Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we investigated the effects of glucocorticoids on in vivo tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate IRS-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cortisone treatment of rats resulted in both hyperglycemia and hyperinsulinemia. Anesthetized animals were injected with 10 U/kg insulin via cardiac puncture and, after 2 min, hindlimb muscles were removed, snap-frozen, and homogenized in SDS. Protein tyrosine phosphorylation was studied by immunoblotting with phosphotyrosine antibody. Insulin receptors and substrate IRS-1 were identified and quantified with specific antibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine phosphorylation (69 +/- 4% of control, P < 0.05). The decreased level of receptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6 +/- 5% of control, P < 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content by 50%, but did not significantly alter the total level of IRS-1 tyrosine phosphorylation. The apparent M(r) of IRS-1 was reduced by approximately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that the lower M(r) likely results from lower phosphoserine and/or phosphothreonine content. To investigate the role of hyperinsulinemia in the glucocorticoid response, rats were made insulin-deficient with streptozotocin (100 mg/kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoid-treated skeletal muscle is characterized by: (a) decreased total tyrosine phosphorylation of insulin receptors as a result of a reduction in the pool of receptors undergoing tyrosine phosphorylation; (b) decreased IRS-1 content and reduced serine and/or threonine phosphorylation of IRS-1. Glucocorticoid-induced hyperinsulinemia appears to be essential for the development of these alterations.
Rat skeletal muscle contains two enzymes which can make epinephrine: phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase. We studied the time-course and mechanism by which the glucocorticoid dexamethasone increases muscle PNMT activity. We also examined the hypothesis that increased muscle E synthesis may contribute to glucocorticoid-induced insulin resistance. Dexamethasone (1 mg/kg s.c. for 12 d) increased muscle PNMT activity seven-fold but did not change NMT activity. Immunotitration with an anti-PNMT antibody indicated that the PNMT elevation was due to increased numbers of PNMT molecules. Dexamethasone rapidly increased PNMT activity and this elevation was largely maintained 6 d after glucocorticoid treatment stopped. Muscle epinephrine levels were transiently elevated by dexamethasone. Dexamethasone-treated rats had elevated insulin levels after a glucose load, and chronic administration of the PNMT inhibitor SKF 64139 reversed this increase. Chronic SKF 64139 improved glucose tolerance in normal rats. Dexamethasone induced muscle synthesis of the epinephrine-forming enzyme PNMT. A PNMT inhibitor lowered insulin levels in glucocorticoid-treated rats and glucose levels in untreated rats. These findings are compatible with antagonism of insulin-mediated glucose uptake by epinephrine synthesized in skeletal muscle.
Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.
Gene profiling; glucocorticoids; metabolic dysfunction
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparation from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparations from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
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 minutes, 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.
insulin resistance; hemorrhage; glucocorticoid receptor; liver; skeletal muscle
Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11β-HSD1 inhibitors improve insulin sensitivity.
RESEARCH DESIGN AND METHODS
Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11β-HSD1 inhibition upon insulin signaling and action.
Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer307 insulin receptor substrate (IRS)-1. 11β-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11β-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer307 IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11β-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer307 IRS1 decreased and pThr308 Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression.
Prereceptor facilitation of glucocorticoid action via 11β-HSD1 increases pSer307 IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11β-HSD1 inhibition decreases pSer307 IRS1, increases pThr308 Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.
Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.
Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of β3-adrenoceptor (β3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective β3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet.
Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the β3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study.
In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet.
These results show that activation of the β3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with β3-AR activation.
Duodenal-jejunal bypass (DJB) has been shown to reverse type 2 diabetes (T2DM) in Goto-Kakazaki (GK) rats, a rodent model of non-obese T2DM. Skeletal muscle insulin resistance is a hallmark decrement in T2DM. The aim of the current work was to investigate the effects of DJB on skeletal muscle insulin signal transduction and glucose disposal. It was hypothesized that DJB would increase skeletal muscle insulin signal transduction and glucose disposal in GK rats.
DJB was performed in GK rats. Sham operations were performed in GK and non-diabetic Wistar-Kyoto (WKY) rats. At two weeks post-DJB, oral glucose tolerance (OGTT) was measured. At three weeks post-DJB, insulin-induced signal transduction and glucose disposal were measured in skeletal muscle.
In GK rats and compared to Sham operation, DJB did not: 1) improve fasting glucose or insulin; 2) improve OGTT; or 3) increase skeletal muscle insulin signal transduction or glucose disposal. Interestingly, skeletal muscle glucose disposal was similar between WKY-Sham, GK-Sham, and GK-DJB.
