Mitochondrial Efflux of Citrate and Isocitrate Is Fully Dispensable for Glucose-Stimulated Insulin Secretion and Pancreatic Islet β-Cell Function

Abstract

The defining feature of pancreatic islet β-cell function is the precise coordination of changes in blood glucose levels with insulin secretion to regulate systemic glucose homeostasis. While ATP has long been heralded as a critical metabolic coupling factor to trigger insulin release, glucose-derived metabolites have been suggested to further amplify fuel-stimulated insulin secretion. The mitochondrial export of citrate and isocitrate through the citrate-isocitrate carrier (CIC) has been suggested to initiate a key pathway that amplifies glucose-stimulated insulin secretion, though the physiological significance of β-cell CIC-to-glucose homeostasis has not been established. Here, we generated constitutive and adult CIC β-cell knockout (KO) mice and demonstrate that these animals have normal glucose tolerance, similar responses to diet-induced obesity, and identical insulin secretion responses to various fuel secretagogues. Glucose-stimulated NADPH production was impaired in β-cell CIC KO islets, whereas glutathione reduction was retained. Furthermore, suppression of the downstream enzyme cytosolic isocitrate dehydrogenase (Idh1) inhibited insulin secretion in wild-type islets but failed to impact β-cell function in β-cell CIC KO islets. Our data demonstrate that the mitochondrial CIC is not required for glucose-stimulated insulin secretion and that additional complexities exist for the role of Idh1 and NADPH in the regulation of β-cell function.

Nutrient metabolism regulates insulin granule formation in the pancreatic islet β-cell via ER redox homeostasis

“…Based on this, we reasoned that NADPH production may be necessary to support ER functions (proinsulin disulfide bond formation and export) via supplying GSH reducing equivalents. To test this, we used shRNA-mediated knockdown of the major cytosolic NADPH-producing enzyme, Idh1, which has been previously shown to support insulin granule exocytosis 35,36,43 . Note that Idh1 loss does not impair mitochondrial function 36 .…”

“…Based on this, we reasoned that NADPH production may be necessary to support ER functions, such as proinsulin disulfide bond formation and export, via supplying cellular (GSH) reducing equivalents (Figure 7A) as previously suggested (Gansemer et al, 2020). To test this, islets were treated with recombinant adenovirus expressing either a non-targeting control shRNA (SAFE) or an shRNA targeting Idh1, the major cytosolic NADPH-producing enzyme in β-cells, which has been previously shown to support insulin granule exocytosis (Bauchle et al, 2021; Ferdaoussi et al, 2015; Ronnebaum et al, 2006). Note that Idh1 loss does not impair mitochondrial function (Ronnebaum et al, 2006).…”

“…Adenovirus containing rat insulin promoter (RIP) expressing Grx1-GFP2 was a kind gift from A. Linneman, Indiana University School of Medicine (Reissaus et al, 2019). Adenoviruses expressing shRNA targeting mouse Idh1 (AdU6-Idh1 shRNA-PGK-mCherry) and non-targeting control shRNA (AdU6-SAFE-shRNA-PGK-mCherry) have been described previously (Bauchle et al, 2021). Recombinant adenoviruses were generated in HEK293 cells and purified by cesium chloride gradient.…”

“…For glutathione (GSSG/GSH) redox measurements, 832/3 cells were treated with AdRIP-Grx1-GFP2 and imaged on an inverted Olympus IX83 microscope with a 20x objective (HC PL APO CS2; 0.75 NA) using Chroma 49002 GFP/CY2 Bandpass (470/535) and custom Chroma BX3-mounted (395/510) filters. Cells were subsequently cultured and re-imaged following DTT (10 mM) and diamide (5 mM) treatment, for 12 min each (Bauchle et al, 2021). Fluorescent intensities of each channel were calculated from masked images (to remove background) of whole cells using macros written for Fiji NIH software.…”

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 β-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions

“…At harvest, β-cell and islet treatment medium was replaced with secretion assay buffer containing 2.8 mM followed by 12 mM glucose, as described previously [ 55 , 96 ]. TMAO treatment concentrations were maintained in the buffer during static stimulation incubations.…”