Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation (Diabetes Metab J 2026;50: 307-19)

We thank the readers for their interest in our work and for their thoughtful and constructive comments regarding our recent article, ‘Lactate-induced lipid accumulation in hepatocytes through GPR81 activation’ [1].

Regarding the physiological relevance of lactate concentrations and exposure duration, while exercise can transiently elevate blood lactate levels to high-millimolar concentrations, these elevations are typically short-lived and rapidly normalize during recovery [2,3]. In contrast, in severe sepsis and septic shock, blood lactate concentrations frequently exceed 4 mmol/L and may reach double-digit millimolar levels in extreme cases, with both peak values and impaired lactate clearance strongly associated with multiorgan failure and mortality [4]. Our study was therefore designed to investigate metabolic alterations induced by chronic exposure to elevated lactate under severe pathological conditions, rather than physiological or adaptive lactatemia. Accordingly, the alpha mouse liver 12 (AML12) hepatocyte model was used as a mechanistic platform to test whether sustained lactate signaling per se (sodium L-lactate 20 or 40 mM for 4 days) is sufficient to promote hepatic lipid accumulation, rather than to recapitulate physiological lactate dynamics observed during exercise or postprandial states. Importantly, the lactate concentrations employed did not compromise cell viability and did not induce overt hepatocellular injury, which supports the interpretation of the observed triglyceride accumulation as a steatogenic metabolic response rather than nonspecific toxicity.

We agree that complementary experimental paradigms—such as lower, high-normal ‘lactate clamp’ conditions or intermittent lactate exposure synchronized with nutrient availability—would further refine physiological relevance and help distinguish amplification of physiological signaling from metabolic effects arising under prolonged supra-physiological exposure. In this regard, we initially attempted to establish chronic lactate elevation in vivo using osmotic pumps. Despite loading the pumps with concentrated lactate solutions, circulating blood lactate levels remained within the normal range throughout the experimental period. Post-explant inspection revealed that a substantial volume of lactate solution remained in the pumps, suggesting limited or ineffective lactate release. Following this unsuccessful attempt, we adopted a zebrafish exposure model, which permits stable and sustained lactate exposure. This approach resulted in hepatic lipid accumulation, providing in vivo support for our central hypothesis that prolonged lactate signaling can drive steatogenic responses.

With respect to AMP-activated protein kinase (AMPK) signaling, we agree that our data indicate a regulatory landscape more complex than a simple linear ‘lactate–G-protein-coupled receptor 81 (GPR81)–AMPK inhibition–steatosis’ axis. In AML12 cells, lactate reduced AMPK phosphorylation, while GPR81 knockdown restored p-AMPK levels and attenuated lipogenic gene expression; pharmacologic AMPK activation with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) similarly reversed lactate-induced lipid accumulation. In contrast, in vivo AMPK regulation differed between dietary models: although both high-fat diet (HFD) and high-fat high-cholesterol (HFHC) feeding increased hepatic lactate levels and GPR81 expression, AMPK phosphorylation decreased in HFD livers but increased in HFHC livers. These findings suggest that AMPK activity in vivo is strongly influenced by dietary composition, nutrient flux, and systemic metabolic stress, with lactate–GPR81 signaling representing only one of several upstream regulatory inputs.

Although the present study was not designed to resolve compartment-specific AMPK pools, our findings are consistent with the concept that lactate–GPR81 signaling may preferentially modulate AMPK functions linked to lipid uptake and lipogenesis, whereas other AMPK-activating pathways engaged under HFHC feeding—such as oxidative or stressadaptive signaling—may remain intact or even be enhanced. We agree that, as suggested by several readers, phosphoproteomic or proximity-labeling approaches centered on liver kinase B1 (LKB1), calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-, and transforming growth factor-β–activated kinase 1 (TAK1)-dependent AMPK signaling would represent a valuable strategy to evaluate whether AMPK pathways linked to lipid metabolism are preferentially affected, while other AMPK-mediated functions remain largely preserved [5,6].

We also agree that lactate biology extends beyond receptor-mediated signaling. In the present study, GPR81 knockdown normalized AMPK phosphorylation and reduced expression of lipogenic genes, whereas pharmacologic inhibition of monocarboxylate transporter 1 (MCT1) did not attenuate lipid accumulation, supporting a dominant role for GPR81-dependent signaling in this setting. Nevertheless, lactate can also function as an epigenetic modifier via histone lysine lactylation, thereby influencing transcriptional programs independently of cell-surface receptors [7]. Although epigenetic mechanisms were not directly examined here, prolonged lactate exposure may plausibly induce chromatin-level alterations at lipogenic loci such as sterol regulatory element-binding transcription factor 1 (SREBF1/SREBP-1), fatty acid synthase (FASN), and stearoyl-CoA desaturase 1 (SCD1). Future studies integrating chromatin immunoprecipitation for lactylated histone marks or epigenomic profiling under chronic low- versus high-millimolar lactate exposure will be required to determine whether receptor-dependent and receptor-independent layers of lactate signaling converge on shared lipogenic networks or act in parallel.

Finally, we appreciate the reviewers’ careful discussion of the translational implications of targeting GPR81. Although increased hepatic GPR81 expression tracks with lactate accumulation and steatosis in both HFD and HFHC models, GPR81 is also highly expressed in adipocytes—where it suppresses lipolysis— and is detectable in immune cells [8,9]. Thus, systemic GPR81 antagonism could potentially relieve hepatic lipid burden at the expense of altered adipose tissue lipid flux, increased circulating non-esterified fatty acids, or unintended effects on immune or tumor microenvironments. In our zebrafish model, 24-hour lactate exposure at 6 days post-fertilization selectively increased hepatic lipid accumulation, without significant lipid deposition in skeletal muscle or mesenteric adipose tissue. Although systemic effects cannot be excluded, these findings suggest a relative hepatic sensitivity to sustained lactate exposure in this experimental context. Accordingly, our findings should be interpreted as supporting a mechanistic role for hepatic lactate–GPR81 signaling in steatosis, rather than as immediate justification for therapeutic inhibition. Any translational strategy would require careful consideration of tissue specificity, metabolic context, and disease endotypes, potentially favoring liver-selective or ligand-biased modulation of GPR81.