Diabetes, a chronic metabolic disease that affects nearly 10% of the world's population can lead to very serious complications such as renal failure, liver cirrhosis, and heart attacks. The most common type is Type 2 diabetes and is diagnosed when a person has elevated amounts of blood glucose due to insulin resistance. This resistance to insulin leads to problems with glucose transport into tissues for subsequent metabolism. Over the years it has been shown that insulin regulates the expression of several key enzymes in both carbohydrate and fatty acid metabolic pathways via the phosphatidyl inositol 3‐kinase (PI3K) pathway. Previously, using glucosamine, a precursor of the hexosamine biosynthetic pathway, we had established a model of insulin resistance in primary rat hepatocytes in culture. Using this primary cell culture model, we showed that under insulin resistant conditions, the expression of glucose 6 phosphate dehydrogenase (G6PDH), a key enzyme in carbohydrate metabolism and fatty acid synthase (FAS), a key enzyme in fat metabolism were differently regulated but the mechanism of this differentiation was unclear. Under this model of insulin resistance, we now show that this differential regulation is due to the liver X receptor (LXR) and insulin induced gene (INSIG).

Insulin‐mimetics are agents that have been shown to mimic the actions of insulin including promoting the entry of glucose into tissues, activating signal proteins, influencing the expression of genes and regulating metabolic processes. Through the years, selenium and vanadium compounds have been shown to mediate a number of insulin‐like actions in models of Type I diabetes both in vivo and in vitro. Few studies, however have ascertained the effectiveness of these mimetics on models of Type II diabetes or insulin resistance. Using glucosamine, a precursor of the hexosamine biosynthetic pathway (HBP) products, models of insulin resistance in a variety of cells in culture have been established. Previously we have demonstrated insulin resistance in primary rat hepatocytes by treating the cells with glucosamine. Using this model we now investigate the effects of the insulin mimetics, selenium and vanadium on insulin signal proteins and expression of the key metabolic genes, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphate dehydrogenase and fatty acid synthase.

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 (4μM) and time (3h) 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 cadmium's 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.

Insulin resistance, a hallmark of type 2 diabetes, is characterized by the inability of a cell or tissue to respond to physiological levels of insulin resulting in problems with glucose transport and metabolism. Several cellular models have been utilized to determine the mechanism of induction of insulin resistance but questions remain unanswered. There is evidence that high glucose induced insulin resistant may be mediated by products of the hexosamine biosynthetic pathway (HBP). The major end product of HBP, UDP-GlcNAC, is the substrate for O-GlcNAC transferase, an enzyme that catalyzes the O-linked transfer of GlcNAC to Ser/Thr residues of numerous proteins. This modification may play a role in induction of insulin resistance and thus needs to be evaluated in different cell types to fully understand its implication. Therefore we developed an insulin resistant model in primary hepatocytes by treating the cells with a precursor of a HBP product, glucosamine, at various concentrations over differing lengths of time. Insulin resistance was considered established when signal proteins such as AKT and the MAPK family were no longer phosphorylated in the presence of insulin. Increased glycosylation of proteins was also observed. Treatment of these cells with selenium, an insulin-mimetic, restored the phosphorylation of the signal proteins. Supported in part by a WMU FRACASF and Monroe Brown award and NSF DBI-0139204.

Faculty, staff, and students at all levels of educational institutions are becoming more aware about ethical issues in the classroom and in research. As educators, it is our responsibility to provide an opportunity to discuss these issues so that future scientists will be prepared to face the many ethical challenges they may encounter. Often, unfortunately, we are reticent to engage in this discussion due solely to the lack of our own repertoire of resources regarding ethics. Thus, this summary of educational resources was compiled to promote ethics discussions in science classrooms and research programs.

Oxidative stress is the result of a cellular imbalance of pro-oxidants and anti-oxidants, with the pro-oxidants being in much higher concentration. This imbalance has been shown to result in disease due to cellular damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Pro-oxidants such as ROS are generated upon exposure to heavy metals, including cadmium (Cd). Cd has become a problematic pollutant due to the industrial production and eventual deposition of batteries and plastics. Cd has also been shown to elevate ROS in a rat hepatocyte model and as a result induce lipid peroxidation, chromosomal damage and LDH leakage. Cd exposure also results in a decrease of reduced glutathione. Glutathione, a ubiquitous tripeptide, serves as an indicator of the redox state of the cell, but also has a protective role in guarding proteins against permanent damage by ROS. This protective role is exemplified in particular with active site Cys residues. Glutathione can bind these residues in a reversible manner, forming a glutathionylated protein (GSSP) when the cell is under attack by ROS, avoiding an irreversible modification that occurs when oxygen radicals such as superoxide and hydrogen peroxide react with the residue. In this study, the effects of Cd induced oxidative stress on the rat hepatoma cell line H4IIE are considered. Western blot analysis is used to probe for the presence of GSSPs. The results indicate that the presence of GSSPs vary with exposure time of Cd. Based on molecular weight comparison of the GSSPs, possible candidates for the proteins that are being modified by GSH include those from the SAPK cascade, which include p38, ERK and JNK. Work is continuing to identify the protein(s) that are glutathionylated, and their role in self-preservation used by the cell to survive cadmium exposure. This work was supported in part by NSF 0136127.

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 whether K-ras mutational status of regional nodes in patients with Dukes B 2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened for K-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B 2 tumors that have mutations in codon 12 or 13 of the K-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) with K-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B 2 colorectal cancers that have mutations in codon 12 or 13 of the K-ras gene are at high risk for the development of nodal metastases. Mutational analysis of the lymph nodes identifies high-risk patients who should he considered for adjuvant chemotherapy. Therefore, K-ras mutational analysis should he considered for molecular staging of colorectal cancer. [

The effect of Cadmium (Cd) on the expression of c‐Jun N‐terminal kinase (JNK), c‐jun, and activator protein‐1 (AP‐1) has been investigated. We previously reported that Cd causes cell damage as indicated by increases in the cytotoxic parameters, lactate dehydrogenase and lipid peroxidation, and this damage was mediated by decreases in cellular concentration of glutathione. In the present study, we investigate the molecular events involved prior to the Cd‐induced cellular toxicity and damage in primary rat hepatocytes. We propose that Cd, through the generation of reactive oxygen species (ROS) and prior to significant cellular damage, activates the stress activated signal protein JNK, regulates c‐jun expression, and promotes the binding of a redox sensitive transcription factor AP‐1. We show JNK activity and c‐jun mRNA level significantly increased at 1 h and AP‐1 DNA binding activity significantly enhanced at 3 h in the presence of 4 μM cadmium chloride. Blocking the Cd induction of JNK activity, c‐jun mRNA level, and AP‐1 binding activity using the antioxidants N‐acetyl cysteine (10 mM) or carnosol (0.5 μg/mL) suggests a role for ROS. Blocking JNK activity and c‐jun mRNA by SP600125 (20 μM), a JNK inhibitor, supports the role of JNK in transmission of signals induced by Cd. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:133–142, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20018

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.

We developed a model of volume contraction in rabbits by using a furosemide/low-salt diet to follow changes, if any, in proximal tubule Na+/H+ exchanger 3 (NHE3) mRNA and brush-border protein. The rabbits' plasma renin, aldosterone, and urine sodium content confirmed the volume-contracted state. RNase protection assays demonstrated increases in treated-animal NHE3 mRNA as a percentage of control with 172 +/- 23, 154 +/- 15, 153 +/- 14, and 141 +/- 7 (SE) % (P < 0.05) at 1, 5, 10, and 31 days, respectively. Western analysis of brush-border membrane with NHE3 antibody revealed increased immunoreactivity in treated animals as a percentage of control with 120 +/- 30, 190 +/- 59, 307 +/- 72, and 427 +/- 41% (P < 0.05) at 1, 5, 10, and 31 days, respectively. There was no significant difference in serum potassium, bicarbonate, and cortisol in control vs. experimental animals. These data suggest that there is chronic upregulation of NHE3 in the volume-contracted state.

. Selenium was first suspected of being an essential dietary trace element in the 1950s. We now know that indeed it is an essential biological element that serves as an integral component of several enzymes, including those in the families of deiodinases and glutathione peroxidases as well as selenoproteins P and W. The multi-author review that follows this introduction concentrates on the important biological role of selenium in enzymes as well as some of the physiological aspects of selenium as either a potential anticarcinogenic agent or insulin mimetic. What should become clear from these contributed articles is the complex and dynamic role that selenium plays in many biological processes and that the investigations in these areas are at the edge of exciting new frontiers.

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 whether K-ras mutational status of regional nodes in patients with Dukes B-2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened for K-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B-2 tumors that have mutations in codon 12 or 13 of the K-rns 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) with K-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B-2 colorectal cancers that have mutations in codon 12 of 13 of the K-rns 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.

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 whether K-ras mutational status of regional nodes in patients with Dukes B 2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened for K-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B 2 tumors that have mutations in codon 12 or 13 of the K-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) with K-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B 2 colorectal cancers that have mutations in codon 12 or 13 of the K-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.

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.