FACTORS CONTROLLING METABOLIC FLUX IN THE LIVER

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 2型糖尿病（T2 DM）的临床定义特征是高血糖症，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，但仍存在与T2 DM中过量葡萄糖产生的来源相关的争议。 最近的研究清楚地表明，葡萄糖的分子来源在糖尿病中受到干扰。 在正常人中，经过一夜的禁食，大约50%的葡萄糖来自糖原，50%来自糖原异生。 但在糖尿病患者中，促血管生成的贡献增加到60%，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重要的认识，但对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，肝脏负责几乎所有的葡萄糖产生。 然而，人们也充分认识到，其他组织有能力在需要时产生葡萄糖。 例如，在人类肝移植的无肝期或动物的全肝切除术中，血浆葡萄糖由肝外来源维持。 我们最近发现，在缺乏肝脏PEPCK的禁食小鼠中，60%的葡萄糖产生来自肝脏以外的组织，而其余的来自肝脏中甘油的代谢。 当整个生物体处于饥饿等强制性条件下时，肾脏和小肠中的葡萄糖产量可能很大。 另一种这样的强制性条件可能发生在急性糖尿病期间，其中胰岛素分泌受到限制或组织对胰岛素的反应能力受损。 在这些条件下，所有产生葡萄糖的组织可能会产生类似于饥饿期间所见的补偿性葡萄糖，即使不需要。 虽然肝外葡萄糖的产生已在长期禁食后的人，狗和大鼠中得到证实，但它在饥饿的影响发生得更快，更明显的小鼠中可能特别重要。 小鼠通常在禁食24小时后进行研究，这可能大约相当于人类禁食72小时。必须在小鼠中理解这种现象，因为越来越多的小鼠模型用于研究人类代谢综合征，并且通常假设肝脏是小鼠中唯一相关的代谢发生部位可能并不总是正确的。60多年来，肾脏一直被认为是产生葡萄糖的器官，但其对全身葡萄糖产生的贡献的重要性仍然是一个争论的主题。 在吸收后状态下，报告的肾脏贡献范围为5 - 28%，而在4-6周的力竭性禁食后报告的肾脏贡献为45%。 在某些情况下，2型糖尿病患者的肾脏再生可能加倍。 广泛报道的肾脏对新生儿造血的贡献反映了进行这种测量在技术上是多么困难。 目前，实验需要肾静脉的导管插入术，通过肾脏的血流量的准确测量，以及葡萄糖浓度的A/V差异的确定。小肠代表了作为葡萄糖产生来源的额外可能性。 在胰岛素缺乏症期间，促胰岛素分泌酶PEPCK、丙酮酸羧化酶、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对葡萄糖生成的贡献可达到总葡萄糖产生的20-25%。 但是像肾脏一样，SI的估计需要严格的侵入性干预，最终的测量可能会带来很大的不确定性。 一种能提供简单、可靠和临床相关指标的方法将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在非侵入性的情况下进行区分，但两者具有与葡萄糖合成的分子起源相关的区别因素。 例如，肝脏储存大量的糖原，而肾脏和小肠等组织仅含有少量的糖原。 因此，它们对葡萄糖产生的贡献必须完全是生糖原的，而不是分解糖原的。 这两个来源很容易区分（见初步数据）稳定同位素示踪方法。 糖尿病患者中的大促血管生成作用（60% vs对照组50%）可能部分是由于肝外葡萄糖的产生。 肝内和肝外血管生成的一个重要区别是底物来源。 肝脏主要从乳酸盐和丙氨酸产生葡萄糖，而小肠和肾脏都强烈地优先于丙氨酸使用乳酸盐和谷氨酰胺。 最近在人类肝移植的无肝期证实了谷氨酰胺对丙氨酸的肝外偏好。 我们正在努力利用底物选择的差异来区分肝脏和肝外对全身葡萄糖产生的贡献。 这些技术将在离体灌注的肾脏和肝脏中进行测试，以建立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.