Felicia V. Nowak

Epigenetic effects of paternal diet on offspring: emphasis on obesity

Overnutrition, obesity, and the rise in associated comorbidities are widely recognized as preventable challenges to global health. Behavioral, metabolic, and epigenetic influences that alter the epigenome, when passed on to offspring, can increase their risk of developing an altered metabolic profile. This review is focused on the role of paternal inheritance as demonstrated by clinical, epidemiological, and experimental models. Development of additional experimental models that resemble the specific epigenetic sensitive situations in human studies will be essential to explore paternally induced trans-generational effects that are mediated, primarily, by epigenetic effects. Further elucidation of epigenetic marks will help identify preventive and therapeutic targets, which in combination with healthy lifestyle choices, can diminish the growing tide of obesity, type 2 diabetes, and other related disorders.

The RhoGAP domain-containing protein, Porf-2, inhibits proliferation and enhances apoptosis in neural stem cells.

Neural stem cells (NSCs) are essential to developing and mature CNS. They shape the structural and functional layouts of the brain in developing CNS and continue to proliferate, generating new neurons in several adult brain regions. Preoptic regulatory factor-2 (Porf-2), a RhoGAP domain-containing protein expressed in CNS, has a role in gender-related brain development and function. Porf-2 expression was knocked down in C17.2, a mouse cerebellar multipotent cell line. This increased proliferation and decreased drug-induced apoptosis without affecting cell type distribution following differentiation induction. It lowered levels of cyclin kinase inhibitor p21, affected G1 to S phase cell cycle transition; partially blocked the elevation in p53 transcriptional activity, p21 and Bcl-2-associated X protein (Bax) levels caused by bleomycin, but had no influence on enhancement of Bax in response to staurosporine. Thus Porf-2 may inhibit NSC proliferation by enhancing p21 protein levels followed by G1 phase arrest; it plays pro-apoptotic roles in response to drug treatment through both p53 transcription-dependent and independent pathways. This is consistent with categorization of Porf-2 as a functional RhoGAP in CNS.

Antioxidant diet, gender and age affect renal expression of nitric oxide synthases in obese diabetic rats.

Development of diabetic nephropathy (DN) is associated with decreased renal nitric oxide production and increased oxidative stress. We studied nitric oxide synthase (NOS) expression in kidney of obese Zucker fa/fa rats, a model of Type 2 obesity-related DN. Male and female rats received a regular (REG) or antioxidant-fortified (AO) diet starting at age 4 weeks. Quantitative PCR and immunoblot analyses were performed on kidney cortex and medulla to determine levels of endothelial, neuronal and inducible NOS at 6, 13 and 20 weeks of age. Multiple antibody-specific proteins were detected for each form. These may represent monomeric splice forms, post-translationally modified forms and their dimers, consistent with the known complexity of regulation of these enzymes. Levels of eNOS and nNOS are higher in males than females at 6 weeks on the REG diet and 13 weeks on either diet; the relationship is reversed in females at 6 weeks on the AO diet. Levels of eNOS and nNOS are lower on the AO diet compared to REG, in males at 6 and 13 weeks and females at 13 weeks; the reverse is seen in 6 week females and 20 week males. All three isoforms show peak levels in the younger animals, at 6 or 13 weeks. Better preservation of kidney function is associated with higher prevalence of dimers with potential to increase production of NO and lower levels of potentially harmful monomers. Differential expression of NOS isoforms may be linked to renal functional and histopathological changes in this rat model of DN.

Expression and characterization of the preoptic regulatory factor-1 and -2 peptides

The preoptic regulatory factors, PORF-1 and PORF-2, were originally detected as cDNAs from the rat preoptic area of the hypothalamus. Expression of the messenger RNAs is dependent on age, gender and hormonal status. PORF-1 is a putative transcription factor while PORF-2 affects cell growth. However, expression of peptides predicted from the PORF-1 and PORF-2 open reading frames has not been reported. The characterization of PORF-1 and PORF-2 peptides expressed in vitro and in vivo is described here. There are three potential PORF-1 peptides of 37, 59 and 86 amino acids, because of the presence of two TGA codons that can code either for termination or for selenocysteine incorporation in the PORF-1 open reading frame. All three peptides are detected after in vitro translation of full-length porf-1 brain mRNA. The two larger ones are found in normal male rat brain. The expression of a given peptide in bacteria depends upon the host strain. In contrast, a single 75 amino acid PORF-2 peptide is detected by in vitro translation, in transformed bacteria, and in rat brain. The PORF-1 and PORF-2 peptides have the charges and hydrophobicities predicted from their brain cDNA open reading frames. These small peptides may represent examples of multiple usage of single genetic loci in eukaryotes, giving rise to both a protein product and its regulatory peptide.

Antioxidant diet and sex interact to regulate NOS isoform expression and glomerular mesangium proliferation in Zucker diabetic rat kidney.

