Susan R. Stapleton

Selenium: an insulin-mimetic.

Abstract. Insulin or agents that can mimic its action (insulin mimetics) are necessary to promote the entry of glucose into tissues where the glucose can either be converted into energy or stored for later use. In recent years, selenium has been shown to mediate a number of insulin-like actions both in vivo and in vitro. These insulin-like actions include stimulating glucose uptake and regulating metabolic processes such as glycolysis, gluconeogenesis, fatty acid synthesis and the pentose phosphate pathway. The mechanism by which selenium is capable of mimicking insulin is not clear; however, reports indicate that selenium does activate key proteins involved in the insulin-signal cascade. Various proteins in the insulin-signal cascade have been shown to be necessary for different insulin-regulated events, and presumably data will be forthcoming soon that illustrate this similarly for selenium. This review compares the action of selenium to that of insulin and discusses the available evidence in support of selenium as an insulin mimetic.

Selenium: potent stimulator of tyrosyl phosphorylation and activator of MAP kinase

Selenium, an essential biological trace element, is an integral component of several enzymes, and its use as a nutritional supplement has been popularized recently due to its potential role in low concentrations as an antioxidant and in higher concentrations as an anticancer agent. Selenium has also been reported to act as an insulin-mimetic agent with regard to normalization of blood glucose levels and regulation of some insulin-mediated metabolic processes. Little work, however, has been done concerning the pathway(s) by which this insulin-mimetic action occurs. In this study, we investigated the mechanism by which selenate exhibits insulin-mimetic properties in two different insulin responsive cell types, primary rat hepatocytes and 3T3 L1 adipocytes. We found that two proteins associated with the insulin signal cascade, the beta-subunit of the insulin receptor and IRS-1, increased in tyrosyl phosphorylation in the presence of selenium. The third identified selenium activated signal protein, MAP kinase, has been implicated not only in the insulin signal transduction pathway but also in other growth factor-mediated responses. Using an in-gel activity assay for MAP kinase, we demonstrated that both the p42 and p44 MAP kinases are activated when either hepatocytes or adipocytes are incubated in the presence of selenate. In addition to the activation of these specific proteins, we found that selenium also eventually profoundly affected overall tyrosyl phosphorylation. Our results therefore show that selenium not only increased the phosphorylation of proteins identified in the insulin signal cascade but also affected the overall phosphorylation state of the cell.

Insulin Regulation of Glucose-6-phosphate Dehydrogenase Gene Expression Is Rapamycin-sensitive and Requires Phosphatidylinositol 3-Kinase

Glucose-6-phosphate dehydrogenase (G6PDH) controls the flow of carbon through the pentose phosphate pathway and also produces NADPH needed for maintenance of reduced glutathione and reductive biosynthesis. Hepatic expression of G6PDH is known to respond to several dietary and hormonal factors, but the mechanism behind regulation of this expression has not been characterized. We show that insulin similarly induces expression of endogenous hepatic G6PDH and a reporter construct containing 935 base pairs of the G6PDH promoter linked to luciferase in transient transfection assays. Using well tested and structurally distinct inhibitors of Ras farnesylation, lovastatin and B581, and a specific inhibitor of mitogen-activated protein kinase kinase activation, PD 98059, we show that the Ras/Raf/mitogen-activated protein kinase pathway is not utilized for the insulin-induced stimulation of G6PDH gene expression in primary rat hepatocytes. Similarly, using well characterized inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002, we show that PI 3-kinase activity is necessary for the induction of G6PDH expression by insulin. Rapamycin, an inhibitor of FRAP protein, which is involved in the activation of pp70 S6 kinase, blocks the insulin induction of G6PDH, suggesting that S6 kinase is also necessary for the insulin induction of G6PDH expression.

Insulin-like effects of vanadate and selenate on the expression of glucose-6-phosphate dehydrogenase and fatty acid synthase in diabetic rats

Insulin is capable of regulating cellular and metabolic processes as well as gene expression. In recent years, enthusiasm has surfaced for using insulin-mimetics to study the mechanism of action of insulin. Vanadate and selenate are two compounds that have been found to mimic the action of insulin on control of blood glucose levels in vivo. Vanadate has also been shown to regulate the expression of several enzymes both in vivo and in vitro, however, studies concerning selenates ability to regulate expression have not been reported. In this study we show that administration of vanadate or selenate to streptozotocin-induced diabetic rats not only normalizes blood glucose levels similarly to insulin but also positively affects the expression of two key metabolic enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS). Both G6PDH and FAS activity are significantly decreased in diabetic animals compared to non-diabetic control. Treatment of the diabetic animals with either insulin, vanadate or selenate restored both activities to about 80-90% of control. All treatment conditions exhibited activities significantly higher than those determined for the diabetic group but did not differ significantly from each other. Increases in G6PDH or FAS activity are due to increases in mRNA level. Increase in both G6PDH and FAS mRNA was comparable to the observed increase in activity suggesting that regulation of expression by the mimetics occurs pretranslationally.

EFFICIENT LIPID-MEDIATED TRANSFECTION OF DNA INTO PRIMARY RAT HEPATOCYTES

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 µg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 µg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.

Molecular staging of colorectal cancer: K-ras mutation analysis of lymph nodes upstages Dukes B patients.

PURPOSE. Multiple attempts have been made to improve the clinical/pathologic staging system of Dukes to focus adjuvant therapy decisions. The purpose of this study was to determine whetherK-ras mutational status of regional nodes in patients with Dukes B2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened forK-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B2 tumors that have mutations in codon 12 or 13 of theK-ras gene were identified. RESULTS: Mutational analysis of the lymph nodes from these patients revealed an 80 percent (16/20) incidence of the same mutations in regional lymph nodes. None of the four patients with mutation-free nodes developed recurrence compared with 37.5 percent (6/16) withK-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B2 colorectal cancers that have mutations in codon 12 or 13 of theK-ras gene are at high risk for the development of nodal metastases. Mutational analysis of the lymph nodes identifies high-risk patients who should be considered for adjuvant chemotherapy. Therefore,K-ras mutational analysis should be considered for molecular staging of colorectal cancer.

