Charlotta S. Olofsson

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic beta-cells.

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β‐cells by a combination of electrophysiological recordings, intracellular Ca2+ ([Ca2+]i) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid‐free albumin) to intact islets exposed to 15 mm glucose increased the [Ca2+]i by ∼30% and insulin secretion 2‐fold. Palmitate remained capable of increasing [Ca2+]i and insulin release in the presence of tolbutamide and in islets depolarized by high K+ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP‐regulated K+ channels (KATP channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single β‐cells and increased the size of the readily releasable pool (RRP) of granules 2‐fold. Whole‐cell peak Ca2+ currents rose by ∼25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 μm isradipine indicating that the free fatty acid specifically acts on L‐type Ca2+ channels. The actions of palmitate on exocytosis and Ca2+ currents were not mimicked by intracellular application of palmitoyl‐CoA. We conclude that palmitate increases insulin secretion by a KATP channel‐independent mechanism exerted at the level of exocytosis and that involves both augmentation of L‐type Ca2+ currents and an increased size of the RRP.

Long-Term Exposure to Glucose and Lipids Inhibits Glucose-Induced Insulin Secretion Downstream of Granule Fusion With Plasma Membrane

Mouse β-cells cultured at 15 mmol/l glucose for 72 h had reduced ATP-sensitive K+ (KATP) channel activity (−30%), increased voltage-gated Ca2+ currents, higher intracellular free Ca2+ concentration ([Ca2+]i; +160%), more exocytosis (monitored by capacitance measurements, +100%), and greater insulin content (+230%) than those cultured at 4.5 mmol/l glucose. However, they released 20% less insulin when challenged with 20 mmol/l glucose. Glucose-induced (20 mmol/l) insulin secretion was reduced by 60–90% in islets cocultured at 4.5 or 15 mmol/l glucose and either oleate or palmitate (0.5 mmol/l). Free fatty acid (FFA)-induced inhibition of secretion was not associated with any major changes in [Ca2+]i or islet ATP content. Palmitate stimulated exocytosis by twofold or more but reduced K+-induced secretion by up to 60%. Basal (1 mmol/l glucose) KATP channel activity was 40% lower in islets cultured at 4.5 mmol/l glucose plus palmitate and 60% lower in islets cultured at 15 mmol/l glucose plus either of the FFAs. Insulin content decreased by 75% in islets exposed to FFAs in the presence of high (15 mmol/l), but not low (4.5 mmol/l), glucose concentrations, but the number of secretory granules was unchanged. FFA-induced inhibition of insulin secretion was not associated with increased transcript levels of the apoptosis markers Bax (BclII-associated X protein) and caspase-3. We conclude that glucose and FFAs reduce insulin secretion by interference with the exit of insulin via the fusion pore.

Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3.

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

PKA‐independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP

The molecular and cellular mechanisms involved in short‐term regulation of white adipocyte adipokine release remain elusive. Here we have examined effects of intracellular cAMP, Ca2+ and ATP on exocytosis and adipokine secretion by a combination of membrane capacitance patch‐clamp recordings and biochemical measurements of secreted adipokines. Our findings show that white adipocyte exocytosis is stimulated by cAMP/Epac (exchange proteins activated by cAMP)‐dependent but Ca2+‐ and PKA‐independent mechanisms and can largely be correlated to release of adiponectin vesicles residing in a readily releasable vesicle pool. A combination of Ca2+ and ATP augments exocytosis/adiponectin secretion via a direct action on the release process and by recruitment of new releasable vesicles. Our results elucidate several previously unknown cellular mechanisms involved in regulation of white adipocyte exocytosis/secretion. The well‐established disturbances of adipokine secretion in obese individuals highlight the significance of understanding how white adipocyte adipokine release is controlled.

White Adipocyte Adiponectin Exocytosis Is Stimulated via β3-Adrenergic Signaling and Activation of Epac1: Catecholamine Resistance in Obesity and Type 2 Diabetes.