Bypassing of the proximal small intestine does not increase skeletal muscle glucose disposal. The lack of skeletal muscle insulin resistance in GK rats questions whether this animal model is adequate to investigate the etiology and treatments for T2DM. Additionally, bypassing of the foregut may lead to different findings in other animal models of T2DM as well as in T2DM patients.
Type 2 diabetes; duodenal-jejunal bypass; skeletal muscle glucose disposal
Sepsis blunts the ability of nutrient signaling by leucine to stimulate skeletal muscle protein synthesis by impairing translation initiation. The present study tested the hypothesis that overproduction of either tumor necrosis factor (TNF)-α or glucocorticoids mediate the sepsis-induced leucine resistance. Prior to producing peritonitis, rats received either vehicle, TNF binding protein (TNFBP) to inhibit endogenous TNFα action, and/or the glucocorticoid receptor antagonist RU486. Leucine was orally administered to all rats 24 h thereafter and the gastrocnemius removed 20 min later to assess protein synthesis and signaling components important in controlling peptide-chain initiation. Muscle protein synthesis was 65% lower in septic rats administered leucine than in leucine-treated control animals. This reduction was not prevented by either TNFBP or RU486 alone, but was completely reversed by the combination. This sepsis-induced leucine resistance was associated with an 80% reduction in the amount of active eIF4E·eIF4G complex, a 5-fold increase in the formation of the inactive eIF4E·4E-BP1 complex as well as markedly reduced (at least 70%) phosphorylation of 4E-BP1, eIF4G, S6K1, S6, and mTOR. Pretreatment of septic rats with either TNFBP or RU486 individually only nominally improved the leucine action as assessed by the above-mentioned endpoints. In contrast, when TNFBP and RU486 were co-administered, the ability of sepsis to impair the leucine-stimulated phosphorylation of 4E-BP1, eIF4G, S6K1, and S6 as well as the redistribution of eIF4E was essentially prevented. No differences in the total amount or phosphorylation of eIF2α and eIF2Bɛ were detected between the different groups, and changes could not be attributed to differences in the prevailing plasma concentration of insulin or leucine. Our data demonstrate the sepsis-induced leucine resistance in skeletal muscle results from the cooperative interaction of both TNFα and glucocorticoids.
Fatty acid-induced insulin resistance and impaired glucose uptake activity in muscle cells are fundamental events in the development of type 2 diabetes and hyperglycemia. There is an increasing demand for compounds including drugs and functional foods that can prevent myocellular insulin resistance.
In this study, we established a high-throughput assay to screen for compounds that can improve myocellular insulin resistance, which was based on a previously reported non-radioisotope 2-deoxyglucose (2DG) uptake assay. Insulin-resistant muscle cells were prepared by treating rat L6 skeletal muscle cells with 750 μM palmitic acid for 14 h. Using the established assay, the impacts of several fatty acids on myocellular insulin resistance were determined.
In normal L6 cells, treatment with saturated palmitic or stearic acid alone decreased 2DG uptake, whereas unsaturated fatty acids did not. Moreover, co-treatment with oleic acid canceled the palmitic acid-induced decrease in 2DG uptake activity. Using the developed assay with palmitic acid-induced insulin-resistant L6 cells, we determined the effects of other unsaturated fatty acids. We found that arachidonic, eicosapentaenoic and docosahexaenoic acids improved palmitic acid-decreased 2DG uptake at lower concentrations than the other unsaturated fatty acids, including oleic acid, as 10 μM arachidonic acid showed similar effects to 750 μM oleic acid.
We have found that polyunsaturated fatty acids, in particular arachidonic and eicosapentaenoic acids prevent palmitic acid-induced myocellular insulin resistance.
Insulin resistance; Glucose uptake; L6 skeletal muscle cells; Palmitic acid; Arachidonic acid; Eicosapentaenoic acid
Fatty acid oversupply is a key mediator of skeletal muscle insulin resistance in obesity, primarily via accumulation of fatty acid metabolites and activation of proinflammatory pathways. Herein, we demonstrate that fatty acid–induced insulin resistance in humans is completely prevented the day after 1 session of endurance exercise. Because skeletal muscle is the primary site for systemic glucose disposal and is highly susceptible to impaired insulin action by elevated fatty acid availability, we obtained skeletal muscle samples to investigate possible mechanisms mediating this protective effect of exercise. Prevention of fatty acid–induced insulin resistance after exercise accompanied enhanced skeletal muscle protein expression of key lipogenic enzymes and an increase in muscle triglyceride synthesis. Partitioning more fatty acids toward triglyceride synthesis within muscle reduced the accumulation of fatty acid metabolites and suppressed the proinflammatory response in skeletal muscle, as evidenced by decreased phosphorylation and activation of JNK and increased abundance of inhibitor of NF-κB α (IκB-α) and IκB-β. We believe this is the first study to demonstrate that 1 session of exercise completely reverses fatty acid–induced insulin resistance in humans. Reversal of insulin resistance accompanied enhanced lipogenic capacity within skeletal muscle, reduced accumulation of highly bioactive fatty acid metabolites, and suppressed activation of proinflammatory pathways known to impair insulin action.
Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser473 (81%) and Thr308 (23%)] and AS160 Thr642 (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr172) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr389 phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.
Angiotensin II; Glucose transport; Red skeletal muscle; Akt; AS160; AMPK; p70 S6K1
Purpose of review
The purpose of this review is to discuss novel insight into mechanisms of glucocorticoid-regulated muscle wasting, in particular the role of transcription factors and nuclear cofactors. In addition, novel strategies that may become useful in the treatment or prevention of glucocorticoid-induced muscle wasting are reviewed.
Studies suggest that glucocorticoid-induced upregulation of the transcription factors FOXO1 and C/EBPβ and downregulation of MyoD and myogenin are involved in glucocorticoid-induced muscle wasting. In addition, glucocorticoid-induced hyperacetylation caused by increased expression of the nuclear cofactor p300 and its histone acetyl transferase activity and decreased expression and activity of histone deacetylases (HDACs) plays an important role in glucocorticoid-induced muscle proteolysis and wasting. Other mechanisms may also be involved in glucocorticoid-induced muscle wasting, including insulin resistance and store-operated calcium entry. Novel potential strategies to prevent or treat glucocorticoid-induced muscle wasting include the use of small molecule HDAC activators, dissociated glucocorticoid receptor agonists, and 11β-hydroxysteroid dehydrogenase type 1 inhibitors.
An increased understanding of molecular mechanisms regulating glucocorticoid-induced muscle wasting will help develop new strategies to prevent and treat this debilitating condition.
Muscle wasting; glucocorticoids; FOXO1; C/EBPβ; MyoD; myogenin; p300; histone deacetylases
Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours.
The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates.
The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to investigate clinically-relevant mechanisms of drug-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.
The clinical use of HIV protease inhibitors (PIs) is associated with the development of peripheral insulin resistance. The incidence and degree of impaired glucose tolerance observed in treated patients vary considerably between drugs, however. To compare the ability of HIV PIs to alter peripheral glucose disposal acutely in a genetically identical model system at therapeutically relevant drug levels, healthy lean male rats previously naive to PI exposure were given ritonavir, amprenavir, lopinavir/ritonavir (4:1), or atazanavir by continuous intravenous infusion to achieve steady state drug levels of 10 or 25 mM rapidly. Under euglycemic hyperinsulinemic clamp conditions, a dose-dependent reduction in the peripheral glucose disposal rate (Rd) was observed with all the PIs except atazanavir. The rank order of sensitivity was ritonavir, lopinavir, and then amprenavir. Changes in skeletal muscle and heart 2-deoxyglucose (2-DOG) uptake correlated with reductions in Rd. All 3 of these PIs also produced significant reductions in 2-DOG uptake into primary rat adipocytes in vitro. Atazanavir had no effect on glucose uptake in vitro or in vivo. The in vivo potency of PIs to impair peripheral glucose disposal acutely correlates with the degree of insulin resistance observed in HIV-infected patients receiving these drugs. Preclinical testing of novel candidate PIs in a rodent model system may be useful in identifying the future risk of altering glucose homeostasis.
glucose clamp; adipokines; HIV protease inhibitors; glucose uptake; glucose transporter; insulin resistance; rodents
Insulin's rate of entry into skeletal muscle appears to be the rate limiting step for muscle insulin action and is slowed by insulin resistance. Despite its obvious importance, uncertainty remains as to whether the transport of insulin from plasma to muscle interstitium is a passive diffusional process or a saturable transport process regulated by the insulin receptor.
To address this, here we directly measure the rate of 125I-insulin uptake by rat hindlimb muscle and examine how that is affected by adding unlabeled insulin at high concentrations. We used mono-iodinated ([125I]TyrA14)insulin and short (5 min) exposure times, combined with trichloroacetic acid precipitation, to trace intact bioactive insulin.
