According to the authors, "transgenerational transmission of metabolic diseases" may be associated with Mfn2 deficiency in the mother's oocytes (immature eggs). Mfn2 refers to mitofusin-2, a protein involved in the regulation of vascular smooth muscle cell proliferation. It is normally found in the outer membrane of mitochondria, the organelles that supply cells with energy. A deficiency leads to mitochondrial swelling and dysfunction, as well as altering the expression of almost 1,000 genes in female gametes.
"A number of studies have found mitofusin-2 to be an important metabolic regulator. There's evidence that weight gain leads to a reduction in levels of the protein in muscle and liver cells, both of which play a key role in regulating blood sugar levels. In the case of diabetics, its expression is reduced in these cells," Marcos Chiaratti, a professor at the Federal University of São Carlos (UFSCar) and principal investigator for the study, which was supported by São Paulo Research Foundation - FAPESP, told.
In the recent publication, Chiaratti and his group report the results of experiments with mice genetically modified so as not to express Mfn2 only in oocytes. The Mfn2 deficiency was expected to affect their fertility, but this was not the case. However, their offspring gained more weight than the offspring of control animals and had become diabetic by the age of 9 months, despite being fed a standard diet.
To investigate the molecular mechanisms associated with this abnormal phenomenon, Chiaratti established a collaboration with researchers at the Center for Research on Redox Processes in Biomedicine (Redoxome) and the Obesity and Comorbidities Research Center (OCRC), both of which are Research, Innovation and Dissemination Centers (RIDCs) funded by FAPESP. Part of the study was conducted during the master's research of Bruna Garcia at UFSCar's Center for Biological and Health Sciences (CCBS), with Chiaratti supervising.
The first step was the identification of the type of dysfunction displayed by Mfn2-deficient oocytes on reaching the stage at which they are ready to be fertilized. The analysis showed a reduced number of mitochondria in these cells and a lower level of ATP (adenosine triphosphate), the molecule that serves as cell fuel.
The researchers also observed that oocyte mitochondria were more aggregated than normal, enlarged to twice the expected size, and further away from the endoplasmic reticulum, an organelle with which they need to interact to import calcium and other substances crucial to their functioning.
According to Chiaratti, one of the known roles of Mfn2 is ensuring that mitochondria stay in contact with the endoplasmic reticulum, a structure that participates in the synthesis and transport of several substances in cells. The results of the study suggest Mfn2 deficiency compromises interaction between the two organelles, impairing the functions of both in oocytes.
"There's evidence that transgenerational transmission of diseases such as diabetes is associated with mitochondrial dysfunction and endoplasmic reticulum stress in oocytes. Our findings corroborate this hypothesis," Chiaratti said. "Mfn2 deficiency appears to affect mitochondrial biogenesis [reducing the number of mitochondria] and the capacity of mitochondria to move about in the cytoplasm in order to meet cellular demand for energy."
The next step consisted of characterizing the Mfn2-deficient oocyte's transcriptome (the full range of messenger RNA molecules expressed) and comparing it with controls. Using RNA sequencing, the researchers found 517 genes that were less expressed in the genetically modified animals' oocytes than in controls and 426 genes that were more expressed.
"We then set out to identify the signaling pathways that belonged to these differentially expressed genes. We found pathways associated with the functioning of the endoplasmic reticulum and mitochondria, as well as pathways associated with endocrine processes such as regulation of blood sugar," Chiaratti said.
Alterations in offspring
Analysis of the offspring of genetically modified females focused on skeletal muscle and liver cells. The aim was to understand why these animals became diabetic even when fed a balanced diet.
Neither muscle cells nor liver cells were found to be in endoplasmic reticulum stress, a condition characterized by an accumulation of proteins that impairs the organelle's functioning, and no mitochondrial alterations were found in muscle cells. Liver cell mitochondria were moderately dysfunctional.
Because this alteration was not sufficient to explain the hyperglycemic phenotype of the offspring, the group decided to study insulin signaling in these animals, as insulin produced by the pancreas enables glucose to enter cells and thereby lowers the level of blood sugar.
Their analysis of pancreas cells showed that insulin production was normal, but the level of insulin in the bloodstream was reduced and the signal it normally sends to muscle and liver cells was weak.
"In these two tissues, insulin causes a biochemical change in the protein Akt [protein kinase B]. The signal sent by insulin makes this molecule become phosphorylated [via addition of phosphate to the protein chain] and this triggers a cascade of biochemical reactions in the cell," Chiaratti explained.
The results of these analyses, therefore, suggested that the offspring's muscle and liver tissue received a small amount of insulin, even though the level of insulin production by the pancreas was normal. This raised the hypothesis, to be confirmed in future studies, that insulin was being broken down faster in the organism of these animals.
Next steps
To deepen their understanding of the molecular mechanisms that led to augmented weight gain and hyperglycemia in Mfn2-deficient pups, the researchers plan to repeat the experiment with some modifications. Mfn2-deficient females will be fed a high-calorie diet to exacerbate the effects of the deficiency in their offspring.
"We also plan to investigate, in animals without any genetic modification, whether a high-calorie diet alone is sufficient to reduce Mfn2 expression and change the way mitochondria function and interact with the reticulum," Chiaratti said.
The knowledge created by these studies, he added, is expected to permit the development of strategies to manipulate Mfn2 expression in the context of obesity and help prevent transgenerational transmission of metabolic diseases.
For Alicia Kowaltowski, a professor at the University of São Paulo's Chemistry Institute (IQ-USP), a member of Redoxome and a co-author of the study, the results obtained so far show that a person's diet and nutritional status influence mitochondrial shape, one of the factors that affect cellular physiology. Proteins that regulate mitochondrial morphology are therefore potential therapeutic targets and should be explored in future research.
"It should be stressed that we didn't find significant alterations to the mitochondria in liver tissue even though the animals were diabetic. This accords with other studies showing that mitochondrial function in the liver is highly resilient," Kowaltowski said. "In our view, there must be protection mechanisms in the liver, given its importance to the metabolism. When mitochondrial dysfunction appears in the liver, the reason is that metabolic syndrome has reached an advanced stage of development."
Infertility and maternal inheritance
Funded by FAPESP via several projects 09/54035-4, 12/50231-6, 17/05899-2, 17/04372-0, 16/11935-9, 16/11942-5, 16/07868-4, 18/06119-3, and 10/51906-1, the study reported in the latest publication is part of a research line that aims to understand how mitochondrial alterations, including DNA mutations, are associated with infertility and transgenerational disease transmission.
"Previous research has shown that mitochondrial dysfunction can compromise egg fertility. We created two animal models with which to investigate this mechanism in more detail: in one we inhibited expression of mitofusin-1 in oocytes, and in the other, we inhibited expression of mitofusin-2," Chiaratti said.
Mfn1 deficiency made females infertile, as reported in an article published in The Faseb Journal.
"In this earlier study we showed that oocyte-specific Mfn1 deletion altered the expression of 161 genes and affected several processes in oocytes, above all communication with ovarian cells," Chiaratti said. "In the case of the Mfn2-deficient animals, we observed other alterations in oocytes and offspring, but fertility was not affected. Curiously, the effects of Mfn1 deletion were attenuated in oocytes when Mfn2 was simultaneously inhibited, suggesting that the action of Mfn1 occurs after that of Mfn2."
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