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.
Highlights
The diabetic etiology in Goto Kakizaki (GK) rats stems from multiple aspects of genetic contribution and gestational metabolic impairment inducing an epigenetic programming of the offspring pancreas transmitted over generations[1], resulting in the reduced β-cell neogenesis and proliferation
Since mitochondrial DNA (mtDNA) exists in the cell in numerous copies contained in the protein complexes, termed nucleoids[18,19,20,21,22,23,24,25,26,27], we aimed to study how the profound reduction in mtDNA is reflected on the level of nucleoids. mtDNA is compacted in nucleoids by the mitochondrial transcription factor A (TFAM) in extreme densities
We reported a 75% decrease of mtDNA in primary β-cell cells sorted from the Accutase-digested pancreatic islets (PIs) of diabetic Goto Kakizaki (GK) rats (48 week old), relatively to samples from age-matched non-diabetic Wistar rats[15]
Summary
The diabetic etiology in Goto Kakizaki (GK) rats stems from multiple aspects of genetic contribution and gestational metabolic impairment inducing an epigenetic programming of the offspring pancreas transmitted over generations[1], resulting in the reduced β-cell neogenesis and proliferation. Since mtDNA exists in the cell in numerous copies contained in the protein complexes, termed nucleoids[18,19,20,21,22,23,24,25,26,27], we aimed to study how the profound reduction in mtDNA is reflected on the level of nucleoids. TFAM-visualized size of nucleoids may change even with constant mtDNA content within nucleoids, or vice versa
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