Compared to saline, high concentrations of unlabeled insulin delivered either continuously (insulin clamp) or as a single bolus significantly raised plasma 125I-insulin, slowed the movement of 125I-insulin from plasma into liver, spleen, and heart (p<0.05, for each) but increased kidney 125I-insulin uptake. High concentrations of unlabeled insulin delivered either continuously (insulin clamp), or as a single bolus significantly decreased skeletal muscle 125I-insulin clearance (p<0.01 for each). Increasing muscle perfusion by electrical stimulation did not prevent the inhibitory effect of unlabeled insulin on muscle 125I-insulin clearance.
This indicates that insulin's trans-endothelial movement within muscle is a saturable process likely involving the insulin receptor. Current findings, together with other recent reports, suggest that trans-endothelial insulin transport may be an important site at which muscle insulin action is modulated in clinical and pathologic settings.
Insulin transport; muscle; insulin; trans-endothelial transport
Glucocorticoids commonly cause drug-induced diabetes. This association is well recognized but available evidence does not answer clinically relevant issues in subjects without diabetes.
Thirty-five individuals without diabetes with a recent diagnosis of acute lymphoblastic leukemia or non-Hodgkin’s lymphoma on high-dose glucocorticoid therapy were studied. Close systematic monitoring of fasting and postprandial glycemia and fasting insulin determinations, HOMA-insulin resistance and HOMA β-cell function were performed. The primary objective was to define the incidence of secondary diabetes in patients treated with high-dose glucocorticoids. Secondary objectives were to specify the intensity, the moment it appears and the evolution of hyperglycemia, in addition to the risk factors, mechanisms and impact of continuous and cyclical glucocorticoids on the development of hyperglycemia.
Mean age of patients was 38.4 ± 18.7 years. The incidence of diabetes was 40.6% and was found after the first week; half the time it occurred between the second and fourth. Two-thirds spontaneously normalized by eight weeks. Continuous glucocorticoid administration had a higher incidence of fasting hyperglycemia (P = 0.003). Mean peak insulin levels were significantly higher in cases of diabetes.
High-dose prednisone for 2 to 3 months produced an elevated incidence of diabetes, usually with mild hyperglycemia occurring between the second and fourth week, normalizing spontaneously in all cases. Hyperglycemia was more frequent with continuous doses and occurred in cases with increased insulin resistance. The clinical and therapeutic characteristics of our participants, who were otherwise healthy, could represent the clinical setting of many patients with illness from other medical areas that might require high doses of GC for six to twelve weeks.
Diabetes; Secondary diabetes; Glucocorticoid-induced diabetes; Glucocorticoid; Hyperglycemia; Drug-induced diabetes
Increased lipid availability reduces insulin-stimulated glucose disposal in skeletal muscle, which is generally explained by fatty acid–mediated inhibition of insulin signaling. It remains unclear whether lipids also impair transcapillary transport of insulin and glucose, which could become rate controlling for glucose disposal. We hypothesized that lipid-induced insulin resistance is induced by inhibiting myocellular glucose uptake and not by interfering with the delivery of insulin or glucose. We measured changes in interstitial glucose and insulin in skeletal muscle of healthy volunteers during intravenous administration of triglycerides plus heparin or glycerol during physiologic and supraphysiologic hyperinsulinemia, by combining microdialysis with oral glucose tolerance tests and euglycemic-hyperinsulinemic clamps. Lipid infusion reduced insulin-stimulated glucose disposal by ∼70% (P < 0.05) during clamps and dynamic insulin sensitivity by ∼12% (P < 0.05) during oral glucose loading. Dialysate insulin and glucose levels were unchanged or even transiently higher (P < 0.05) during lipid than during glycerol infusion, whereas regional blood flow remained unchanged. These results demonstrate that short-term elevation of free fatty acids (FFAs) induces insulin resistance, which in skeletal muscle occurs primarily at the cellular level, without impairment of local perfusion or transcapillary transport of insulin and glucose. Thus, vascular effects of FFAs are not rate controlling for muscle insulin-stimulated glucose disposal.
Glucocorticoids are a mainstay of anti-inflammatory therapy, but significant adverse effects ultimately limit their utility. Previous efforts to design glucocorticoid structures with an increased therapeutic window have focused on dissociating anti-inflammatory transcriptional repression from adverse effects primarily driven by transcriptional activation. An alternative to this medicinal chemistry approach is a systems biology based strategy that seeks to amplify selectively the anti-inflammatory activity of very low dose glucocorticoid in immune cells without modulating alternative cellular networks that mediate glucocorticoid toxicity.
