Regulation of glucose responsive protein (GRP) gene expression by insulin.

While screening for insulin-induced genes, we identified two members of a family of stress-induced genes referred to as glucose-regulated proteins (GRPs). GRPs are members of the stress-responsive superfamily of genes which also includes heat shock proteins (HSPs). The GRP proteins are not normally heat-inducible, but are overproduced when cells are starved of glucose. The two major GRP proteins, GRP78 and GRP94, are highly conserved among vertebrates. We have found that physiological concentrations of insulin stimulate the transcription of GRP78 and GRP94 in rat H4IIE hepatoma cells. The regulation of GRP78 transcription was rapid, with the first induction within minutes, and a further induction after several hours, and both occurred in the presence of glucose. GRP78 transcription was more greatly induced by insulin in the presence of SB202190, a specific p38-MAPK inhibitor. Transcription of GRP94 was also induced, but only after several hours. Calcimycin (A23187) and anisomycin were used to induce endoplasmic reticulum (ER)/cellular stress, and both induced GRP78 and GRP94 transcription.

Insulin attenuates TNFα-induced hemopexin mRNA: An anti-inflammatory action of insulin in rat H4IIE hepatoma cells.

Proinflammatory cytokines, including TNF-α and IL-6, can contribute to insulin resistance. Conversely, insulin has some actions that can be considered anti-inflammatory. Hemopexin is a Class 2 acute phase reactant and control of its transcription is predominantly regulated by IL-6, with TNF-α and IL-1ß also inducing hemopexin gene expression. Thus, we asked whether insulin could inhibit the ability of TNF-α to stimulate hemopexin mRNA expression. In cultured rat hepatoma (H4IIE) cells, TNF-α significantly increased hemopexin mRNA accumulation. The TNF-α-induced increase of hemopexin mRNA was dramatically attenuated by insulin, even though TNF-α reduced peak insulin activation of ERK. Thus, even though TNF-α can contribute to insulin resistance, the residual insulin response was still able to counteract TNF-α actions.

Polytrauma-induced hepatic stress response and the development of liver insulin resistance.

Insulin resistance and metabolic dysfunction are common following injury. Polytrauma is defined as combined injuries to more than one body part or organ system, and is common in modern warfare, as well as automobile and industrial accidents. Polytrauma can include any combination of burn injury, fracture, hemorrhage, trauma to the extremities, and blunt or penetrating trauma. Multiple minor injuries are often more deleterious than a more severe single injury. To investigate the mechanisms of development of insulin resistance following injury, we have developed a rat model of polytrauma which combined soft tissue trauma with burn injury and penetrating gastrointestinal (GI) trauma. Male Sprague-Dawley rats were subjected to a laparotomy plus either a 15-18% total body surface area scald burn or a single puncture of the cecum (CLP) with a G30 needle, or the combination of both burn and CLP injuries (polytrauma). We examined the effects of polytrauma which increased markers of hepatic endoplasmic reticulum (ER) stress, and increased hepatic Trib3 mRNA levels coincident with reduced insulin-inducible insulin signaling. Phosphorylation/activation of the insulin receptor (IR) and AKT were decreased at 24, but not 6h following polytrauma. These results demonstrate a complex, time-dependent development of hepatic ER-stress and a diminished response to insulin, which were among the pathological sequelae following polytrauma.

Prenylation differentially inhibits insulin-dependent immediate early gene mRNA expression.

Increased activity of prenyl transferases is observed in pathological states of insulin resistance, diabetes, and obesity. Thus, functional inhibitors of farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) may be promising therapeutic treatments. We previously identified insulin responsive genes from a rat H4IIE hepatoma cell cDNA library, including ß-actin, EGR1, Pip92, c-fos, and Hsp60. In the present study, we investigated whether acute treatment with FTase and GGTase inhibitors would alter insulin responsive gene initiation and/or elongation rates. We observed differential regulation of insulin responsive gene expression, suggesting a differential sensitivity of these genes to one or both of the specific protein prenylation inhibitors.

Skeletal muscle atrogene expression and insulin resistance in a rat model of polytrauma.

