Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Abstract

Aims/HypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/InterpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Precise coupling efficiencies are determined at constant ⌬p, requiring at least qualitative estimates of this parameter. There is general agreement that ␤-cell mitochondria possess a high endogenous proton leak (5,17,163,542), consistent with the "leaky bucket" analogy discussed above.…”

“…In contrast, Ishihara et al (235) C]glucose was unaffected by LDH overexpression, although the proportion metabolized to lactate was not determined. Finally, it should be noted that HIT-T15, RINm5F, and EndoC-␤H1 cells express relatively high levels of LDH (17,477). Thus RINm5F cells show a greatly enhanced lactate production on addition of a mitochondrial inhibitor, while INS-1 cells respond with a further reduction of an already low level (477).…”

“…In 2000, Hohmeier et al (226) stably transfected INS-1 cells with a plasmid containing the human proinsulin gene, and derived the strongly responsive INS-1 832/13, together with poorly responsive clones such as 832/2 (24). A human-derived ␤-cell line, EndoC-␤H1, has been developed (433), using targeted lentiviral oncogenesis in human fetal tissue, and characterized (17,197,433). These cells possess the advantage that their proteins are encoded by human genes, overcoming the criticism that the ion channels involved in generating plasma membrane potential responses differ between rodent and human cells.…”

“…Basic "housekeeping" ATP turnover also responds to "⌬p," which stabilizes at a suboptimal level at which the restricted input corresponding to low glucose balances the sum of the two outputs (FIGURE 3Bi). Cell respirometry indicates that high glucose roughly doubles respiration (FIGURE 3C) (17,147,184,331). To model this, we double the rate of water inflow (FIGURE 3Bii), and as a result, the water level rises until the increased hydrostatic head has increased the two outflows sufficiently to balance in the input.…”

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

“…INS-1E cells were preincubated for 1 h in assay medium containing 1.7 mM glucose at 37°C in air after which respiration was measured following sequential injections of 17 mM glucose, 5 M oligomycin, 4 M FCCP, and 1 M rotenone as described (77).…”