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. 2021 Sep 24;7(39):eabi7514.
doi: 10.1126/sciadv.abi7514. Epub 2021 Sep 24.

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance

Giulia Rossetti  1   2   3 , Judith A Ermer  1   2   3 , Maike Stentenbach  1   2   3 , Stefan J Siira  1   2   3 , Tara R Richman  1   2   3 , Dusanka Milenkovic  4 , Kara L Perks  1   2   3 , Laetitia A Hughes  1   2   3 , Emma Jamieson  5 , Gulibaikelamu Xiafukaiti  6 , Natalie C Ward  7 , Satoru Takahashi  6 , Nicola Gray  8 , Helena M Viola  9 , Livia C Hool  9   10 , Oliver Rackham  1   2   11   12   13 , Aleksandra Filipovska  1   2   3   13   14
Affiliations

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance

Giulia Rossetti et al. Sci Adv. .

Abstract

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet β cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population.

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Figures

Fig. 1.
Fig. 1.. The N434S mice develop insulin resistance when fed on a HFD.
(A) Frequencies of the MRPP3 N437S single-nucleotide polymorphism (chr14:35266761 A>G, GRCh38.p12)(rs11156878) across different human populations. (B) Conservation of the MRPP3 protein sequence surrounding the N437S variant amino acid (red) is highlighted with residues identical to those in human sequence boxed in blue. (C) The location of N437 (red) is shown within the modeled structure of the human MRPP3 enzyme in complex with a tRNA substrate. The catalytic domain of MRPP3 is shown in blue, its four active site aspartates in orange, the PPR domain in green, and the bound tRNA in purple. (D) Close-up views of the N437 residue or N437S variant of MRPP3. The surface charge distribution is colored according to the local electrostatic potential (blue, +5 kT; red, −5 kT). (E) The N434S mutation in the Mrpp3 gene was confirmed by Sanger sequencing of PCR amplicons from homozygous N434S and wt littermates. (F) Body weight of NCD and HFD-fed wt and N434S mice measured at 20 weeks of age. (G) Glucose tolerance tests (GTT) and (H) insulin tolerance tests (ITT) were carried out at 12 and 13 weeks of NCD or HFD, respectively. Quantitative values are area under the curve (AUC) for GTT or area above the curve (AAC) for ITT ± SEM *P < 0.05 and **P < 0.01, Student’s t test.
Fig. 2.
Fig. 2.. The N434S variant leads to reduced circulating insulin and hepatosteatosis in HFD-fed mice.
(A) H&E staining of pancreata from 20-week-old mice fed NCD show enzymatic necrosis of the acinar cells (yellow circles) in the N434S mice. Sections were visualized at ×10 (left), ×20 (right), or ×40 magnification (bottom). Scale bars, 100 and 25 μm. (B) Circulating insulin levels were measured in serum obtained from fasted 20-week-old N434S and wt (n = 4) mice fed either a NCD or a HFD. (C) H&E staining of liver sections from 20-week-old wt and N434S mice fed a NCD and a HFD (left) and Oil Red O staining (right). Magnification is ×10 or ×20. Scale bars, 100 μm. (D) Sera of 20-week-old wt and N434S mice fed a NCD or a HFD (n = 4) were analyzed for circulating albumin, ALT, TAGs, LDL, HDL, and cholesterol levels. (E) Sera of 20-week-old wt and N434S mice fed a NCD or a HFD (n = 9) were analyzed for short-chain fatty acids (SCFA) levels: acetate, butyrate, and propionate. Values are means ± SEM *P < 0.05 and ***P < 0.001, Student’s t test.
Fig. 3.
Fig. 3.. Changes in Akt and mTOR signaling but not the expression of glucose transporters in Mrpp3-N434S mice.
Liver and pancreas whole tissue lysates (30 μg) from 20-week-old N434S mice and wt mice fed a NCD or a HFD were immunoblotted against antibodies to investigate the endogenous levels of Akt and its phosphorylated form (Ser473) under basal conditions (A) and following insulin exposure (terminal insulin) (B), where the mice were injected with insulin (1 U/kg) and euthanized after 5 min before the tissue was collected. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Immunoblotting against the glucose transporter proteins (GLUT2) in livers and pancreata lysates (30 μg) from 20-week-old N434S mice and wt mice fed a NCD or a HFD. GAPDH was used as a loading control. (D) Immunoblotting against the S6 and phospho-S6 in pancreata lysates (30 μg) from 20-week-old N434S mice and wt mice fed a NCD or a HFD. GAPDH was used as a loading control. Relative abundance of proteins was measured using Image Studio Lite and normalized to GAPDH. The data are representative of results obtained from at least six mice from each genotype and diet. Error bars *P < 0.05, Student’s t test.