Polytrauma is a combination of injuries to more than one body part or organ system. Polytrauma is common in warfare, and in automobile and industrial accidents. The combination of injuries can include burn, fracture, hemorrhage, and trauma to the extremities or specific organ systems. Resistance to anabolic hormones, loss of muscle mass, and metabolic dysfunction can occur following injury. To investigate the effects of combined injuries, we have developed a highly reproducible rodent model of polytrauma. This model combines burn injury, soft tissue trauma, and penetrating injury to the gastrointestinal (GI) tract. Adult, male Sprague-Dawley rats were anesthetized with pentobarbital and subjected to a 15-20% total body surface area scald burn, or laparotomy and a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). In the current studies, the inflammatory response to polytrauma was examined in skeletal muscle. Changes in skeletal muscle mRNA levels of the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 were observed following single injuries and polytrauma. Increased expression of the E3 ubiquitin ligases Atrogin-1/FBX032 and TRIM63/MuRF-1 were measured following injury, as was skeletal muscle insulin resistance, as evidenced by decreased insulin-inducible insulin receptor (IR) and AKT/PKB (Protein Kinase B) phosphorylation. Changes in the abundance of IR and insulin receptor substrate-1 (IRS-1) were observed at the protein and mRNA levels. Additionally, increased TRIB3 mRNA levels were observed 24 h following polytrauma, the same time when insulin resistance was observed. This may suggest a role for TRIB3 in the development of acute insulin resistance following injury.

A rat model of concurrent combined injuries (polytrauma).

Polytrauma, a combination of injuries to more than one body part or organ system, is common in modern warfare and in automobile and industrial accidents. The combination of injuries can include burn injury, fracture, hemorrhage, trauma to the extremities, and trauma to specific organ systems. To investigate the effects of combined injuries, we have developed a new and highly reproducible model of polytrauma. This model combines burn injury with soft tissue and gastrointestinal (GI) tract trauma. Male Sprague Dawley rats were subjected to a 15-20% total body surface area scald burn, or a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). Unlike many 'double hit' models, the injuries in our model were performed simultaneously. We asked whether multiple minor injuries, when combined, would result in a distinct phenotype, different from single minor injuries or a more severe single injury. There were differences between the single injuries and polytrauma in the maintenance of blood glucose, body temperature, body weight, hepatic mRNA and circulating levels of TNF-α, IL-1ß and IL-6, and hepatic ER-stress. It has been suggested that models utilizing combinatorial injuries may be needed to more accurately model the human condition. We believe our model is ideal for studying the complex sequelae of polytrauma, which differs from single injuries. Insights gained from this model may suggest better treatment options to improve patient outcomes.

Aging and energetics' 'Top 40' future research opportunities 2010-2013.

BACKGROUND: As part of a coordinated effort to expand our research activity at the interface of Aging and Energetics a team of investigators at The University of Alabama at Birmingham systematically assayed and catalogued the top research priorities identified in leading publications in that domain, believing the result would be useful to the scientific community at large. OBJECTIVE: To identify research priorities and opportunities in the domain of aging and energetics as advocated in the 40 most cited papers related to aging and energetics in the last 4 years. DESIGN: The investigators conducted a search for papers on aging and energetics in Scopus, ranked the resulting papers by number of times they were cited, and selected the ten most-cited papers in each of the four years that include 2010 to 2013, inclusive. RESULTS:   Ten research categories were identified from the 40 papers.  These included: (1) Calorie restriction (CR) longevity response, (2) role of mTOR (mechanistic target of Rapamycin) and related factors in lifespan extension, (3) nutrient effects beyond energy (especially resveratrol, omega-3 fatty acids, and selected amino acids), 4) autophagy and increased longevity and health, (5) aging-associated predictors of chronic disease, (6) use and effects of mesenchymal stem cells (MSCs), (7) telomeres relative to aging and energetics, (8) accretion and effects of body fat, (9) the aging heart,  and (10) mitochondria, reactive oxygen species, and cellular energetics. CONCLUSION: The field is rich with exciting opportunities to build upon our existing knowledge about the relations among aspects of aging and aspects of energetics and to better understand the mechanisms which connect them.

Regulation of hepatic insulin receptor activity following injury.

Impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of injury or critical illness and Type 2 diabetes. However, mechanisms that modulate IR function remain unclear. With an animal model of critical-illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 min following trauma and hemorrhage. Possible mechanisms for this defect were examined, including IR protein levels and IR posttranslational modifications. The total amounts of hepatic IRα and IRß subunits and the membrane localization of the IR were not altered by trauma and hemorrhage, and, likewise, no change in IR tyrosine nitration was found in the liver. However, there was a decrease in the level of protein O-linked ß-N-acetlyglucosamine (O-GlcNac) modification on Ser/Thr in the liver following trauma and hemorrhage. Inhibition of JNK increased IR O-GlcNac modification, implicating an involvement of JNK. These findings suggest that a balance between O-GlcNac modification and JNK-induced phosphorylation may exist, with decreased Ser/Thr O-GlcNac modification following trauma and hemorrhage, allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits IR activity. The present studies suggest potential mechanisms of hemorrhage-induced defects in IR activity and a potential role for acutely decreased O-GlcNac and increased serine phosphorylation of the IR.

Acute psychological stress results in the rapid development of insulin resistance.

In recent years, the roles of chronic stress and depression as independent risk factors for decreased insulin sensitivity and the development of diabetes have been increasingly recognized. However, an understanding of the mechanisms linking insulin resistance and acute psychological stress are very limited. We hypothesized that acute psychological stress may cause the development of insulin resistance, which may be a risk factor in developing type 2 diabetes. We tested the hypothesis in a well-established mouse model using 180 episodes of inescapable foot shock (IES) followed by a behavioral escape test. In this study, mice that received IES treatment were tested for acute insulin resistance by measuring glucose metabolism and insulin signaling. When compared with normal and sham mice, mice that were exposed to IES resulting in escape failure (defined as IES with behavioral escape failure) displayed elevated blood glucose levels in both glucose tolerance and insulin tolerance tests. Furthermore, mice with IES exposure and behavioral escape failure exhibited impaired hepatic insulin signaling via the insulin-induced insulin receptor/insulin receptor substrate 1/Akt pathway, without affecting similar pathways in skeletal muscle, adipose tissue, and brain. Additionally, a rise in the murine growth-related oncogene KC/GRO was associated with impaired glucose metabolism in IES mice, suggesting a mechanism by which psychological stress by IES may influence glucose metabolism. The present results indicate that psychological stress induced by IES can acutely alter hepatic responsiveness to insulin and affect whole-body glucose metabolism.

Hepatic growth hormone resistance after acute injury.

Severe injury and infection are often followed by accelerated protein catabolism and acute insulin resistance. This results in several effects that complicate and prolong recovery, including weakness, immobility, impaired wound healing, and organ dysfunction. Recent studies have demonstrated the development of GH resistance during severe inflammation, providing a potential mechanism for the protein loss that follows injury and infection. To understand this GH resistance, we recently developed a murine model of acute injury. Mice were subjected to soft-tissue injury, alone or combined with hemorrhage, and injected iv with GH 30, 60, or 90 minutes later. Hepatic GH signaling was measured via Western analysis. GH-induced signal transducer and activator of transcription 5 phosphorylation was decreased immediately after completion of the trauma procedure, and at 30 and 60 minutes, but further decreased by 90 minutes after trauma. Combined trauma and hemorrhage resulted in severely decreased GH-induced signal transducer and activator of transcription 5 phosphorylation compared with trauma alone, and this was true at all time points studied. Western analysis revealed an apparent decrease in the molecular weight of the hepatic GH receptor (GHR) after trauma and hemorrhage, but not trauma alone. Additional studies determined that the hemorrhage-induced decrease in receptor size was not due to changes in GHR N-linked glycosylation. These results suggest that GH sensitivity is rapidly impaired after acute injury and that trauma combined with hemorrhage results in a more severe form of GH resistance resulting from alteration or inactivation of hepatic GHR.

Injury-induced insulin resistance in adipose tissue.

Hyperglycemia and insulin resistance are common findings in critical illness. Patients in the surgical ICU are frequently treated for this 'critical illness diabetes' with intensive insulin therapy, resulting in a substantial reduction in morbidity and mortality. Adipose tissue is an important insulin target tissue, but it is not known whether adipose tissue is affected by critical illness diabetes. In the present study, a rodent model of critical illness diabetes was used to determine whether adipose tissue becomes acutely insulin resistant and how insulin signaling pathways are being affected. There was a reduction in insulin-induced phosphorylation of IR, IRS-1, Akt and GSK-3ß. Since insulin resistance occurs rapidly in adipose tissue, but before the insulin resistance in skeletal muscle, it may play a role in the initial development of critical illness diabetes.