Isolation and sequence of a rat glucose-6-phosphate dehydrogenase promoter.

A 935 bp fragment of the rat glucose-6-phosphate dehydrogenase (G6PDH) gene containing promoter activity was isolated using the polymerase chain reaction (PCR). This fragment was sequenced and primer extension analysis showed a transcription initiation site in agreement with the human and mouse genes. Computer analysis of the sequence showed a 60% and 78% similarity to the human and mouse G6PDH sequences, respectively. A TATA box element, TTAAAT, was found and shown to be 100% similar to the human and mouse TATA box elements. Based on sequence comparison, some putative transcriptional regulatory elements were also found.

Exogenous nitric oxide downregulates MIP-2 and MIP-1α chemokines and MAPK p44/42 after ischemia and reperfusion of the rat kidney

The mechanisms by which nitric oxide (NO) exerts its protective effect in the ischemia/reperfusion (I/R) injury of the kidney have not been fully determined. The hypothesis of this study was based on the assumption that I/R upregulates some chemokines (MIP-2 and MIP-1 f ) as well as certain protein kinases (MAPK p44/42), and therefore we aimed in this work at recognizing if an exogenous NO donor would downregulate these effects in rat ischemic kidneys at the same time that it would offer functional protection as measured by serum creatinine. Sprague-Dawley rats were subjected to renal warm ischemia (75 min) and contralateral nephrectomy. Animals were divided into 3 groups ( n = 8 per group): sham, ischemic control, and ischemic group treated with sodium nitroprusside (NaNP 5 mg/kg) given 15 min prior to reperfusion. Serum creatinine (SCr), serum chemokines (MIP-2 and MIP-1 f ), kidney tissue MAPK p44/42, kidney neutrophil infiltration determined by myeloperoxidase (MPO), and light histology were evaluated 4 h after reperfusion began. There were significant improvements in SCr and better histopathological features in the I/R-NaNP group compared with the I/R group. Similarly, the I/R-NaNP kidneys exhibited a downregulating effect of serum chemokines (MIP-2 and MIP-1 f ) and kidney tissue MAPK p44/42 that was not observed in the I/R group alone. The MPO levels were lower in the I/R-NaNP group compared with the I/R untreated group. We can conclude from these experiments that I/R of the rat kidney upregulated the production of MIP-2 and MIP-1 f chemokines and the activation of MAPKp44/42. It also had a detrimental effect on the function and structure of the ischemic kidney. Exogenous NO had a temporal protective effect in organ function and histology and exerted a downregulating response in the production of MIP-2 and MIP-1 f chemokines and the activation of MAPK p44/42 following I/R.

Early sensing and gene expression profiling under a low dose of cadmium exposure.

Cadmium (Cd) has been shown to have various detrimental effects on health. In recent years progress has been made in dissecting apart the molecular mechanisms underlying the effects of exposure to this toxic metal. In this paper we investigated changes in gene expression using a global transcript profiling approach to better understand the early molecular events that occur in primary rat hepatocytes when exposed to Cd at a concentration (4 microM) and time (3 h) that is prior to any significant increase in cytotoxic parameters. Gene expression changes were most dramatically noticed for proteins involved in transcriptional regulation, zinc finger protein production, and heat shock protein expression. Other genes whose expression changed significantly were those associated with maintaining cellular redox homeostasis such as increasing glutathione synthesis and antioxidant capacity, facilitating the survival or death response, and repairing damage or stimulating degradation. Expression changes were confirmed for selected genes in various groups utilizing qRT-PCR. Various times of Cd incubation were also used to assess the extent of the impact. To define whether or not any of these changes were associated with cadmiums ability to disturb the redox balance, we also tested the effects of Cd in the presence of a blocker of glutathione synthesis, D,L-buthionine-(S,R)-sulfoximine (BSO), and an antioxidant, N-acetylcysteine (NAC). The results show that the Cd induction of some genes can be categorized as occurring primarily in response to changes in the redox state as measured by attenuation of the response by the addition of NAC or to the availability of reduced glutathione as measured by the increase in response in the presence of BSO.

Effects of acetaldehyde on glucose-6-phosphate dehydrogenase activity and mRNA levels in primary rat hepatocytes in culture

Ethanol has been shown to induce the activity of glucose-6-phosphate dehydrogenase (G6PDH). To clarify the mechanism behind this induction, we examined the role of acetaldehyde (AA), the first product of ethanol metabolism. In primary adult rat hepatocytes maintained in chemically defined medium, we examined the effect of AA on G6PDH activity, mRNA levels and lipid synthesis. We observe a 40% increase in G6PDH activity and a similar increase in mRNA levels, following exposure to 100 microM AA. The increase in activity was found to be maximal at 24 h while mRNA levels increased over controls as early as 3 h. The induction in G6PDH by AA was found to occur at lower concentrations and earlier time points than those reported using ethanol. The role of insulin, a known inducer of G6PDH activity was studied alone and in combination with AA on both G6PDH activity and mRNA levels as well as lipid biosynthesis. Insulin (300 ng/ml) was found to increase G6PDH activity, mRNA levels and [14C]-acetate incorporation into lipid. It was also shown to have an additive effect with AA on G6PDH activity, suggesting their actions are mediated via different mechanistic pathways. No change in [14C]-acetate incorporation into lipid, however, was observed with acetaldehyde alone.