Lukas N. Groschner

Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes

Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic β-Cells

Background: The molecular contributors of the mitochondrial Ca2+ uptake, which is essential for metabolism-secretion coupling in β-cells, are unknown. Results: Knockdown of MICU1 and MCU reduced agonist- and depolarization-induced mitochondrial Ca2+ sequestration, ATP production, and d-glucose-stimulated insulin secretion. Conclusion: MICU1 and MCU are integral to metabolism-secretion coupling in β-cells. Significance: The presented data identify MICU1 and MCU as important contributors to pancreatic β-cell function. In pancreatic β-cells, uptake of Ca2+ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca2+ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca2+ uptake 1 (MICU1), mitochondrial Ca2+ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca2+ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca2+ uptake upon both intracellular Ca2+ release and Ca2+ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca2+ uptake in response to either intracellular Ca2+ release or Ca2+ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca2+ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca2+ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca2+ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.

Inhibition of Autophagy Rescues Palmitic Acid-induced Necroptosis of Endothelial Cells

Background: Accumulation of palmitic acid in endothelial cells induces cellular dysfunction and death. Results: Palmitic acid triggers Ca2+-dependent autophagy, which results in programmed necrotic death (necroptosis) of endothelial cells. Conclusion: Autophagy promotes lipotoxic signaling of palmitic acid in endothelial cells leading to necroptosis. Significance: Showing a new molecular mechanism of palmitic acid-induced cytotoxicity may reveal novel strategies in the treatment of diseases related to lipid overload. Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca2+ triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Endothelial mitochondria—less respiration, more integration

Lining the inner surface of the circulatory system, the vascular endothelium accomplishes a vast variety of specialized functions. Even slight alterations of these functions are implicated in the development of certain cardiovascular diseases that represent major causes of morbidity and mortality in developed countries. Endothelial mitochondria are essential to the functional integrity of the endothelial cell as they integrate a wide range of cellular processes including Ca2+ handling, redox signaling and apoptosis, all of which are closely interrelated. Growing evidence supports the notion that impairment of mitochondrial signaling in the endothelium is an early event and a causative factor in the development of diseases such as atherosclerosis or diabetic complications. In this review, we want to outline the significance of mitochondria in both physiology and pathology of the vascular endothelium.

ATP Increases within the Lumen of the Endoplasmic Reticulum Upon Intracellular Ca2+-Release

Real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor within the lumen of the ER reveal a local Ca2+-controlled ATP signal that is restored during energy stress. The data highlight a novel Ca2+-controlled process that supplies the ER with additional energy upon cell stimulation.

Na+ current properties in islet α‐ and β‐cells reflect cell‐specific Scn3a and Scn9a expression

α‐ and β‐cells express both Nav1.3 and Nav1.7 Na+ channels but in different relative amounts. The differential expression explains the different properties of Na+ currents in α‐ and β‐cells. Nav1.3 is the functionally important Na+ channel α subunit in both α‐ and β‐cells. Islet Nav1.7 channels are locked in an inactive state due to an islet cell‐specific factor.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

ABSTRACT Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains.

NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes

Background: NAT8L (N-acetyltransferase 8-like) synthesizes N-acetylaspartate and is required for myelination in the brain. Its function in other tissues was undefined. Results: Nat8l is highly expressed in adipose tissues and impacts adipogenic marker gene expression, lipid turnover, and energy metabolism in brown adipocytes. Conclusion: Nat8l expression influences cellular bioenergetics in adipocytes. Significance: These findings establish a novel pathway in brown adipocyte metabolism. NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.

Molecularly distinct routes of mitochondrial Ca2+ uptake are activated depending on the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).

Background: Mitochondria may utilize different proteins to decode high and low cytosolic Ca2+. Results: Inhibition of SERCA shifts mitochondrial Ca2+ uptake from being UCP3-dependent to Letm1-dependent. Conclusion: Depending on the mode of intracellular Ca2+ release, two different mitochondrial Ca2+ uptake pathways are engaged. Significance: The dissection of two molecularly distinct mitochondrial Ca2+ uptake routes depending on SERCA activity points to the complexity of the mitochondrial Ca2+ uptake machinery. The transfer of Ca2+ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca2+ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca2+ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca2+ uptake pathway or establish distinct routes for mitochondrial Ca2+ sequestration. In this study, we show that a modulation of Ca2+ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca2+ signals and consequently switches mitochondrial Ca2+ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca2+ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca2+ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca2+ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca2+ rises.

The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by preventing STIM1-Orai1 interaction.

Summary The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca2+ entry (SOCE), a ubiquitous Ca2+ entry pathway regulating many cellular functions, is unknown. Here we show that NAGly, but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic &bgr;-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca2+ depletion of the endoplasmic reticulum, whereas it had no effect on Ca2+ entry through L-type voltage-gated Ca2+ channels. Enhanced Ca2+ entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca2+ depletion of the endoplasmic reticulum. In contrast, independently from its slow depolarizing effect on mitochondria, NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings revealed the STIM1–Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.