FACTORS CONTROLLING METABOLIC FLUX IN THE LIVER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 2型糖尿病(T2 DM)的临床特征是高血糖，因此，葡萄糖代谢是T2 DM研究最广泛的方面。尽管做出了相当大的努力，但关于T2 DM中过量葡萄糖产生的来源仍然存在争议。最近的研究清楚地表明，糖尿病患者的葡萄糖分子来源受到干扰。在正常人中，在隔夜禁食后，大约50%的葡萄糖来自糖原，50%来自糖异生。但在糖尿病患者中，糖异生的贡献率增加到60%，这表明对这一途径失去了控制。尽管对糖尿病中葡萄糖的产生有了这一重要的见解，但关于葡萄糖的解剖起源仍存在一些分歧。人们普遍认为，在正常的吸收后状态下，几乎所有葡萄糖的产生都由肝脏负责。然而，人们也很好地认识到，在需要的时候，其他组织也有能力产生葡萄糖。例如，在人类肝移植的无肝期或动物的全肝切除术中，血糖由肝外来源维持(S)。我们最近发现，在缺乏肝脏PEPCK的空腹小鼠中，60%的葡萄糖来自肝脏以外的组织，其余的来自肝脏甘油的糖异生。当整个生物体处于饥饿等令人信服的条件下时，肾脏和小肠中的葡萄糖产量可能会很大。另一种令人信服的情况可能发生在急性糖尿病期间，即胰岛素分泌受限或组织对胰岛素的反应能力受损。在这种情况下，所有产生葡萄糖的组织可能会产生与饥饿时相似的反应，产生补偿葡萄糖，即使不需要。虽然在长期禁食后，人、狗和大鼠都已经证明了肝外葡萄糖的产生，但在饥饿影响发生得更快、更明显的小鼠中，这一点可能尤其重要。老鼠通常在禁食24小时后进行研究，这可能大约相当于人类72小时的禁食。这一现象必须在小鼠身上得到理解，因为越来越多的小鼠模型被用于研究人类代谢综合征，而通常认为肝脏是小鼠糖异生的唯一相关部位的普遍假设可能并不总是正确的。60多年来，肾脏一直被认为是葡萄糖产生的器官，但它们对全身葡萄糖产生的贡献的重要性仍然是一个有争议的话题。报告的肾脏贡献在吸收后状态下为5%-28%，而在精疲力竭的禁食4-6周后报告为45%。在一定条件下，T2 DM患者肾脏糖异生可加倍。已报道的肾脏对糖异生的广泛贡献反映了进行这一测量的技术难度有多大。目前，实验需要对肾静脉进行插管，准确测量通过肾脏的血流量，并确定葡萄糖浓度的A/V差。作为葡萄糖生产的来源，小肠代表了另外一种可能性。在胰岛素缺乏时，糖异生酶PEPCK、丙酮酸羧基酶、果糖-1，6-二磷酸酶和葡萄糖-6磷酸酶上调。据估计，在糖尿病期间，SI对糖异生的贡献可能达到总葡萄糖产量的20%-25%。但就像肾脏一样，SI糖异生的估计需要严格的侵入性干预，最终的测量可能带有重大的不确定性。提供一种简单、可靠和临床相关的肝外糖异生指标的方法将非常有助于最终理解这一现象的重要性。肝脏和肝外葡萄糖的产生很难无创地区分，但两者都有与葡萄糖合成的分子起源有关的区别因素。例如，肝脏储存了大量的糖原，而肾脏和小肠等组织只含有少量的糖原。因此，它们对葡萄糖生产的贡献必须完全是糖异生的，而不是糖原分解的。这两个来源很容易通过稳定同位素示踪方法区分(见初步数据)。糖尿病患者糖异生的巨大贡献(对照组为60%比50%)可能部分是由于肝外葡萄糖的产生。肝糖异生和肝外糖异生的一个重要区别是底物来源。肝脏主要由乳酸和丙氨酸产生葡萄糖，而小肠和肾脏都热衷于使用乳酸和谷氨酰胺，而不是丙氨酸。在肝移植的无肝期，这种谷氨酰胺对丙氨酸的肝外偏好最近在人类身上得到了证实。我们正在努力利用底物选择的这种差异来区分肝脏和肝外对全身葡萄糖产生的贡献。这些技术将在分离的灌流肾脏和肝脏中进行测试，以确定底物偏好并验证体内实验。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The clinically defining feature of type 2 diabetes (T2DM) is hyperglycemia, and as such, glucose metabolism the most widely studied aspect of T2DM. Despite this considerable effort, there remains controversy related to the source of superfluous glucose production in T2DM. Recent work clearly demonstrates that the molecular source of glucose is disturbed in diabetes. In the normal person, after an overnight fast, about 50% of glucose is derived from glycogen and 50% from gluconeogenesis. But in the diabetic person, the gluconeogenic contribution increases to 60%, suggesting a loss of control over this pathway. Notwithstanding this important insight into glucose production in diabetes, there is some disagreement about the anatomical origin of glucose. It is widely accepted that the liver is responsible for nearly all glucose production in the normal post-absorptive state. However, it is also well recognized that other tissues have the capacity to produce glucose in times of need. For instance during the anhepatic phase of liver transplantation in humans or in the total hepatectomy of animals, plasma glucose is maintained by extrahepatic source(s). We've recently shown that in fasted mice lacking hepatic PEPCK, 60% of glucose production comes from tissues other than liver, while the remainder comes from gluconeogenesis from glycerol in liver. Glucose production can be significant in the kidney and small intestine when the whole organism is subjected to compelling conditions such as starvation. Another such compelling condition may occur during acute diabetes where insulin secretion is restricted or the tissues' ability to respond to insulin is impaired. Under these conditions, all glucose producing tissues may respond similarly to that seen during starvation by producing compensatory glucose even though none is needed. While