Pulsatile Insulin Research Articles

Thermodynamic Optimality of Glycolytic Oscillations.

Temporal order in living matters reflects the self-organizing nature of dynamical processes driven out of thermodynamic equilibrium. Because of functional reasons, the period of a biochemical oscillation must be tuned to a specific value with precision; however, according to the thermodynamic uncertainty relation (TUR), the precision of the oscillatory period is constrained by the thermodynamic cost of generating it. After reviewing the basics of chemical oscillations using the Brusselator as a model system, we study the glycolytic oscillation generated by octameric phosphofructokinase (PFK), which is known to display a period of several minutes. By exploring the phase space of glycolytic oscillations, we find that the glycolytic oscillation under the cellular condition is realized in a cost-effective manner. Specifically, over the biologically relevant range of parameter values of glycolysis and octameric PFK, the entropy production from the glycolytic oscillation is minimal when the oscillation period is (5-10) min. Furthermore, the glycolytic oscillation is found at work near the phase boundary of limit cycles, suggesting that a moderate increase of glucose injection rate leads to the loss of oscillatory dynamics, which is reminiscent of the loss of pulsatile insulin release resulting from elevated blood glucose level.

Insulin Pulse Characteristics and Insulin Action in Non-diabetic Humans.

Pulsatile insulin secretion is impaired in diseases such as type 2 diabetes that are characterized by insulin resistance. This has led to the suggestion that changes in insulin pulsatility directly impair insulin signaling. We sought to examine the effects of pulse characteristics on insulin action in humans, hypothesizing that a decrease in pulse amplitude or frequency is associated with impaired hepatic insulin action. We studied 29 nondiabetic subjects on two occasions. On 1 occasion, hepatic and peripheral insulin action was measured using a euglycemic clamp. The deuterated water method was used to estimate the contribution of gluconeogenesis to endogenous glucose production. On a separate study day, we utilized nonparametric stochastic deconvolution of frequently sampled peripheral C-peptide concentrations during fasting to reconstruct portal insulin secretion. In addition to measuring basal and pulsatile insulin secretion, we used approximate entropy to measure orderliness and Fourier transform to measure the average, and the dispersion of, insulin pulse frequencies. In univariate analysis, basal insulin secretion (R2 = 0.16) and insulin pulse amplitude (R2 = 0.09) correlated weakly with insulin-induced suppression of gluconeogenesis. However, after adjustment for age, sex, and weight, these associations were no longer significant. The other pulse characteristics also did not correlate with the ability of insulin to suppress endogenous glucose production (and gluconeogenesis) or to stimulate glucose disappearance. Overall, our data demonstrate that insulin pulse characteristics, considered independently of other factors, do not correlate with measures of hepatic and peripheral insulin sensitivity in nondiabetic humans.

Pharmacokinetic\u2013pharmacodynamic modelling of the hypoglycaemic effect of pulsatile administration of human insulin in rats

The relationship between the plasma insulin (INS) concentration–time course and plasma glucose concentration–time course during and after pulsatile INS administration to rats was characterized using a pharmacokinetic–pharmacodynamic (PK–PD) model. A total INS dose of 0.5 IU/kg was intravenously injected in 2 to 20 pulses over a 2-h period. Compared with the single bolus administration, the area under the effect-time curve (AUE) increased depending on the number of pulses, and the AUEs for more than four pulses plateaued at a significantly larger value, which was similar to that after the infusion of a total of 0.5 IU/kg of INS over 2 h. No increase in plasma INS concentration occurred after pulsatile administration. Two indirect response models primarily reflecting the receptor-binding process (IR model) or glucose transporter 4 (GLUT4) translocation (GT model) were applied to describe the PK–PD relationship after single intravenous bolus administration of INS. These models could not explain the observed data after pulsatile administration. However, the IR-GT model, which was a combination of the IR and GT models, successfully explained the effects of pulsatile administration and intravenous infusion. These results indicate that the receptor-binding process and GLUT4 translocation are responsible for the change in AUE after pulsatile administration.

Reducing Glucokinase Activity to Enhance Insulin Secretion: A Counterintuitive Theory to Preserve Cellular Function and Glucose Homeostasis.