We investigated the physiological regulation of adiponectin exocytosis in health and metabolic disease by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of short-term (30-min incubations) adiponectin secretion. Epinephrine or the β3-adrenergic receptor (AR) agonist CL 316,243 (CL) stimulated adiponectin exocytosis/secretion in cultured 3T3-L1 and in primary subcutaneous mouse adipocytes, and the stimulation was inhibited by the Epac (Exchange Protein directly Activated by cAMP) antagonist ESI-09. The β3AR was highly expressed in cultured and primary adipocytes, whereas other ARs were detected at lower levels. 3T3-L1 and primary adipocytes expressed Epac1, whereas Epac2 was undetectable. Adiponectin secretion could not be stimulated by epinephrine or CL in adipocytes isolated from obese/type 2 diabetic mice, whereas the basal (unstimulated) adiponectin release level was elevated twofold. Gene expression of β3AR and Epac1 was reduced in adipocytes from obese animals, and corresponded to a respective ∼35% and ∼30% reduction at the protein level. Small interfering RNA–mediated knockdown of β3AR (∼60%) and Epac1 (∼50%) was associated with abrogated catecholamine-stimulated adiponectin secretion. We propose that adiponectin exocytosis is stimulated via adrenergic signaling pathways mainly involving β3ARs. We further suggest that adrenergically stimulated adiponectin secretion is disturbed in obesity/type 2 diabetes as a result of the reduced expression of β3ARs and Epac1 in a state we define as “catecholamine resistance.”

Cooling reduces cAMP-stimulated exocytosis and adiponectin secretion at a Ca2+-dependent step in 3T3-L1 adipocytes.

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca2+ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca2+] to ~1.5 μM (9 mM Ca2+ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca2+-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.

Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics.

AIMS To establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. METHODS HL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA+reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca(2+)measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells. RESULTS A time-dependent drop in viability was shown after HEDA (P<0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca(2+) peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca(2+) oscillations with a decreased frequency. CONCLUSION A threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum.

Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1

In the present study, we have applied ratiometric measurements of intracellular Ca2+ concentrations ([Ca2+]i) to show that extracellularly applied ATP (adenosine triphosphate) (100 µM) stimulates store-operated Ca2+ entry (SOCE) in 3T3-L1 adipocytes. ATP produced a rapid increase in [Ca2+]i consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+. Gene expression data and [Ca2+]i recordings with uridine-5′-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+]i elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si)RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.

Inhibition of FOXO1 transcription factor in primary human adipocytes mimics the insulin resistant state of type 2 diabetes

Type 2 diabetes is characterized by insulin resistance in the expanding adipose tissue of obesity. The insulin resistance manifests in human adipocytes as system-wide impairment of insulin signalling. An exception is the regulation of transcription factor FOXO1 (forkhead box protein O1), which is phosphorylated downstream of mTORC2 (mammalian/mechanistic target of rapamycin in complex with raptor) and is therefore not exhibiting impaired response to insulin. However, the abundance, and activity, of FOXO1 is reduced by half in adipocytes from patients with diabetes. To elucidate the effect of reduced FOXO1 activity, we here transduced human adipocytes with a dominant-negative construct of FOXO1 (DN-FOXO1). Inhibition of FOXO1 reduced the abundance of insulin receptor, glucose transporter-4, ribosomal protein S6, mTOR and raptor. Functionally, inhibition of FOXO1 induced an insulin-resistant state network-wide, a state that qualitatively and quantitatively mimicked adipocytes from patients with type 2 diabetes. In contrast, and in accordance with these effects of DN-FOXO1, overexpression of wild-type FOXO1 appeared to augment insulin signalling. We combined experimental data with mathematical modelling to show that the impaired insulin signalling in FOXO1-inhibited cells to a large extent can be explained by reduced mTORC1 activity - a mechanism that defines much of the diabetic state in human adipocytes. Our findings demonstrate that FOXO1 is critical for maintaining normal insulin signalling of human adipocytes.

Escherichia coli outer membrane vesicles can contribute to sepsis induced cardiac dysfunction

Sepsis induced cardiac dysfunction (SIC) is a severe complication to sepsis which significantly worsens patient outcomes. It is known that bacteria have the capacity to release outer membrane vesicles (OMVs), which are nano-sized bilayered vesicles composed of lipids and proteins, that can induce a fatal inflammatory response. The aim of this study was to determine whether OMVs from a uropathogenic Escherichia coli strain can induce cardiac dysfunction, and to elucidate any mechanisms involved. OMVs induced irregular Ca2+ oscillations with a decreased frequency in cardiomyocytes through recordings of intracellular Ca2+ dynamics. Mice were intraperitoneally injected with bacteria-free OMVs, which resulted in increased concentration of pro-inflammatory cytokine levels in blood. Cytokines were increased in heart lysates, and OMVs could be detected in the heart after OMVs injection. Troponin T was significantly increased in blood, and echocardiography showed increased heart wall thickness as well as increased heart rate. This study shows that E. coli OMVs induce cardiac injury in vitro and in vivo, in the absence of bacteria, and may be a causative microbial signal in SIC. The role of OMVs in clinical disease warrant further studies, as bacterial OMVs in addition to live bacteria may be good therapeutic targets to control sepsis.