Investigation of Gut-Derived Circulating Succinate in Metabolism and Liver Signaling

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2022

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Abstract

The tricarboxylic acid (TCA) cycle is the epicenter of cellular aerobic metabolism and accounts for the majority of cellular ATP production. In addition to its use in anabolic and catabolic processes, TCA cycle intermediates may serve as signaling molecules. The TCA cycle intermediates succinate and α-ketoglutarate are specific natural ligands for previously orphan receptors GPR91 (Sucnr1) and GPR99 (Oxgr1), respectively. The function of Sucnr1 is more extensively studied and its activation by succinate regulates pleiotropic biological processes. The physiological range of circulating succinate coincides with the pharmacological range (EC50) of Sucnr1 activation, suggesting that changes in circulating succinate levels have the potential to affect the activity of Sucnr1 and its downstream targets. This supports the potential for a signaling role of succinate in health and disease.Despite the potential importance of circulating TCA cycle metabolites as signaling molecules, the source of circulating TCA cycle intermediates remains uncertain. Although prior studies showed that exogenous succinate via activating Sucnr1 regulates biological function in various tissues, succinate concentrations used in such studies typically far exceed circulating levels. Thus, it is uncertain whether circulating succinate signals through Sucnr1 and whether there are specific cell-types or tissue-types where this is more or less likely to occur. As a result, the physiological roles for circulating succinate as a signaling molecule remains speculative. In my thesis work, we set out to address three major questions. First, we explored the major source of circulating TCA cycle metabolites and found the gut plays a major role in this. Second, we investigated the role of intestinal-derived circulating succinate in signaling with a focus on the role of Sucnr1 signaling in the liver. Third, using our new knowledge that the intestine is a major production site of circulating TCA cycle metabolites including succinate, we used this knowledge to develop a method to further probe intestinal and systemic metabolism. Our work elucidating the site of circulating TCA cycle metabolites began when we first observed that TCA cycle intermediates levels are much higher in the portal vein compared with tail vein plasma. This observation led directly to our hypothesis that the gut might be a major contributor to circulating TCA cycle metabolites. With a focus on succinate as a representative TCA cycle intermediate with known signaling activities and using a combination of germ-free mice and isotopomer tracing, we demonstrated that intestinal microbiota are not major contributors to circulating TCA cycle metabolites. Moreover, we demonstrated that endogenous succinate production is markedly higher than intestinal succinate absorption in normal physiological conditions. Altogether, these results showed that endogenous succinate production within the intestinal tissue is a major physiological source of circulating succinate. We next sought to determine whether gut-derived succinate may function as a metabolic signal and regulate biological function. This could occur through activation of the previously described succinate receptor, Sucnr1, or through other, unknown mechanism. Sucnr1 is highly expressed in multiple metabolic tissues including the liver. This combined with our evidence that the intestine is a major source of circulating succinate led to the hypothesis that cells within the liver expressing Sucnr1 might be a major target of intestine-derived, circulating succinate. By analyzing single-cell RNA-sequencing data, we found that Sucnr1 is expressed in both hepatocytes and liver non-parenchymal cells including hepatic stellate cell, endothelial cells and the Kupffer cells. Using distinct methods to generate liver-selective versus hepatocyte-selective Sucnr1 knockout mice, we demonstrated that Sucnr1 is predominantly expressed in hepatocytes and to a much lesser degree in non-parenchymal cells. As Sucnr1 is a Gi coupled GPCR, we further hypothesized that succinate mediated activation of Sucnr1 might antagonize the glucagon receptor (GCGR), the most abundant Gs-coupled GPCR expressed in hepatocytes. Initially, succinate infusion experiments and assessment of its effects on glucagon signaling in mouse liver appeared to support the possibility that succinate might antagonize hepatocyte glucagon signaling. However, subsequent experiments in liver specific Suncr1 knockout mice did not strongly support this hypothesis. Therefore, to investigate the potential role of gut-derived succinate and its effects on liver Sucnr1 from a more unbiased perspective, we conducted RNA-sequencing of liver samples from liver-specific Sucnr1 knockout mice and their littermate controls which suggested that hepatic Sucnr1 may play modest regulatory role in regulating amino acid metabolism and in some infections. Altogether, our work indicates that hepatocytes are a major site of Sucnr1 expression. However, whether it has a major role in responding to circulating succinate and what that role might be remains less certain. Our results suggest that hepatic Suncr1 does not play a major role in antagonizing hepatic glucagon signaling. Hepatic RNA-seq analysis suggests that hepatic Sucnr1 may have a role in infection and this may be consistent with a putative role for Sucnr1 in responding to intestinal parasitic infections and in immunity more generally. In the course of this work, we demonstrated that the intestine tissue is a major contributor to circulating TCA cycle intermediates. Additionally, recent studies from our lab and others showed that fructose is largely metabolized in the intestine tissue and readily incorporated into the TCA cycle intermediates. Therefore, we hypothesized that we could use intestinal fructose metabolism and its incorporation into circulating TCA cycle metabolites as a methodology to investigate intestinal metabolism in live, conscious animals. Metformin is the most widely prescribed anti-diabetic drug and concentrates at high levels within enterocytes. Its mechanism of action to enhance systemic metabolism and reduce or delay the progression to type 2 diabetes remains highly controversial. Therefore, we hypothesized that isotope-tracing metabolomics performed after gavaging U-C-13 fructose could be used to investigate the role of metformin in intestinal and systemic metabolism. Consistent with this, we have now demonstrated that metformin significantly diminished intestinal fructose metabolism, flux of metabolites into the TCA cycle within the intestine, and intestinal glucose production. Metformin also reduced the delivery of ingested fructose to the liver. Altogether, these results indicate that isotopomer tracing of intestinal fructose metabolism and TCA cycle intermediate production may serve as a non-invasive method to investigate intestinal metabolism and the mechanism of action of metformin. Overall, our work provides insight into the major source of circulating TCA cycle intermediates, potential function of the succinate sensing receptor, Sucnr1, in hepatocytes, and the role of metformin in regulating intestinal metabolism and TCA cycle production. These data provide a foundation for further investigation into the role of the intestine in regulating circulating TCA cycle metabolites and related signaling effects in health and disease. Furthermore, these data clarify that Sucnr1 is predominantly expressed in the liver hepatocyte, in contrast with prior findings that suggested that Sucnr1 within the liver was predominantly expressed in non-parenchymal cells. The contributions and novel insight provided here will be important to fully understand the regulatory role of succinate-Sucnr1 axis in liver functions and provide a novel approach for using intestinal fructose metabolism and TCA cycle intermediates production to probe intestinal and systemic metabolism.

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Tong, Wenxin (2022). Investigation of Gut-Derived Circulating Succinate in Metabolism and Liver Signaling. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25213.

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