AMPK-PDZD8 Drives Glutamine Metabolism in the Early Stage of Low Glucose
Carbohydrates, fats, and proteins are the three major energy sources for the human body. When glucose levels decrease, the body or cells will turn to consume other molecules to make up for the lack of Glucose. AMPK plays a key role in this metabolic conversion adaptation process, but its mechanism is still unclear.
Glutamine is the most abundant circulating amino acid, accounting for more than 50% of free amino acids in the body during fasting. Palmitic acid is the most common saturated fatty acid, accounting for about 20-30% of total fatty acids in the body. Therefore, the researchers first pre-labeled mouse embryonic fibroblasts with [U-13C]-palmitic acid and [U-13C]-glutamine, respectively, to measure the utilization of glutamine and fatty acids by cells under glucose starvation conditions. Simply put, after fatty acids and glutamine enter the TCA cycle, their labeled carbon atoms (U-13C) will participate in the synthesis of downstream products such as citric acid, α-ketoglutaric acid, succinic acid, and malic acid. Therefore, the changes in the U-13C content in these downstream molecules can reflect the utilization of fatty acids and glutamine by cells.
They found that when cells were starved of glucose for just 2 hours, U-13C in [U-13C]-glutamine increased significantly in downstream molecules of the TCA cycle. In contrast, U-13C in [U-13C]-palmitate did not appear significantly in downstream molecules until 12 hours. In addition, when the researchers knocked down GLS1, the rate-limiting enzyme in glutamine metabolism, and treated with low sugar for 2 hours, the increase in cellular oxygen consumption rates (OCR) was significantly inhibited compared with non-starvation, while knocking out CPT1 had no effect. These data indicate that when glucose is scarce, cells first use glutamine to compensate for the lack of glucose rather than fatty acids in the early stage.
Fig. 1 In the early stages of glucose starvation, cells switch to utilizing glutamine. (Li, et al., 2024)
AMPK plays a central role in maintaining energy homeostasis and is very sensitive to drops in glucose levels. Previous studies have shown that when glucose is scarce, AMPK is activated, and it promotes the catabolism of amino acids by inhibiting the targets of TORC1, thereby providing energy to cells. Therefore, the researchers speculated that when glucose is scarce, whether cells first use glutamine through the above pathway? They knocked out AMPKα and the LKB1, AXIN or LAMTOR1 molecules required for AMPK activation and found that under low glucose conditions, glutamine metabolism in cells within 2 hours could be significantly inhibited. In contrast, under low glucose conditions, when TORC1 was knocked out or Torin1 was used for inhibition, glutamine metabolism in cells within 2 hours was not affected. This suggests that when glucose is scarce, the first decomposition and utilization of glutamine by cells is mediated independently by AMPK and does not rely on TORC1.
So how does AMPK mediate the metabolic conversion of cells to glutamine under low-glucose conditions? Before this study, researchers found that AMPK under low-glucose conditions would lead to a decrease in the production of pure mitochondria in cells, and proved that this was caused by the interaction between the mitochondria-associated membrane (MAM) and the endoplasmic reticulum membrane. Therefore, their goal in this study was very clear, which was to find MAM proteins that might interact with AMPK.
Subsequently, they separated and purified the MAM and related components of cells under low-glucose conditions, and obtained the substrates of AMPK through protein pull-down combined with mass spectrometry analysis. One of the substrates, PDZD8, can not only be phosphorylated by AMPK under low-glucose conditions, but also promote the metabolism of glutamine in the TCA cycle.
Through the analysis of PDZD8 phosphorylation sites and in vitro kinase experiments, it was found that AMPK can phosphorylate the T527 site of PDZD8. This shows that PDZD8 is a new substrate of AMPK. More importantly, in cells with PDZD8 knockout, the introduction of the T527A mutant only prevents the early utilization of glutamine by cells under low-glucose conditions. This shows that in the early stage of low-glucose starvation, cells accelerate the consumption of glutamine by phosphorylating the PDZD8 T527 site through AMPK to make up for the loss of capacity caused by glucose deficiency.
So how does PDZD8 promote the utilization of glutamine? The first step of glutamine metabolism in mitochondria is the decomposition into glutamate catalyzed by GLS1. Therefore, the researchers suspected that PDZD8 activated GLS1 under low-sugar conditions. The results showed that in glucose-starved cells, the enzymatic activity of GLS1 was significantly enhanced; and knocking out PDZD8 blocked the enhancement of GLS1 activity. Enzymes generally require the help of cofactors when activated. But interestingly, in an in vitro system, even when GLS1 does not have a cofactor, phosphate, the PDZD8 T527 phosphorylated form can independently activate GLS1. This shows that PDZD8 does not require the participation of cofactors to activate GLS1. Further, biochemical experiments showed that the C-terminal domain of PDZD8 can directly interact with GLS1. In addition, PDZD8 Phosphorylation also enhances the affinity of GLS1 for the substrate glutamine. Therefore, these data suggest that cells activate PDZD8 through AMPK in the early stage of low-glucose starvation, thereby enhancing GLS1 function and accelerating the degradation and utilization of glutamine.
Finally, the researchers evaluated the effects of the AMPK-PDZD8-GLS1 axis on cellular physiological functions.
Muscles are the part of the human body that requires the most energy. After knocking out PDZD8 in mouse muscle cells, they re-expressed exogenous PDZD8 wild-type or T527A mutant, and then starved for 8 hours of glucose. The results showed that compared with PDZD8 Knockout cells, re-expression of PDZD8 wild-type significantly promoted the decomposition and utilization of glutamine and increased ATP production, while the mutant had no effect.
Carbohydrates have a promoting effect on inflammation. Therefore, the researchers observed the effects of the AMPK-PDZD8-GLS1 axis on macrophage-involved inflammation in macrophages. They induced inflammation in mice by lipopolysaccharide induction. Compared with PDZD8 wild-type macrophages, T527A mutant macrophages significantly reduced the levels of proinflammatory cytokines in mouse serum, such as TNFα and IL-6, and the mortality rate of mice was significantly reduced, accompanied by improved lung injury.
Fig. 2 AMPK-PDZD8 plays a crucial role in the switch of carbon utilization from glucose to glutamine. (Li, et al., 2024)
These data indicate that under low glucose conditions, enhanced glutamine metabolism by the AMPK-PDZD8-GLS1 axis is important for maintaining muscle energy production. At the same time, this signaling axis also plays a pivotal role in macrophage-mediated proinflammatory responses.
In the early stages of glucose starvation, cells activate AMPK, which in turn phosphorylates the PDZD8 T527 site. Activated PDZD8 directly interacts with GLS1 through its C-terminal domain, thereby enhancing its enzymatic activity and affinity for glutamine, rapidly promoting the catabolism of glutamine in the TCA cycle, and compensating for the energy deficiency caused by glucose deficiency. This mechanism plays an important role in the maintenance of energy in muscle cells under low glucose conditions, as well as the LPS-induced inflammatory response involving macrophages.
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