Role of tissue macrophages in the development of critical illness diabetes.

Macrophage-derived factors, including TNF-α, are known as important inducers of insulin resistance. However, the role of macrophages in insulin resistance in the liver is unclear. Hyperglycemia and insulin resistance commonly occur following acute injuries or critical illness, referred to as "critical illness diabetes." In the present studies, the roles of macrophages in hepatic insulin resistance following surgical trauma and hemorrhage were investigated. Intravenous administration of gadolinium chloride or clodronate-liposome resulted in depletion of macrophages in both liver and spleen of rats. Macrophage depletion by either gadolinium chloride or clodronate-liposome did not prevent the development of trauma and hemorrhage-induced insulin resistance in the liver of rats, as indicated by impaired hepatic insulin signaling following a 90-minute hemorrhage period. Similarly, hepatic insulin resistance still developed in rats after removal of the spleen (splenectomy). In contrast, macrophage depletion significantly reversed the hepatic insulin resistance several hours later, following resuscitation. As a comparison, splenectomy resulted in improvement in hepatic insulin signaling following resuscitation, but to a lesser extent, suggesting that both liver and spleen resident macrophages have a role in the continuation of hepatic insulin resistance following resuscitation. These studies demonstrated that the initial development of insulin resistance in liver is macrophage-independent in a rodent model of critical illness diabetes, whereas both liver and spleen macrophages have a role in the later maintenance of the insulin-resistant state, following resuscitation.

Role of inhibitory κB kinase and c-Jun NH2-terminal kinase in the development of hepatic insulin resistance in critical illness diabetes.

Hyperglycemia and insulin resistance induced by acute injuries or critical illness are associated with increased mortality and morbidity, as well as later development of type 2 diabetes. The molecular mechanisms underlying the acute onset of insulin resistance following critical illness remain poorly understood. In the present studies, the roles of serine kinases, inhibitory κB kinase (IKK) and c-Jun NH(2)-terminal kinase (JNK), in the acute development of hepatic insulin resistance were investigated. In our animal model of critical illness diabetes, activation of hepatic IKK and JNK was observed as early as 15 min, concomitant with the rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate 1. Inhibition of IKKα or IKKß, or both, by adenovirus vector-mediated expression of dominant-negative IKKα or IKKß in liver partially restored insulin signaling. Similarly, inhibition of JNK1 kinase by expression of dominant-negative JNK1 also resulted in improved hepatic insulin signaling, indicating that IKK and JNK1 kinases contribute to critical illness-induced insulin resistance in liver.

Role of reactive oxygen species in injury-induced insulin resistance.

Acute insulin resistance is common after injury, infection, and critical illness. To investigate the role of reactive oxygen species (ROS) in critical illness diabetes, we measured hepatic ROS, which rapidly increased in mouse liver. Overexpression of superoxide dismutase 2, which decreased mitochondrial ROS levels, protected mice from the development of acute hepatic insulin resistance. Insulin-induced intracellular signaling was dramatically decreased, and cellular stress signaling was rapidly increased after injury, resulting in the hyperglycemia of critical illness diabetes. Insulin-induced intracellular signaling, activation of stress (c-Jun N-terminal kinase) signaling, and glucose metabolism were all normalized by superoxide dismutase 2 overexpression or by pretreatment with antioxidants. Thus, ROS play an important role in the development of acute hepatic insulin resistance and activation of stress signaling after injury.

Adenovirus infection results in alterations of insulin signaling and glucose homeostasis.

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents.

Anti-Inflammatory Action of Insulin via Induction of Gadd45-ß Transcription by the mTOR Signaling Pathway.