Pancreatic beta-cells are the only cells in the body that can synthesize and secrete insulin. Through the process of glucose-stimulated insulin secretion, beta-cells release insulin into circulation, stimulating GLUT4-dependent glucose uptake into peripheral tissue. Insulin is normally secreted in pulses that promote signaling at the liver. Long before type 2 diabetes is diagnosed, beta-cells become oversensitive to glucose, causing impaired pulsatility and overstimulation in fasting levels of glucose. The resulting hypersecretion of insulin can cause poor insulin signaling and clearance at the liver, leading to hyperinsulinemia and insulin resistance. Continued overactivity can eventually lead to beta-cell exhaustion and failure at which point type 2 diabetes begins. To prevent or reverse the negative effects of overstimulation, beta-cell activity can be reduced. Clinical studies have revealed the potential of beta-cell rest to reverse new cases of diabetes, but treatments lack durable benefits. In this perspective, we propose an intervention that reduces overactive glucokinase activity in the beta-cell. Glucokinase is known as the glucose sensor of the beta-cell due to its high control over insulin secretion. Therefore, glycolytic overactivity may be responsible for hyperinsulinemia early in the disease and can be reduced to restore normal stimulus-secretion coupling. We have previously reported that reducing glucokinase activity in prediabetic mouse islets can restore pulsatility and enhance insulin secretion. Building on this counterintuitive finding, we review the importance of pulsatile insulin secretion and highlight how normalizing glucose sensing in the beta cell during prediabetic hyperinsulinemia may restore pulsatility and improve glucose homeostasis.

1787-P: Insulin Pulse Characteristics Do Not Alter Hepatic Glucose Metabolism and Insulin Action

Insulin is secreted into the portal vein in a pulsatile manner. Portal vein insulin concentrations directly and rapidly alter endogenous glucose production (EGP). Pulse frequency has been inversely correlated with hepatic insulin action in some, but not all, human studies. Indeed, increased pulse disorderliness, decreased secretory burst mass and decreased pulse frequency have been observed in type 2 diabetes, in aging and in obesity. In dogs, disruption of pulsatile insulin secretion is associated with defects in hepatic insulin action. As part of a series of experiments examining the mechanisms underlying prediabetes, we studied 29 nondiabetic subjects (BMI 28 ± 1 Kg/m2) on 2 occasions. On one occasion, EGP was measured using [3-3H]glucose after an overnight fast (14.2 ± 0.5μmol/kg/min) and during a euglycemic clamp (6.8 ± 1.1μmol/kg/min). The deuterated water method was used to estimate the contribution of gluconeogenesis to EGP. On a separate study day we utilized nonparametric stochastic deconvolution of frequently sampled peripheral C-peptide concentrations during fasting to reconstruct portal insulin secretion. In addition to measuring basal and pulsatile insulin secretion, we used Approximate Entropy (ApEn) to measure orderliness and a Fourier transform to measure the average and the dispersion of insulin pulse frequency. The gradient of Loge EGP vs. Loge insulin in each individual (-1.2 ± 0.5) was used as a measure of hepatic insulin action. Basal secretion (126 ± 8 pmol/min, R2=0.04), pulse amplitude (41 ± 38 pmol/min, R2=0.02), ApEn (1.14 ± 0.03, R2=0.01), pulse interval (5.7 ± 0.4 min, R2<0.01) and frequency dispersion (0.47 ± 0.01, R2<0.01) did not correlate (all p > 0.10) with hepatic insulin action. Suppression of gluconeogenesis by insulin also did not correlate with fasting insulin pulse characteristics. Overall, our data demonstrate that insulin pulse characteristics do not directly alter the sensitivity of EGP and gluconeogenesis to insulin in humans. Disclosure M.C. Laurenti: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. R.T. Varghese: None. J. Jones: None. C. Barosa: None. R.A. Rizza: None. A. Matveyenko: None. G. De Nicolao: None. C. Cobelli: None. A. Vella: Advisory Panel; Self; vTv Therapeutics, Zealand Pharma A/S. Research Support; Self; Novo Nordisk A/S, Xeris Pharmaceuticals, Inc. Funding National Institutes of Health (DK78646, DK116231)

