Dietary fat absorption is one of the most energy-demanding processes in nutrient intake. Fatty acid activation, triglyceride synthesis, and the transport of chylomicrons through the secretory pathway all require ATP. However, it remains unclear how intestinal cells cope with the surge in ATP consumption following fat intake. This study demonstrates that the purine biosynthesis/salvage pathway provides the necessary ATP and that ankyrin repeat domain 9 (ANKRD9) couples ATP synthesis with lipoprotein transport. Ankrd9 knockout mice, despite having intact mitochondrial and glycolytic functions, exhibit reduced intestinal ATP levels, altered Golgi morphology, delayed ApoB/chylomicron transport, resulting in intracellular lipid accumulation and a lean phenotype. These findings reveal a novel mechanism regulating lipid absorption in intestinal cells and identify ANKRD9 as a core component of this mechanism.

Introduction

Energy production and utilization are fundamental properties of every cell. Processes by which cells generate ATP, such as glycolysis, mitochondrial respiration, and the purine de novo synthesis/salvage pathway, have been well characterized. However, during dietary fat absorption, ATP consumption significantly increases to meet the energy demands of chylomicron production and transport, and the mechanism by which the intestine rapidly responds to this surge in energy demand remains unclear. This study explores the role of the ANKRD9 protein in the small intestine and uncovers its critical function in maintaining high ATP levels and facilitating the intracellular processing of chylomicrons.

ANKRD9 is expressed in metabolically active tissues

To understand the physiological function of ANKRD9, we used Ankrd9 knockout mice (Ankrd9-/-). In these mice, the third exon of the Ankrd9 gene was replaced with an FRT-LacZ-loxp-Neo-FRT-loxp cassette, resulting in the loss of Ankrd9 function while allowing LacZ to be expressed under the endogenous Ankrd9 promoter. Using LacZ expression as a surrogate marker for Ankrd9 function, we found that LacZ signals were most abundant in the intestine, heart, and skeletal muscle, all of which are highly metabolically active tissues, suggesting that Ankrd9 may be involved in the regulation of energy supply and/or utilization.

Ankrd9 is crucial for efficient dietary fat handling

Adult Ankrd9-/- mice appeared healthy, with body weights and random blood glucose levels similar to those of control Ankrd9+/+ mice, but exhibited lower fasting blood glucose levels. Despite unchanged insulin sensitivity, male Ankrd9-/- mice had significantly lower total body fat mass compared to controls. Further analysis revealed that while triglyceride (TG) levels in the liver and blood of Ankrd9-/- mice were similar to those of controls, TG levels in the small intestine (jejunum) were eight times higher than in controls, indicating that the further processing of dietary fat after its entry into jejunal enterocytes was affected in Ankrd9-/- mice.

Fatty acid uptake is unaffected by Ankrd9 deficiency

Ankrd9-/- mice had normal intestinal length and morphology, suggesting that the defect may lie within enterocytes. By examining lipid uptake, ApoB abundance, and the transport of ApoB/chylomicrons through the secretory pathway, we found that Ankrd9 deficiency did not affect the rate of fatty acid uptake but delayed the transport of ApoB/chylomicrons.

Ankrd9 deficiency alters ApoB transport

ApoB is the major protein component of chylomicrons. Ankrd9 deficiency did not affect the protein and mRNA levels of ApoB but significantly altered its localization and transport. In wild-type mice, ApoB was mainly concentrated in the perinuclear region of enterocytes, dispersed into the cytoplasm after lipid uptake, and eventually appeared at the apical and lateral membranes. In contrast, in Ankrd9-/- mice, ApoB was initially located in the perinuclear region but its transport to the apical membrane was delayed after lipid uptake, leading to ApoB accumulation within the cells.

ANKRD9 does not directly interact with ApoB but accumulates near the cis-Golgi and lateral membranes

In human differentiated Caco-2 cells, ANKRD9 did not colocalize with ApoB, indicating that ANKRD9 does not regulate ApoB through direct protein-protein interactions. However, in differentiated intestinal Caco-2 cells and human organoids, ANKRD9 accumulated near the cis-Golgi marker GM130 and below the apical membrane. Ankrd9 deficiency led to abnormal Golgi morphology, particularly a significant enlargement of the cis-Golgi, suggesting that ANKRD9 may facilitate the docking of ApoB-containing vesicles with the cis-Golgi and/or the transport from the cis-Golgi to the trans-Golgi.

Ankrd9 deficiency upregulates proteins involved in nucleotide synthesis and lipid transport

By comparing the proteomes of Ankrd9+/+ and Ankrd9-/- jejunal organoids, we found that Ankrd9 deficiency significantly affected the levels of proteins involved in nucleotide homeostasis, glucose metabolism, lipid transport, and protein transport. In particular, the level of IMPDH2, a key enzyme in the purine synthesis pathway, was significantly increased, while the level of adenylate kinase AK4 was decreased. The levels of lipid transport proteins (such as fatty acid-binding proteins and ApoAI, ApoAIV) were also significantly elevated, consistent with intracellular lipid accumulation and delayed chylomicron maturation in enterocytes.

Ankrd9-/- jejunal organoids have low ATP levels despite normal mitochondrial respiration and glycolysis

Ankrd9 deficiency significantly reduced ATP and GTP levels in jejunal organoids, although mitochondrial respiration and glycolysis remained unaffected. This suggests that Ankrd9 may coordinate nucleotide homeostasis and chylomicron transport by regulating the purine biosynthesis/salvage pathway.

Ankrd9 deficiency disrupts purine biosynthesis in the intestine

Ankrd9 deficiency resulted in decreased adenine nucleotide synthesis and increased guanine nucleotide synthesis in the purine biosynthesis pathway, but the total GTP level was still lower than that of controls. This indicates that Ankrd9 deficiency not only affects the stability of IMPDH2 but also impacts other enzymes in the purine biosynthesis pathway. PRPS1 is a key enzyme in the purine biosynthesis pathway. Ankrd9 deficiency did not affect the total abundance of PRPS1 but inhibited its ability to form larger protein complexes.

Lipid uptake triggers changes in ANKRD9 and purine homeostasis

Lipid uptake triggered the disappearance of ANKRD9 from large puncta near the cis-Golgi and its recovery after 30 minutes. Meanwhile, lipid processing increased ATP levels, a response that was lost in Ankrd9-/- enterocytes. In addition, lipid processing induced the formation of rod-like structures (cytoophidia) by IMPDH2, a response that was also less pronounced in Ankrd9-/- enterocytes.

Discussion

This study demonstrates that the purine biosynthesis/salvage pathway in the small intestine plays an important role in providing the ATP required for dietary fat absorption and that ANKRD9 plays a central role in coupling purine biosynthesis with ApoB transport. Ankrd9 deficiency leads to dysregulation of the purine biosynthesis/salvage pathway, reduced ATP levels, and delayed chylomicron transport. ANKRD9 may regulate local nucleotide pools by forming different spatially segregated protein complexes to meet the high energy demands in the vicinity of the Golgi apparatus.