疫沉淀作用,然后进行免疫印迹,其中100-150μg总蛋白直接用于免疫印迹(Hirosumi等,Nature 420:333(2002);通过引用结合到本文中)。