1902-P: Insulin Pulses Do Not Inhibit Glucagon Secretion in Nondiabetic Humans

Type 2 diabetes (T2DM) is characterized by defects in insulin secretion and glucagon suppression. It has been assumed that defective glucagon secretion arises due to impaired insulin secretion. Indeed, prior work suggests that pulsatile insulin secretion has an inverse relationship with post-prandial glucagon secretion. However, more recent data suggest that dysregulation of glucagon secretion in prediabetes occurs early, and independently, of changes in insulin secretion. During a series of experiments examining the mechanisms underlying prediabetes, we studied 29 nondiabetic subjects (age = 44 ± 2, BMI 28 ± 1 Kg/m2). Hepatic vein catheterization allowed sampling of insulin and glucagon at 2 minute intervals during fasting and during a hyperglycemic clamp (150mg/dL). Mean insulin concentrations rose from fasting (33 ± 1 vs. 144 ± 2 pmol/L, p<0.01) while glucagon was suppressed (96 ± 1 vs. 62 ± 1 ng/L, p<0.01) during the clamp. A cross-correlation analysis of the two hormones showed, on average, a small in-phase coordination between insulin and glucagon pulses during fasting, which inverted during hyperglycemia (0.2 ± 0.1 vs. -0.4 ± 0.1, p<0.01). Subsequently, Cross-Approximate Entropy (Cross-ApEn), a technique for comparing synchronicity between two time series, was used to evaluate their interaction. This showed a moderate level of synchronicity, unaffected by glucose concentrations, whether calculated using glucagon as the template to assess pattern reproducibility in insulin concentration (0.78 ± 0.03 vs. 0.76 ± 0.03, fasting vs. hyperglycemia), or when using insulin as the template for the glucagon pattern (0.78 ± 0.02 vs. 0.76 ± 0.03, fasting vs. hyperglycemia). The values of Cross-ApEn did not differ between the two scenarios, suggesting the interaction between the two hormones is not driven by either. Overall these data suggest that, on a minute-to-minute basis, direct control and secretion of glucagon is not mediated (or restrained) by insulin. Disclosure M.C. Laurenti: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. R.T. Varghese: None. R.A. Rizza: None. A. Matveyenko: None. C. Cobelli: None. A. Vella: Advisory Panel; Self; vTv Therapeutics, Zealand Pharma A/S. Research Support; Self; Novo Nordisk A/S, Xeris Pharmaceuticals, Inc. Funding National Institutes of Health (DK78646, DK116231)

Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye.

Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

Effectiveness and safety of pulsatile intravenous insulin therapy for the improvement of respiratory quotient in Chinese patients with diabetes mellitus.

Pulsatile intravenous insulin therapy (PIVIT) is a means of imitating naturally occurring insulin pulses artificially. It is thought to improve carbohydrate metabolism, which can be assessed using the respiratory quotient (RQ). The aim of this present study was to assess the efficacy and safety of PIVIT for the improvement of RQ in Chinese patients with diabetes mellitus (DM). This 12-week, multi-center, prospective, randomized, open-label, parallel-group study involved 110 DM patients (both type 1 and type 2) whose RQ was <0.8. Of these, 53 patients formed the control group, in which standard anti-diabetic therapy was maintained, and 54 patients formed the treatment group, which underwent weekly PIVIT in addition to the administration of standard anti-diabetic therapy. RQ was evaluated monthly in control subjects, and before and after every PIVIT treatment in the treatment group. After weekly PIVIT for 12 weeks, the mean RQ increased from 0.70 to 0.90 in the treatment group, but did not change in the control group. The percentage of subjects reporting adverse events (AEs) was 31.5% (17/54) in the treatment group and 9.43% (5/53) in the control group (P=0.0053). The most frequently reported AE (by 12 subjects) was a gastroenteric reaction when these individuals were receiving 50% glucose during the PIVIT treatment. The majority of AEs were mild and did not interfere with the ongoing treatment. Thus, PIVIT can be viewed as tolerated and effective for the improvement of RQ in Chinese DM patients. This study was retrospectively registered with the Chinese Clinical Trial Registry (http://www.chictr.org.cn) on November 13th 2019 (registration no. ChiCTR1900027510).

Spatially Compartmentalized Phase Regulation in the Ca2+-cAMP-PKA-Oscillatory Circuit

Signaling networks are spatiotemporally organized in order to sense diverse inputs, process information, and carry out specific cellular tasks. In pancreatic β cells, Ca2+, cyclic adenosine monophosphate (cAMP), and Protein Kinase A (PKA) exist in an oscillatory circuit characterized by a high degree of feedback, and this circuit is instrumental in mediating and potentiating pulsatile insulin secretion. Here, we describe a novel mode of regulation within this circuit involving a spatial dependence of the relative phase between cAMP, PKA, and Ca2+. We show that nanodomain clustering of Ca2+-sensitive adenylyl cyclases drives oscillations of local cAMP levels within the membrane nanodomain to be precisely in-phase with Ca2+ oscillations, whereas Ca2+-sensitive phosphodiesterases maintains out-of-phase oscillations within the general plasma membrane, providing a striking example and novel mechanism of cAMP compartmentation. Disruption of this precise in-phase relationship perturbs Ca2+ oscillations, suggesting that the relative phase within an oscillatory circuit can encode specific functional information. This example of a signaling nanodomain utilized for localized tuning of an oscillatory circuit has broad implications for the spatiotemporal coordination of many biological networks.