Insulin regulates a large number of genes in a tissue-specific manner. We have identified genes modulated by insulin in the liver and in liver-derived cells that had not yet been characterized as insulin regulated, and these previous studies indicate that numerous genes are induced by insulin via the MEK-ERK pathway. We now describe new studies indicating that Gadd45-ß can be induced by acute insulin treatment. Although other regulators of Gadd45-ß expression may utilize the MEK-ERK pathway, the data indicate that insulin utilizes signaling pathways separate from either MEK-ERK, PI3-K or p38 signaling pathways in the regulation of Gadd45-ß transcription. Our findings show that activation of a downstream effector of multiple signaling pathways, mTOR, was required for insulin-induction of Gadd45-ß gene transcription. Increased expression of Gadd45-ß can inhibit c-Jun N-terminal kinase (JNK) activity. Since TNFα is increased during inflammation, and acts, at least in part, via the JNK signaling pathway, insulin induction of Gadd45-ß suggests a mechanism for the anti-inflammatory actions of insulin.

Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance.

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes.

Acute insulin resistance following injury.

Hyperglycemia and insulin resistance often occur following injury and/or critical illness. Whereas intensive insulin treatment reduces hyperglycemia, mortality and morbidity in certain patients, little is known regarding the pathophysiology of acute insulin resistance following injury and infection. Studies suggest that acute insulin resistance is complex and might differ in a tissue-specific manner, involving multiple causative factors and intracellular signaling pathways. Therefore, the advantages of intensive insulin therapy might not be uniform to all injuries or critical illnesses. Clearly, the increased incidence of hypoglycemic incidents following intensive insulin therapy indicates a need for understanding the underlying molecular mechanisms of the acute development of insulin resistance, which will allow a more targeted approach to treat altered glucose metabolism of critically ill patients.

Age and tissue specific differences in the development of acute insulin resistance following injury.

Injuries, hemorrhage, sepsis, burn, and critical illnesses all induce insulin resistance, and insulin resistance is strongly associated with advancing age. However, the effect of age on injury induced insulin resistance is not well studied. We performed surgical trauma in male rats of three different ages (3-, 6-, and 10-weeks old). Rats were either hemorrhaged to a mean arterial pressure of 35-40 mmHg and subsequently maintained at that pressure for up to 90 min, or maintained without hemorrhage as controls. Results indicate that insulin-induced intracellular signaling was diminished in liver and skeletal muscle of 6- and 10-week old rats following trauma and hemorrhage. In even younger rats, immediately post-weaning ( approximately 3 weeks of age), insulin signaling was lost in liver, but not in skeletal muscle. Glucocorticoids can play a role in the chronic development of insulin resistance. Our results demonstrate that corticosterone levels were increased in 6- and 10-week old animals following hemorrhage, but little change was measured in 3-week old animals. Blockade of glucocorticoid synthesis prevented the development of insulin resistance in skeletal muscle, but not in liver of 6- and 10-week old rats. Moreover, skeletal muscle glucocorticoid receptor levels increased dramatically between 3 and 6 weeks of age. These results indicate that trauma and hemorrhage-induced hepatic insulin resistance occurs at all ages tested. However, there is no development of insulin resistance following trauma and hemorrhage in skeletal muscle of post-weaning rats. In skeletal muscle of 6- and 10-week old rats, inhibition of glucocorticoid levels prevents the development of insulin resistance.

Crosstalk between growth hormone and insulin signaling.

Growth Hormone (GH) is a major growth-promoting and metabolic regulatory hormone. Interaction of GH with its cell surface GH receptor (GHR) causes activation of the GHR-associated cytoplasmic tyrosine kinase, JAK2, and activation of several signaling pathways, including the STATs, ERK1/2, and PI3K pathways. Insulin is also a key hormone regulating metabolism and growth. Insulin binding to the insulin receptor (IR) results in phosphorylation/activation of the IR, and activates the PI3K/Akt and ERK1/2 pathways. Due to their important roles in growth and metabolism, GH and insulin can functionally interact with each other, regulating cellular metabolism. In addition, recent data suggests that GH and insulin can directly interact by signaling crosstalk. Insulin regulation of GH signaling depends on the duration of exposure to insulin. Transient insulin exposure enhances GH-induced activation of MEK/ERK pathway through post-GHR mechanisms, whereas prolonged insulin exposure inhibits GH-induced signaling at both receptor and postreceptor levels. Chronic excessive GH interferes with insulin's activation of the IR/IRS/PI3K pathway and several proteins are involved in the mechanisms underlying GH-induced insulin resistance.