Oxidative stress contributes substantially to the pathophysiology of diabetic nephropathy (DN). Consumption of an antioxidant-fortified (AO) diet from an early age prevents or delays later development of DN in the Zucker rat female with type 2 diabetes. We hypothesize this is due to effects on mesangial matrix and renal nitric oxide synthase (NOS) distribution and to sex-specific differences in NOS responses in the diabetic kidney. Total glomerular tuft area (GTA) and PAS-positive tuft area (PTA), endothelial (e), neuronal (n) and inducible (i) NOS were quantified in males and females on AO or regular (REG) diet at 6 and 20 weeks of age. eNOS was observed in glomeruli and tubules. nNOS predominantly localized to tubular epithelium in both cortex and medulla. iNOS was expressed in proximal and distal tubules and collecting ducts. Sex, diabetes duration and AO diet affected the distribution of the three isoforms. GTA and PTA increased with duration of hyperglycemia and showed a negative correlation with renal levels of all NOS isoforms. AO diet in both genders was associated with less PAS-positive staining and less mesangial expansion than the REG diet, an early increase in cortical iNOS in males, and sex-specific changes in cortical eNOS at 20 weeks. These effects of AO diet may contribute to sex-specific preservation of renal function in females.

Preoptic Regulatory Factor-2, a Rhogap Domain Protein that Modifies Cell Cycle Progression and Apoptosis in the CNS

RhoGAPS are proteins that control Rho GTPases, small enzymes that regulate diverse cellular functions. Preoptic regulatory factor-2 (Porf-2) is a RhoGAP domain-containing protein that is both pro-apoptotic and anti-proliferative. It is widely distributed among vertebrate taxa and expressed in multiple organs and cell types. Porf-2 expression is regulated at the level of transcription by age, gender, location, gonadal hormones, and pituitary and growth regulating factors. It may help coordinate sexually dimorphic development and be a target for prevention or treatment of diseases that exhibit apoptotic or proliferative imbalance.

Porf-2 = Arhgap39 = Vilse: A Pivotal Role in Neurodevelopment, Learning and Memory

Abstract Small GTP-converting enzymes, GTPases, are essential for the efficient completion of many physiological and developmental processes. They are regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Arhgap39, also known as preoptic regulatory factor-2 (Porf-2) or Vilse, a member of the Rho GAP group, was first identified in 1990 in the rat CNS. It has since been shown to regulate apoptosis, cell migration, neurogenesis, and cerebral and hippocampal dendritic spine morphology. It plays a pivotal role in neurodevelopment and learning and memory. Homologous or orthologous genes are found in more than 280 vertebrate and invertebrate species, suggesting preservation through evolution. Not surprisingly, loss of the Arhgap39/Porf-2 gene in mice manifests as an embryonic lethal condition. Although Arhgap39/Porf-2 is highly expressed in the brain, it is also widely distributed throughout the body, with potential additional roles in oncogenesis and morphogenesis. This review summarizes, for the first time, the known information about this gene under its various names, in addition to considering its transcripts and proteins. The majority of findings described have been made in rats, mice, humans, and fruit flies. This work surveys the known functions, functional mediators, variables modifying expression and upstream regulators of expression, and potential physiological and pathological roles of Arhgap39/Porf-2 in health and disease.

Paternal high‐fat diet enhances offspring whole‐body insulin sensitivity and skeletal muscle insulin signaling early in life

Evidence suggests that paternal diet can predispose offspring to metabolic dysfunction. Despite this knowledge, little is known regarding the effects of paternal high‐fat feeding on offspring insulin sensitivity. The purpose of this study was to investigate for the first time the effects of paternal high‐fat feeding on whole‐body and skeletal muscle insulin action in young and adult offspring. At 4 weeks of age, founder C57BL6/N males (F0) were fed a high‐fat diet or control diet for 12 weeks and then bred with females on a control diet. Offspring (F1) were euthanized at 6 weeks, 6 months, or 12 months and insulin‐stimulated insulin signaling was measured ex vivo in isolated soleus muscle. At 6 weeks of age, paternal high fat offspring (HFO) had enhanced whole‐body insulin sensitivity (35%, P < 0.05), as well as, increased insulin‐stimulated skeletal muscle phosphorylation of Akt threonine 308 (70%, P < 0.05) and AS160 threonine 642 (80%, P < 0.05) compared to paternal control fed offspring (CFO), despite both offspring groups consuming standard chow. At 6 months of age, HFO had increased percent body fat compared to CFO (74%, P < 0.005) and whole‐body and skeletal muscle insulin signaling normalized to CFO. Body fat was inversely related with insulin signaling in HFO, but not CFO. These findings suggest that paternal high‐fat feeding contributes to enhanced whole‐body and skeletal muscle insulin sensitivity in HFO early in life; however, these benefits are lost by early adulthood, potentially due to premature increases in body fat.