Fig. 4.
Fig. 4.. Mitochondrial RNA processing and posttranslational tRNA modification are not functionally affected by the N434S variant.
(A) A complete map of mtRNA abundance and mean log2 fold change (FC) [(KOmean/Ctrlmean)] determined by RNA sequencing coverage in pancreatic islets from 20-week-old N434S and wt mice fed a NCD or HFD (n = 3). Increases are shown in red and decreases in blue. The log2 fold changes range from −2.5 to 1. (B) qRT-PCR was used to measure mitochondrial RNA junctions in pancreata from 20-week-old N434S and wt mice either fed a NCD or a HFD (n = 3). Data were normalized to 18S rRNA. Error bars are means ± SEM. (C) Relative rate of nonreference bases, indicative of modification, at the ninth mt-tRNA position relative to a nonreference tRNA position and (D) relative tRNA abundance, determined by small RNA sequencing in pancreatic islets from 20-week-old N434S and wt mice fed a HFD (n = 3). Data were normalized to total library size. (E) Pancreatic mitochondrial proteins (30 μg) were isolated from 20-week-old N434S mice and wt mice fed a NCD or a HFD and immunoblotted against OXPHOS proteins and normalized to SDHA.
Fig. 5.
Fig. 5.. The MRPP3 N434S variant reduces its association with LETM1 via MRPP2.
(A) BioID of BirA*-tagged wt or N434S MRPP3 expressed in HeLa cells and the associated proteins were detected by mass spectrometry. Mitochondrial-targeted BirA* (mt-BirA*) was used as a control. (B) Average Venus intensities normalized to red fluorescent protein (RFP) intensity for interactions between LETM1 and the wt or N434S MRPP3, as well as MRPP1 and MRPP2 in NIH-3T3 cells determined by fluorescence-activated cell sorting. GST1 was used as a negative control. Data are shown as a percent change compared to the respective control. (C) Average Venus intensities normalized to RFP intensity comparing interactions between the wt and N434S MRPP3 with MRPP1 or MRPP2. GST1 was used as a negative control. (D) Immunoprecipitation of HA-tagged MRPP3 and MRPP3-N434S expressed in NIH-3T3 cells and immunoblots of the fractions from the immunoprecipitation against LETM1 and MRPP2. HA-tagged SOD2 was used as a control bait. (E) The levels of MRPP2 and LETM1 were measured by immunoblotting of pancreatic mitochondria and normalized to porin. Amino acid content was measured in pancreata (F) or serum (G) by mass spectrometry. All data are representative of results obtained from at least three experiments or three mice from 20-week-old N434S mice and wt mice fed an NCD or an HFD. Error bars are SEM *P < 0.05 and **P < 0.01, Student’s t test.
Fig. 6.
Fig. 6.. The MRPP3 N434S variant results in reduced mitochondrial calcium and insulin secretion.
Cellular (A) and mitochondrial (B) calcium levels were analyzed in isolated pancreatic β cells using Fura-2 or Rhod-2 fluorescent microscopy, respectively, under basal conditions and after exposure to glucose. (C) Potassium levels were measured in serum and (D) insulin levels were measured in pancreata by enzyme-linked immunosorbent assay (ELISA). (E) Insulin and glucagon levels were measured in pancreata by fluorescent immunohistochemistry. Hoechst staining was used to visualize the nuclei. (F) Glucose-stimulated insulin secretion was measured in pancreatic islet cells in the presence of low and high glucose, and total insulin levels were measured in the cell pellets as controls using an ELISA. All data are representative of results obtained from at least three mice from 20-week-old N434S mice and wt mice fed a NCD or a HFD. Error bars are SEM *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test. (G) Reduced RNase P association with LETM1 may affect calcium transport into mitochondria and the cellular calcium availability required for insulin secretion. The increased calorie intake from the HFD causes an increased requirement for insulin secretion, and the calcium levels are not sufficient to trigger increased insulin secretion that may eventually lead to insulin resistance.
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