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In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart., Highlights • GFAT2 is not expressed in cardiac myocytes. • GFAT2 is abundantly expressed in cardiac fibroblasts. • GFAT1 is expressed in both cardiac myocytes and fibroblasts. • GFAT1 regulates the stress-induced increase in cardiac O-GlcNAcylation.

Protein posttranslational modifications (PTMs) by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) rise during pressure‐overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O‐GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O‐GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O‐GlcNAc levels. We hypothesize that induction of key enzymes augment protein O‐GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O‐GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1‐week TAC (1wTAC) for early hypertrophy, or 6‐week TAC (6wTAC) for established hypertrophy. We used western blots to determine O‐GlcNAc regulation. To assess the effect of increased protein O‐GlcNAcylation with established hypertrophy, mice received thiamet‐g (TG) starting 4 weeks after TAC. Protein O‐GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O‐GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate‐limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O‐GlcNAc levels but did not affect cardiac mass. In summary, protein O‐GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O‐GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O‐GlcNAc levels during established hypertrophy did not restart hypertrophic growth., Protein O‐GlcNAc levels temporally vary during pressure‐overload hypertrophy (POH) with the highest levels during active hypertrophic growth early after transverse aortic constriction. While it is commonly hypothesized that O‐GlcNAc levels reflect increased glucose utilization during POH, our study confirms the importance of non‐nutrient regulation through enzymes like GFAT1 and OGA.

Esophageal squamous cell carcinomas (ESCCs) as well as adenocarcinomas (EACs) were developed in rat duodenal contents reflux models (reflux model). The present study aimed to shed light on the mechanism by which bile acid stimulation causes cancer onset and progression. Metabolomics analyses were performed on samples of neoplastic and nonneoplastic tissues from reflux models, and K14D, cultivated from a nonmetastatic, primary ESCC, and ESCC‐DR, established from a metastatic thoracic lesion. ESCC‐DRtca2M was prepared by treating ESCC‐DR cells with taurocholic acid (TCA) to accelerate cancer progression. The lines were subjected to comprehensive genomic analyses. In addition, protein expression levels of glucose‐6‐phosphate dehydrogenase (G6PD), nuclear factor kappa B (NF‐κB) (p65) and O‐linked N‐Acetylglucosamine (O‐GlcNAc) were compared among lines. Cancers developed in the reflux models exhibited greater hexosamine biosynthesis pathway (HBP) activation compared with the nonneoplastic tissues. Expression of O‐GlcNAc transferase (OGT) increased considerably in both ESCC and EAC compared with nonneoplastic squamous epithelium. Conversely, cell line‐based experiments revealed the greater activation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. G6PD overexpression in response to TCA exposure was observed. Both NF‐κB (p65) and O‐GlcNAc were expressed more highly in ESCC‐DRtca2M than in the other cell lines. Moreover, ESCC‐DRtca2M cells had additional chromosomal abnormalities in excess of ESCC‐DR cells. Overall, glucose metabolism was upregulated in both esophageal cancer tissue and cell lines. While bile acids are not mutagenic, chronic exposure seems to trigger NF‐κB(p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression. Glucose metabolism was upregulated in both esophageal cancer tissue and cell lines, and the HBP was activated in the former. The cell line‐based experiments demonstrated upregulation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. While bile acids are not mutagenic, chronic exposure seems to trigger G6PD overexpression and NF‐κB (p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression.

Lung cancer is one of the three most common cancers among men and women and causes more cancer-related deaths than any other type of carcinoma. Of the two subtypes, Non-small cell lung cancer (NSCLC) is the most common. These tumors are characteristically high grade, PET-positive, indicating heightened glucose uptake and altered cancer metabolism. Sirtuin 6 (SIRT6) is a member of the sirtuin family of histone deacetylases and has previously been shown to be a key regulator of glucose metabolism, DNA damage repair, and aging. SIRT6 has been demonstrated to act as a tumor suppressor in colon, pancreatic and liver cancer. However, its role in NSCLC remains elusive. Here, we have developed a mouse model to study the role of SIRT6 in NSCLC, utilizing a KRASG12D driver mutation and p53flox/flox to drive lung tumorigenesis in response to intranasal deliver of adenovirus expressing Cre recombinase. Work described here indicates that SIRT6 expression is necessary for lung tumor initiation and progression. Loss of SIRT6 expression resulted in a delay of tumor growth leading to decreased tumor burden and prolonged survival compared to animals expressing both wild-type alleles of SIRT6. Utilizing NSCLC developed mouse cell lines, we were able to show that cells lacking SIRT6 exhibited decreased proliferation, decreased formation of colonies of soft agar and increased senescence, providing a possible mechanism for the delay in tumor initiation and formation in vivo. Since NSCLC maintain SIRT6 mRNA expression in primary tumors, we sought to identify a potential mechanism by which cancer cells post-translationally regulate SIRT6 activity. In this study, we propose a mechanism where by SIRT6 can be lost in response to the metabolic state of the cell. Data presented here shows that SIRT6 undergoes a glucose-responsive N-terminal cleavage that is predicted to result in loss of its chromatin-localization sequence (CLS), causing it to dissociate from chromatin. More specifically, we found that SIRT6 is directly O-GlcNAcylated and that the O-GlcNAc transferase (OGT) is necessary for this cleavage to occur. Our lab has previously shown that OGT also O-GlcNAcylates the NF-κB subunit, p65, to allow for its full transcriptional activation. As SIRT6 and NF-κB have been previously shown to physically interact and antagonize one another in transcription of key metabolic genes, loss of the CLS of SIRT6 and transcriptional activation of p65 in response to O-GlcNAcylation may serve as a mechanism for p65-induced transcription of metabolic target genes. Work described in this thesis underscores the importance of SIRT6 as an essential enzyme required for NSCLC development, yet once carcinoma is established it becomes dynamically misregulated by altered metabolic flux of the cancer cell.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. Our genome-wide association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood–brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction. DOI: http://dx.doi.org/10.7554/eLife.04790.001, eLife digest Traumatic brain injury (TBI) caused by a violent blow to the head or body and the resultant collision of the brain against the skull is a major cause of disability and death in humans. Primary injury to the brain triggers secondary injuries that further damage the brain and other organs, generating many of the detrimental consequences of TBI. However, despite decades of study, the exact nature of these secondary injuries and their origin are poorly understood. A better understanding of secondary injuries should help to develop novel therapies to improve TBI outcomes in affected individuals. To obtain this information, in 2013 researchers devised a method to inflict TBI in the common fruit fly, Drosophila melanogaster, an organism that is readily amenable to detailed genetic and molecular studies. This investigation demonstrated that flies subjected to TBI display many of the same symptoms observed in humans after a brain injury, including temporary loss of mobility and damage to the brain that becomes worse over time. In addition, many of the flies die within 24 hr after brain injury. Now Katzenberger et al. use this experimental system to investigate the secondary injuries responsible for these deaths. First, genetic variants were identified that confer increased or decreased susceptibility to death after brain injury. Several of the identified genes affect the structural integrity of the intestinal barrier that isolates the contents of the gut—including nutrients and bacteria—from the circulatory system. Katzenberger et al. subsequently found that the breakdown of this barrier after brain injury permits bacteria and glucose to leak out of the intestine. Treating flies with antibiotics did not increase survival, whereas reducing glucose levels in the circulatory system after brain injury did. Thus, Katzenberger et al. conclude that high levels of glucose in the circulatory system, a condition known as hyperglycemia, is a key culprit in death following TBI. Notably, these results parallel findings in humans, where hyperglycemia is highly predictive of death following TBI. Similarly, individuals with diabetes have a significantly increased risk of death after TBI. These results suggest that the secondary injuries leading to death are the same in flies and humans and that further studies in flies are likely to provide additional new information that will help us understand the complex consequences of TBI. Important challenges remain, including understanding precisely how the brain and intestine communicate, how injury to the brain leads to disruption of the intestinal barrier, and why elevated glucose levels increase mortality after brain injury. Answers to these questions could help pave the way to new therapies for TBI. DOI: http://dx.doi.org/10.7554/eLife.04790.002