The combination of prednisolone and the antithrombotic drug dipyridamole was profiled using in vitro and in vivo models of anti-inflammatory activity and glucocorticoid-induced adverse effects to demonstrate a dissociated activity profile.
The combination synergistically suppresses release of proinflammatory mediators, including tumour necrosis factor-α, IL-6, chemokine (C-C motif) ligand 5 (RANTES), matrix metalloproteinase-9, and others, from human peripheral blood mononuclear cells and mouse macrophages. In rat models of acute lipopolysaccharide-induced endotoxemia and delayed-type hypersensitivity, and in chronic models of collagen-induced and adjuvant-induced arthritis, the combination produced anti-inflammatory activity that required only a subtherapeutic dose of prednisolone. The immune-specific amplification of prednisolone anti-inflammatory activity by dipyridamole did not extend to glucocorticoid-mediated adverse effects, including corticosterone suppression or increased expression of tyrosine aminotransferase, in vivo after repeat dosing in rats. After 8 weeks of oral dosing in mice, treatment with the combination did not alter prednisolone-induced reduction in osteocalcin and mid-femur bone density, which are markers of steroid-induced osteoporosis. Additionally, amplification was not observed in the cellular network of corticotroph AtT-20/D16v-F2 cells in vitro, as measured by pro-opiomelanocortin expression and adrenocorticotropic hormone secretion.
These data suggest that the multi-target mechanism of low-dose prednisolone and dipyridamole creates a dissociated activity profile with an increased therapeutic window through cellular network selective amplification of glucocorticoid-mediated anti-inflammatory signaling.
Insulin resistance is a common finding in chronic kidney disease (CKD) and is manifested by mild fasting hyperglycemia and abnormal glucose tolerance testing. Circulating levels of glucocorticoids are high. In muscle, changes in the insulin signaling pathway occur. An increase in the regulatory p85 subunit of Class I phosphatidylinositol 3-Kinase enzyme leads to decreased activation of the downstream effector protein kinase B (Akt). Mechanisms promoting muscle proteolysis and atrophy are unleashed. The link of Akt to the ubiquitin proteasome pathway, a major degradation pathway in muscle, is discussed. Another factor associated with insulin resistance in CKD is angiotensin II (Ang II) which appears to induce its intracellular effects through inflammatory cytokines or reactive oxygen species. Skeletal muscle ATP is depleted and the ability of AMP-activated protein kinase (AMPK) to replenish energy stores is blocked. How this can be reversed is discussed. Interleukin-6 (IL-6) levels are elevated in CKD and impair insulin signaling at the level of IRS-1. With exercise, IL-6 levels are reduced; glucose uptake and utilization are increased. For patients with CKD, exercise may improve insulin signaling and build up muscle. Treatment strategies for preventing muscle atrophy are discussed.
Although widely prescribed for their potent antiinflammatory actions, glucocorticoid drugs (e.g., dexamethasone) cause undesirable side effects that are features of the metabolic syndrome, including hyperglycemia, fatty liver, insulin resistance, and type II diabetes. Liver x receptors (LXRs) are nuclear receptors that respond to cholesterol metabolites and regulate the expression of a subset of glucocorticoid target genes. Here, we show LXRβ is required to mediate many of the negative side effects of glucocorticoids. Mice lacking LXRβ (but not LXRα) were resistant to dexamethasone-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, but remained sensitive to dexamethasone-dependent repression of the immune system. In vivo, LXRα/β knockout mice demonstrated reduced dexamethasone-induced expression of the key hepatic gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK). In perfused liver and primary mouse hepatocytes, LXRβ was required for glucocorticoid-induced recruitment of the glucocorticoid receptor to the PEPCK promoter. These findings suggest a new avenue for the design of safer glucocorticoid drugs through a mechanism of selective glucocorticoid receptor transactivation.
Evidence demonstrates that exogenously administered nitric oxide (NO) can induce insulin resistance in skeletal muscle. We have investigated the modulatory effects of two NO donors, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) on the early events in insulin signaling in rat skeletal myocytes.
Skeletal muscle cells from 6–8 week old Sprague-Dawley rats were treated with SNAP or GSNO (25 ng/ml) in the presence or absence of glucose (25 mM) and insulin (100 nM). Cellular insulin receptor-β levels and tyrosine phosphorylation in IRS-1 were significantly reduced, while serine phosphorylation in IRS-1 was significantly increased in these cells, when compared to the insulin-stimulated control. Reversal to near normal levels was achieved using the NO scavenger, 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO).
These data suggest that NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus.