High throughput generation of a resource of the human secretome in mammalian cells.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic ß-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity.

AIMS/HYPOTHESIS: During the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-ßH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment. METHODS: EndoC-ßH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity. RESULTS: EndoC-ßH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-ßH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: The present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity. DATA AVAILABILITY: Microarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code.

SGLT2 inhibition with empagliflozin improves coronary microvascular function and cardiac contractility in prediabetic ob/ob-/- mice.

BACKGROUND: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) is the first class of anti-diabetes treatment that reduces mortality and risk for hospitalization due to heart failure. In clinical studies it has been shown that SGLT2i's promote a general shift to fasting state metabolism characterized by reduced body weight and blood glucose, increase in glucagon/insulin ratio and modest increase in blood ketone levels. Therefore, we investigated the connection between metabolic changes and cardiovascular function in the ob/ob-/- mice; a rodent model of early diabetes with specific focus on coronary microvascular function. Due to leptin deficiency these mice develop metabolic syndrome/diabetes and hepatic steatosis. They also develop cardiac contractile and microvascular dysfunction and are thus a promising model for translational studies of cardiometabolic diseases. We investigated whether this mouse model responded in a human-like manner to empagliflozin treatment in terms of metabolic parameters and tested the hypothesis that it could exert direct effects on coronary microvascular function and contractile performance. METHODS: Lean, ob/ob-/- untreated and ob/ob-/- treated with SGLT2i were followed for 10 weeks. Coronary flow velocity reserve (CFVR) and fractional area change (FAC) were monitored with non-invasive Doppler ultrasound imaging. Food intake, urinary glucose excursion and glucose control via HbA1c measurements were followed throughout the study. Liver steatosis was assessed by histology and metabolic parameters determined at the end of the study. RESULTS: Sodium-glucose cotransporter 2 inhibitors treatment of ob/ob-/- animals resulted in a switch to a more catabolic state as observed in clinical studies: blood cholesterol and HbA1c were decreased whereas glucagon/insulin ratio and ketone levels were increased. SGLT2i treatment reduced liver triglyceride, steatosis and alanine aminotransferase, an indicator for liver dysfunction. L-Arginine/ADMA ratio, a marker for endothelial function was increased. SGLT2i treatment improved both cardiac contractile function and coronary microvascular function as indicated by improvement of FAC and CFVR, respectively. CONCLUSIONS: Sodium-glucose cotransporter 2 inhibitors treatment of ob/ob-/- mice mimics major clinical findings regarding metabolism and cardiovascular improvements and is thus a useful translational model. We demonstrate that SGLT2 inhibition improves coronary microvascular function and contractile performance, two measures with strong predictive values in humans for CV outcome, alongside with the known metabolic changes in a preclinical model for prediabetes and heart failure.

Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis.

One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin:insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1α and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre-treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1α and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses.

Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells.

Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na(+)/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans.

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell (due to the firing of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell KATP channel conductance (GK,ATP) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC50 of approximately 4mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca2+]i waves to spread with a speed of approximately 80 microms-1, similar to that observed experimentally in confocal [Ca2+]i imaging.

Modelling the electrical activity of pancreatic alpha-cells based on experimental data from intact mouse islets.

Detailed experimental data from patch clamp experiments on pancreatic alpha-cells in intact mouse islets are used to model the electrical activity associated with glucagon secretion. Our model incorporates L- and T-type Ca(2+) currents, delayed rectifying and A-type K(+) currents, a voltage-gated Na(+) current, a KATP conductance, and an unspecific leak current. Tolbutamide closes KATP channels in the alpha-cell, leading to a reduction of the resting conductance from 1.1 nS to 0.4 nS. This causes the alpha-cell to depolarise from -76 mV to 33 mV. When the basal membrane potential passes the range between -60 and -35 mV, the alpha-cell generates action potentials. At higher voltages, the alpha-cell enters a stable depolarised state and the electrical activity ceases. The effects of tolbutamide are simulated by gradually reducing the KATP conductance (g(K,ATP)) from 500 pS to 0 pS. When g(K,ATP ) is between 72 nS and 303 nS, the model generates action potentials in the same voltage range as the alpha-cell. When g(K,ATP) is lower than 72 nS, the model enters a stable depolarised state, and firing of action potentials is inhibited due to voltage-dependent inactivation of the Na(+) and T-type Ca(2+) currents. This is in accordance with experimental results. Changing the inactivation parameters to those observed in somatostatin-secreting delta-cells abolishes the depolarised inactive state, and leads to beta-cell like electrical activity with action potentials generated even after complete closure of the KATP channels.

Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium.

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.

Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet.

We review a new method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. By using low-resistance patch electrodes, rapid current responses can be monitored under voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting beta-cells. First, the role of a K(+)-current dependent on Ca(2+)-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second, the conductance of gap junctions (approximately 1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may have implications for neuroscience research such as the mechanism of oscillatory electrical activity in general and processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration.