Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet β-cells of diabetic Goto Kakizaki rats

Abstract

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured “nucleoids” which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The β-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic β-cells.

Organization of DNA in Mammalian Mitochondria

“…The recent development of super-resolution techniques has enabled a better characterization of nucleoid size, shape, and organization, and also a more precise counting of mtDNA molecules present within a nucleoid. Several high-resolution microscopy techniques such as FPALM [fluorescence photoactivation localization microscopy], dSTORM [direct stochastic optical reconstruction microscopy], SIM [structured illumination microscopy], and STED [stimulated emission depletion] microscopy, and also cryo-electron tomography and rotary-shadowing electron microscopy have been employed [40,41,42,43,44,45]. These techniques have allowed the demonstration that nucleoids are rather small structures, with a mean size of ~110 nm [40,41,42].…”

“…Moreover, a consensus has emerged regarding the shape of the mammalian mitochondrial nucleoid, which appears to be slightly ellipsoid, with an average shape varying between the studies from slightly elongated (80 × 80 × 100 nm) [43] to truly ellipsoid (25 × 45 × 100 nm) [42,44]. Lastly, concerning the number of mtDNA molecules per nucleoid, nucleoids reconstituted in vitro and also nucleoids from mouse embryonic fibroblasts typically contain a single copy of mtDNA [43], even though there have been some findings supporting the idea of multiple mtDNA copies per nucleoid [45].…”

Contribution of Oxidative Stress and Impaired Biogenesis of Pancreatic β-Cells to Type 2 Diabetes

“…Oxidative stress leads undoubtedly to impaired maintenance and biogenesis of pancreatic β-cells (see the Impaired Biogenesis of Pancreatic β-Cells section), including the putative “insulin resistance of β-cells,” that is, the dysregulation of autocrine insulin pathway, if it exists. This leads to decreased mtDNA copy number (262) and further GSIS deterioration by such a vicious cycle. Factors of mtDNA replication and transcription are essentially involved and their dysregulation or impairment leads to β-cell dysfunction including GSIS failure (214).…”

Nicotinamide promotes differentiation of pancreatic endocrine progenitors from human pluripotent stem cells through poly (ADP-ribose) polymerase inhibition

“…Consistent with this observation, we showed that cells increase their oxygen consumption rate as they transition from Nkx6.1 negative pancreatic progenitors on day 2 of nicotinamide treatment to Nkx6.1 positive endocrine progenitors on day 5 of nicotinamide treatment. In a similar but converse finding, Nkx6.1 expression decline was correlated with mitochondrial DNA reduction in diabetic Goto Kakizaki rats [29].…”