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

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.

Targeted Derivation of Organotypic Glucose- and GLP-1-Responsive β Cells Prior to Transplantation into Diabetic Recipients.

SummaryGeneration of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy.

Leader β-cells coordinate Ca2+ dynamics across pancreatic islets in vivo.

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader β-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory β-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.

Wirelessly Controlled Implantable System for On-demand and Pulsatile Insulin Administration

We propose a wirelessly controlled implantable system for on-demand and pulsatile insulin delivery with a more convenient and safer strategy than currently available strategies. The system is a combined entity of a magnetically driven pump (i.e., an MDP), external control device (i.e., an ECD) and mobile app. The MDP for implantation consists of a plunger, barrel and drug reservoir, where an accurate amount of insulin can be infused in a pulsatile manner only at the time when a magnetic force is applied to actuate the plunger in the barrel. The ECD at the outside body can modulate the MDP actuation with an electromagnet and its control circuit, and this modulation can be wirelessly controlled by the mobile app. As a safety feature, the mobile app is programmed to pre-set the restrictions for the insulin dose and administration schedule to avoid overdose. The system is shown to infuse insulin in a highly reproducible manner, but it does not allow for insulin infusion when the pre-set restrictions are violated. When tested with diabetic rats, the profiles of insulin plasma concentration and blood glucose level are similar to those of animals treated with a subcutaneous injection of the same dose of insulin.

Assessment of pulsatile insulin secretion derived from peripheral plasma C-peptide concentrations by nonparametric stochastic deconvolution.

The characteristics of pulsatile insulin secretion are important determinants of type 2 diabetes pathophysiology, but they are understudied due to the difficulties in measuring pulsatile insulin secretion noninvasively. Deconvolution of either peripheral C-peptide or insulin concentrations offers an appealing alternative to hepatic vein catheterization. However, to do so, there are a series of methodological challenges to overcome. C-peptide has a relatively long half-life and accumulates in the circulation. On the other hand, peripheral insulin concentrations reflect relatively fast clearance and hepatic extraction as it leaves the portal circulation to enter the systemic circulation. We propose a method based on nonparametric stochastic deconvolution of C-peptide concentrations, using individually determined C-peptide kinetics, to overcome these limitations. The use of C-peptide (instead of insulin) concentrations allows estimation of portal (and not post-hepatic) insulin pulses, whereas nonparametric stochastic deconvolution allows evaluation of pulsatile signals without any a priori assumptions of pulse shape and occurrence. The only assumption required is the degree of smoothness of the (unknown) secretion rate. We tested this method first on simulated data and then on 29 nondiabetic subjects studied during euglycemia and hyperglycemia and compared our estimates with the profiles obtained from hepatic vein insulin concentrations. This method produced satisfactory results both in the ability to fit the data and in providing reliable estimates of pulsatile secretion, in agreement with hepatic vein measurements. In conclusion, the proposed method enables reliable and noninvasive measurement of pulsatile insulin secretion. Future studies will be needed to validate this method in people with type 2 diabetes.

Synchronization of pancreatic islets by periodic or non-periodic muscarinic agonist pulse trains.

Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet β-cells through actions on M3 muscarinic receptors which are coupled to Gq type G-proteins. In addition, the neurons secrete several peptide hormones that act on β-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.

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 β-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.

Transitions between bursting modes in the integrated oscillator model for pancreatic β-cells

Insulin-secreting β-cells of pancreatic islets of Langerhans produce bursts of electrical impulses, resulting in intracellular Ca2+ oscillations and pulsatile insulin secretion. The mechanism for this bursting activity has been the focus of mathematical modeling for more than three decades, and as new data are acquired old models are modified and new models are developed. Comprehensive models must now account for the various modes of bursting observed in islet β-cells, which include fast bursting, slow bursting, and compound bursting. One such model is the Integrated Oscillator Model (IOM), in which β-cell electrical activity, intracellular Ca2+, and glucose metabolism interact via numerous feedforward and feedback pathways. These interactions can produce metabolic oscillations with a sawtooth time course or a pulsatile time course, reflecting very different oscillation mechanisms. In this report, we determine conditions favorable to one form of oscillations or the other, and examine the transitions between modes of bursting and the relationship of the transitions to the patterns of metabolic oscillations. Importantly, this work clarifies what can be expected in experimental measurements of β-cell oscillatory activity, and suggests pathways through which oscillations of one type can be converted to oscillations of another type.