extra-hepatic glucose production has been demonstrated in man, dog and rats after long-term fasting, it may be especially important in mice where the affects of starvation occur more rapidly and are more pronounced. Mice are commonly studied after fasts as long as 24-hours, which may be approximately equivalent to a 72-hour fast in man. This phenomenon must be understood in mice because a growing number of mouse models are used to study human metabolic syndromes and the common assumption that the liver is the only relevant site of gluconeogenesis in mice may not always be true. The kidneys have been recognized as glucose-producing organs for over 60 years, but the significance of their contribution to whole body glucose production remains a subject of debate. Reports of the renal contribution range from 5 - 28% in the post-absorptive state while a 45% contribution was reported after an exhaustive fast of 4-6 weeks. Under certain conditions, renal gluconeogenesis may be doubled in T2DM. The wide range of reported renal contributions to gluconeogenesis reflects how technically difficult it is to make this measurement. Currently, experiment requires catheterization of the renal vein, accurate measurement of blood flow through the kidneys, and determination of A/V differences in glucose concentration. The small intestine represents and additional possibility as a source of glucose production. During insulinopenia the gluconeogenic enzymes PEPCK, pyruvate carboxylase, fructose-1,6-bisphosphatase and glucose-6 phosphatase are up-regulated. It has been estimated that the contribution of the SI to gluconeogenesis may reach 20-25% of total glucose production during diabetes. But like the kidney, estimations of SI gluconeogenesis requires rigorous invasive intervention and the final measurement may carry significant uncertainty. A method which supplies a simple, reliable and clinically relevant indicator of extra-hepatic gluconeogenesis would be extremely helpful to finally understanding the importance of this phenomenon. Hepatic and extra-hepatic glucose productions are difficult to differentiate non-invasively, but the two have distinguishing factors related to the molecular origin of glucose synthesis. For instance, the liver stores large amounts of glycogen while tissues such as the kidneys and small intestine contain only minor amounts of glycogen. Thus their contribution to glucose production must be entirely gluconeogenic rather than glycogenolytic. These two sources are easily distinguished (see preliminary data) by stable isotope tracer methods. The large gluconeogenic contribution in diabetics (60% vs 50% in controls) may be partly due to extra-hepatic glucose production. An important difference between hepatic and extra-hepatic gluconeogenesis is the substrate source. The liver produces glucose largely from lactate and alanine, whereas both the small intestine and kidneys avidly use lactate and glutamine in large preference to alanine. This extra-hepatic preference of glutamine over alanine was recently demonstrated in humans during the anhepatic phase of liver transplantation. We are working to take advantage of this difference in substrate selection to distinguish between hepatic and extra-hepatic contributions to systemic glucose production. These techniques will be tested in the isolated perfused kidney and liver to establish substrate preference and validate in vivo experiments.