A primary hepatopancreatic cell platform enables cellular dissection of glucose metabolism and endocrine-metabolic regulation in Litopenaeus vannamei.

The lack of robust continuous cell lines remains a major bottleneck limiting cell-level mechanistic dissection of endocrine and metabolic regulation in crustaceans. In this context, experimentally tractable primary cell systems provide a practical and physiologically relevant strategy for interrogating regulatory processes under controlled conditions. Here, we established and functionally validated a primary hepatopancreatic cell platform from the Pacific white shrimp Litopenaeus vannamei for investigating glucose metabolism and endocrine-metabolic regulation. Morphological characterization and systematic optimization of key culture parameters showed that the isolated cell population retained representative features of major hepatopancreatic cell types and could be maintained under short-term culture conditions suitable for functional analyses. Using this system, we showed that primary hepatopancreatic cells rapidly sensed exogenous glucose and initiated a coordinated metabolic response characterized by enhanced glucose transport and glycolysis-related transcription, together with suppression of gluconeogenesis-related genes. Exogenous insulin further amplified intracellular glucose accumulation and reinforced this glucose-utilizing transcriptional program, indicating that the platform preserves hormone-responsive metabolic properties in vitro. Conversely, starvation-mimicking conditions induced a distinct adaptive response, marked by reduced intracellular glucose availability, repression of glycolysis-related genes, and activation of gluconeogenesis-related transcription. Linsitinib treatment substantially blunted glucose responsiveness and shifted the transcriptional profile toward impaired glucose utilization. To extend the applicability of the platform beyond acute in vitro stimulation, in vivo endocrine perturbations were coupled with subsequent in vitro glucose-loading assays. Primary cells derived from insulin-degrading enzyme (IDE)-suppressed shrimp displayed enhanced glucose accumulation, whereas prolonged insulin exposure produced a delayed glucose-response profile, indicating an altered temporal response to glucose loading. In addition, optimized cytochemical staining enabled visualization of glycogen and lipid storage differences under distinct nutritional states, providing complementary phenotypic readouts of cellular energy status. Collectively, this study establishes a controllable and expandable primary hepatopancreatic cell platform for dissecting nutrient sensing, glucose metabolic homeostasis, and endocrine-metabolic regulation in L. vannamei. This system provides a valuable cellular tool for crustacean endocrinology and metabolism research and offers a useful framework for future functional studies in economically important crustacean species.