Coordinated Cross‐talk between calcium and cAMP in regulating pulsatile insulin secretion: A novel role for the unique inhibitory G‐protein, Gαz, in regulating β‐cell function

Cyclic adenosine monophosphate (cAMP) is an important secondary messenger involved in a variety of signal transduction mechanisms. In the pancreatic β‐cell, cAMP amplifies pulsatile insulin secretion through protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC). Inhibition of adenylyl cyclase (AC) limits glucose stimulated insulin secretion, even in the absence of activated stimulatory proteins, suggesting an essential role of cAMP in β‐cell function. It has been proposed that cAMP may coordinate with calcium oscillations to regulate pulsatile insulin secretion; however, this has not been demonstrated in intact islets under physiological conditions. In this study we utilize a β‐cell‐specific, genetically encoded cAMP biosensor expressed in intact isolated mouse islets to demonstrate for the first time an anti‐phasic relationship between cAMP and Ca2+ oscillations. This suggests that intracellular cAMP levels are governed by the Ca2+ sensitive phosphodiesterase, which act to degrade intracellular cAMP during the active phase of insulin exocytosis. Furthermore, loss of the inhibitory G‐protein alpha‐subunit, Gαz, an important contributor to diabetes pathology, increased the amplitude of cAMP oscillations and sensitivity to glucagon like peptide‐1 (GLP‐1) receptor stimulation. Ligand activation of E‐prostanoid receptor 3 (EP3), a G‐protein coupled receptor (GPCR) up regulated in the diabetic state that couples to Gαz, reduces cAMP oscillations but also dramatically blunts Ca2+ oscillations. These results suggest a coordinated cross‐talk between Ca2+ and cAMP dynamics in the β‐cell, as well as a novel role for Gz signaling which acts on both Ca2+ dynamics and inhibition of the cAMP‐mediated amplifying pathway of insulin secretion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extraordinary Rapid Wound Healing Time in Diabetic patients treated with Microburst Insulin Infusion

Importance: Non-healing diabetic ulcerations of the foot lead to amputations and premature mortality. Microburst Insulin Infusion has demonstrated an ability to accelerate healing in otherwise non-healing wounds. Objective: To demonstrate the benefit of adding Microburst Insulin Infusion to conventional diabetic wound care to significantly decrease wound healing time. Methods: A case series of five patients received Microburst Insulin Infusion therapy for significant peripheral diabetic ulcerations after failing conventional wound care. These five cases were treated from 4 independent Microburst Insulin Infusion clinics. Four of the wounds exceeded 6 cm². Patient charts were reviewed and relevant data was collected. All individuals in this manuscript have given written informed consent to publish these case details. Setting: Multicenter referral clinics providing Microburst Insulin Infusion Therapy Exposure: Microburst Insulin Infusion is an adjunctive treatment to conventional diabetic therapy performed in an outpatient clinic setting. A weight based oral glucose dose is administered while pulsatile intravenous insulin is delivered by the Bionica Microdose pump. The treatment is comprised of three 1 hour sessions and occurs up to five times a week, when the patient presents with un-healing wounds. Main Outcomes and Measures: Time to wound healing. A secondary outcome was failure of wound healing. Hypothesis for this study was formulated post treatment. Results: A significant decrease in wound healing time was observed when utilizing Microburst Insulin Infusion. The mean time to complete healing was 84.2 days compared to 133 days in published literature(1). The patient with a foot wound measuring an amazing 228.6 cm² healed in 102 days and amputation was not necessary. There were no treatment failures in this small series. As a result of decreased healing time, there was an absence of infection and no amputations were required for these wounds. Conclusion and Relevance: The addition of Microburst Insulin Infusion therapy resulted in a shortening of the time to healing by 49 days (7 weeks) for patients with poor response to conventional wound care. This is a 37% reduction in the time to heal thus reducing risk of infection and additional healthcare costs. The mechanism of action of Microburst Insulin Infusion in healing diabetic wounds is still unclear, but may involve an improved cellular metabolism, decreased inflammation and increased levels of Nitric oxide that enhance peripheral vascular supply and tissue healing.