Aruna Asipu

Mutations involved in Aicardi-Goutieres syndrome implicate SAMHD1 as regulator of the innate immune response

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.

Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice

Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Fructose intake also causes features of metabolic syndrome in laboratory animals and humans. The first enzyme in fructose metabolism is fructokinase, which exists as two isoforms, A and C. Here we show that fructose-induced metabolic syndrome is prevented in mice lacking both isoforms but is exacerbated in mice lacking fructokinase A. Fructokinase C is expressed primarily in liver, intestine, and kidney and has high affinity for fructose, resulting in rapid metabolism and marked ATP depletion. In contrast, fructokinase A is widely distributed, has low affinity for fructose, and has less dramatic effects on ATP levels. By reducing the amount of fructose for metabolism in the liver, fructokinase A protects against fructokinase C-mediated metabolic syndrome. These studies provide insights into the mechanisms by which fructose causes obesity and metabolic syndrome.

High‐fat and high‐sucrose (western) diet induces steatohepatitis that is dependent on fructokinase

Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high‐fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild‐type or fructokinase knockout mice were fed a low‐fat (11%), high‐fat (36%), or high‐fat (36%) and high‐sucrose (30%) diet for 15 weeks. Both wild‐type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high‐fat diet compared to a low‐fat diet. In contrast, wild‐type mice fed a high‐fat and high‐sucrose diet developed more severe hepatic steatosis with low‐grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein‐1, alpha‐smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. Conclusion: An additive effect of high‐fat and high‐sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high‐fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis. (Hepatology 2013;58:1632–1643)

Endogenous fructose production and metabolism in the liver contributes to the development of metabolic syndrome

Carbohydrates with high glycemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations glucose are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously-produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase deficient mice were protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism whereby glucose promotes the development of metabolic syndrome.

Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.

Endogenous Fructose Production and Fructokinase Activation Mediate Renal Injury in Diabetic Nephropathy

Diabetes is associated with activation of the polyol pathway, in which glucose is converted to sorbitol by aldose reductase. Previous studies focused on the role of sorbitol in mediating diabetic complications. However, in the proximal tubule, sorbitol can be converted to fructose, which is then metabolized largely by fructokinase, also known as ketohexokinase, leading to ATP depletion, proinflammatory cytokine expression, and oxidative stress. We and others recently identified a potential deleterious role of dietary fructose in the generation of tubulointerstitial injury and the acceleration of CKD. In this study, we investigated the potential role of endogenous fructose production, as opposed to dietary fructose, and its metabolism through fructokinase in the development of diabetic nephropathy. Wild-type mice with streptozotocin-induced diabetes developed proteinuria, reduced GFR, and renal glomerular and proximal tubular injury. Increased renal expression of aldose reductase; elevated levels of renal sorbitol, fructose, and uric acid; and low levels of ATP confirmed activation of the fructokinase pathway. Furthermore, renal expression of inflammatory cytokines with macrophage infiltration was prominent. In contrast, diabetic fructokinase-deficient mice demonstrated significantly less proteinuria, renal dysfunction, renal injury, and inflammation. These studies identify fructokinase as a novel mediator of diabetic nephropathy and document a novel role for endogenous fructose production, or fructoneogenesis, in driving renal disease.

TRPM2-mediated intracellular Zn2+ release triggers pancreatic β-cell death.

Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating transient receptor potential (melastatin) 2 (TRPM2) channels. Cell death has been attributed to the ability of these channels to raise cytosolic Ca2+. Recent studies however revealed that TRPM2 channels can also conduct Zn2+, but the physiological relevance of this property is enigmatic. Given that Zn2+ is cytotoxic, we asked whether TRPM2 channels can permeate sufficient Zn2+ to affect cell viability. To address this, we used the insulin secreting (INS1) β-cell line, human embryonic kidney (HEK)-293 cells transfected with TRPM2 and pancreatic islets. H2O2 activation of TRPM2 channels increases the cytosolic levels of both Ca2+ and Zn2+ and causes apoptotic cell death. Interestingly, chelation of Zn2+ alone was sufficient to prevent β-cell death. The source of the cytotoxic Zn2+ is intracellular, found largely sequestered in lysosomes. Lysosomes express TRPM2 channels, providing a potential route for Zn2+ release. Zn2+ release is potentiated by extracellular Ca2+ entry, indicating that Ca2+-induced Zn2+ release leads to apoptosis. Knockout of TRPM2 channels protects mice from β-cell death and hyperglycaemia induced by multiple low-dose streptozotocin (STZ; MLDS) administration. These results argue that TRPM2-mediated, Ca2+-potentiated Zn2+ release underlies ROS-induced β-cell death and Zn2+, rather than Ca2+, plays a primary role in apoptosis.

Stable and temperature-sensitive transformation of baby rat kidney cells by SV40 suppresses expression of membrane dipeptidase

Membrane dipeptidase (MDP) is a zinc metalloenzyme located in the lungs and on the brush border membranes of the kidney and intestine. The gene for MDP (also termed DPEP1) is both frequently lost in Wilms tumours and is located on human chromosome 16q24.3, a region of the genome known to contain a tumour suppressor gene(s). We now report on the regulation of MDP gene expression in normal and transformed cells. MDP enzyme activity and mRNA was detected in primary baby rat kidney (BRK) cells maintained in culture for up to 4 weeks. In contrast all stable transformed cell lines that were tested, derived either by transformation with the DNA tumour viruses SV40 or adenovirus, or in human tumour cell lines, contained very low levels of or no detectable MDP mRNA or enzyme activity. In BRK cells transformed by the temperature-sensitive tsA58 mutant of SV40 T antigen, MDP activity was not detectable, in cell lines grown at the permissive temperature (33°C) but after 5 – 14 days of incubation at the non-permissive temperature (39.5°C), MDP protein and enzyme activity could be readily detected. Taken together, these results indicate that MDP expression is characteristic of differentiated kidney epithelial cells and is down-regulated in proliferating, transformed cells.

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development.

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

Cell-type specific factors bind to regulatory elements located downstream of the TATA-box element in the mouse myelin basic protein (MBP) gene promoter

Cell-type specific transcription of the myelin basic protein (MBP) gene in primary oligodendrocytes (OL) is regulated by cis-acting regulatory elements located at both upstream and downstream of the TATA-box region of the MBP promoter. To identify cell-type specific factors that bind to the downstream regulatory elements, we utilised DNase I footprinting analysis and gel retardation assays with nuclear extracts from myelin-forming OL as well as a non-myelin forming cell line, C6 glioma (C6) cells. Several regions of DNA were protected from DNAse I digestion by nuclear extracts of both cell types. However, two regions, from -17 to +17 and from +47 to +58 were protected specifically in OL, while three regions, from + 17 to + 22, from +43 to +49 and from +58 to +64 were protected only with C6 nuclear extracts. Inspection of the protected regions for homology with known transcription factor binding sites revealed that sequences at from +47 to +58 and from +56 to +68 showed extensive homology to the negative regulatory element (NRE1), of the mouse renin gene and to the interferon (IFN) consensus sequence of major histocompatibility complex class I genes (MHC I-ICS), respectively. Gel retardation assays using a MHC I-ICS oligonucleotide and transient transfection assays using MBP-CAT constructs were used to study the effect of IFNs on MBP promoter activity in OL and C6 cells. In OL, IFN-alpha/beta caused little induction of CAT activity, but IFN-gamma resulted in a 2-3.5-fold decrease in CAT activity. In contrast, in C6 cells both IFN-alpha/beta and IFN-gamma induced a 1.5-2.5-fold increase in CAT activity. The cooperative effects of factors binding to NREs and ICS may be responsible for the cell-type specific regulation of MBP gene transcription.