Intact pancreatic islets and dispersed beta-cells both generate intracellular calcium oscillations but differ in their responsiveness to glucose.

Pancreatic islets produce pulses of insulin and other hormones that maintain normal glucose homeostasis. These micro-organs possess exquisite glucose-sensing capabilities, allowing for precise changes in pulsatile insulin secretion in response to small changes in glucose. When communication among these cells is disrupted, precision glucose sensing falters. We measured intracellular calcium patterns in 6-mM-steps between 0 and 16 mM glucose, and also more finely in 2-mM-steps from 8 to 12 mM glucose, to compare glucose sensing systematically among intact islets and dispersed islet cells derived from the same mouse pancreas in vitro. The calcium activity of intact islets was uniformly low (quiescent) below 4 mM glucose and active above 8 mM glucose, whereas dispersed beta-cells displayed a broader activation range (2-to-10 mM). Intact islets exhibited calcium oscillations with 2-to-5-min periods, yet beta-cells exhibited longer 7-10 min periods. In every case, intact islets showed changes in activity with each 6-mM-glucose step, whereas dispersed islet cells displayed a continuum of calcium responses ranging from islet-like patterns to stable oscillations unaffected by changes in glucose concentration. These differences were also observed for 2-mM-glucose steps. Despite the diversity of dispersed beta-cell responses to glucose, the sum of all activity produced a glucose dose-response curve that was surprisingly similar to the curve for intact islets, arguing against the importance of "hub cells" for function. Beta-cells thus retain many of the features of islets, but some are more islet-like than others. Determining the molecular underpinnings of these variations could be valuable for future studies of stem-cell-derived beta-cell therapies.

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and ß-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies.

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.