Mapping Metabolic Gradients in Liver and Gut

Among the strongest spatial enzyme gradients in the mouse liver is periportal localization of the glutamine catabolic enzyme glutaminase (Extended Data Fig. 8b,c). Consistent with such localization, we observed increased labelling of both glutamate and malate from glutamine periportally (Fig. 3a,b and Extended Data Fig. 8c,d). Glutamine resynthesis from glutamate was pericentral, consistent with the enzyme glutamine synthetase (Fig. 3a,b and Extended Data Fig. 8c). TCA labelling from lactate was more modestly periportal (Fig. 3a,b). Lactate enters the TCA cycle via pyruvate through two different routes. Oxidative catabolism to acetyl-CoA through pyruvate dehydrogenase generates M+2 TCA intermediates and supports TCA turning and energy generation (Fig. 3a). By contrast, pyruvate carboxylation to oxaloacetate generates M+3 TCA intermediates (Fig. 3a,b). These can either feed gluconeogenesis or be converted into M+2 TCA intermediates by TCA turning (Fig. 3a,b). Thus, the malate M+3/M+2 ratio reflects pyruvate carboxylase flux relative to TCA turning. Consistent with gluconeogenesis being periportal, the malate M+3/M+2 ratio was greater periportally (Extended Data Fig. 8f–h). Tracing with [1-¹³C]lactate, which selectively labels malate made through pyruvate carboxylase, further supported periportal localization of pyruvate carboxylase activity (Extended Data Fig. 8i). Overall, these tracing data confirmed expected periportal localization of both gluconeogenesis and glutamine catabolism, as well as the expected pericentral localization of glutamine resynthesis from glutamate.

In the intestine, we were particularly interested in whether isotope tracing could shed light on the differential localization of malate (tips) and citrate (crypts). Citrate synthesis from malate involves two enzymatic reactions: malate oxidation to oxaloacetate followed by addition of a two-carbon unit from acetyl-CoA, typically coming from glucose, lactate or fat oxidation (Fig. 3c). As both of these steps from malate to citrate depend on oxidative capacity, they logically might be hindered in the less-well-perfused villus tips. If this were the case, we would expect the TCA cycle to be fed through oxidative lactate metabolism (M+2 TCA intermediates from lactate) preferentially in the crypts and for the major alternative fuel, glutamine, to predominate at the tips (Fig. 3c). Indeed, we observed preferential lactate contribution to intestinal glutamate in the crypts and glutamine contribution in the villus tips (Fig. 3d). The dual sources of glutamate further align with its dual crypt-tip localization (Extended Data Fig. 6d). Thus, TCA fuel use varies spatially in both the liver and intestine and helps to explain observed metabolite concentration gradients.