Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet ß-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.

High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria.

Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.

NCLX prevents cell death during adrenergic activation of the brown adipose tissue.

A sharp increase in mitochondrial Ca2+ marks the activation of brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca2+ deleterious effects are poorly understood. Here, we show that adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca2+ extrusion via the mitochondrial Na+/Ca2+ exchanger, NCLX. Adrenergic stimulation of NCLX-null brown adipocytes (BA) induces a profound mitochondrial Ca2+ overload and impaired uncoupled respiration. Core body temperature, PET imaging of glucose uptake and VO2 measurements confirm a thermogenic defect in NCLX-null mice. We show that Ca2+ overload induced by adrenergic stimulation of NCLX-null BAT, triggers the mitochondrial permeability transition pore (mPTP) opening, leading to a remarkable mitochondrial swelling and cell death. Treatment with mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT, while calcium overload persists. Our findings identify a key pathway through which BA evade apoptosis during adrenergic stimulation of uncoupling. NCLX deletion transforms the adrenergic pathway responsible for thermogenesis activation into a death pathway.

Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin-related protein 1 (DRP1)] markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)-2 strongly reduced the mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1-DN-promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts cellular Ca2+ homeostasis.-Kowaltowski, A. J., Menezes-Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral-Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni-Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal ß Cell Proliferation.

Pancreatic ß cell mass for appropriate blood glucose control is established during early postnatal life. ß cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of ß cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of ß cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative ß cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature ß cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal ß cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal ß cells and paves the way for the identification of novel therapeutic targets to stimulate ß cell regeneration.

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional ß cells.

Insulin secretion from ß cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing ß cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues ß cell function in T2D.

cis-Diamminedichloridoplatinum(II) N,N-dimethyl-formamide monosolvate.

In the title compound, cis-[PtCl2(NH3)2]·C3H7NO, the metal complex mol-ecules are stacked parallel to the b axis, forming close Pt⋯Pt inter-actions of 3.4071 (7) and 3.5534 (8) Šand weak N-H⋯Cl hydrogen bonds between the ammine ligand and the Cl atoms of the neighboring complex. Conventional N-H⋯O hydrogen bonds are formed between ammine ligands and the O atom of adjacent N,N-dimethyl-formamide mol-ecules. The crystal was found to be a split crystal and was analyzed using two domains related by a rotation of ca 4.4° about the reciprocal axis (-0.351 1.000 0.742) and refined to give a minor component fraction of 0.084 (6).