Michael D. Karlstad

Effects of eicosapentaenoic and γ-linolenic acid on lung permeability and alveolar macrophage eicosanoid synthesis in endotoxic rats

OBJECTIVES Proinflammatory eicosanoids (cyclooxgenase and lipoxygenase metabolites of arachidonic acid) released by alveolar macrophages play an important role in endotoxin-induced acute lung injury. We investigated the effect of prefeeding rats for 21 days with enteral diets that provided the anti-inflammatory fatty acids, eicosapentaenoic acid and gamma-linolenic acid (derived from fish oil and borage oil, respectively), as compared with an n-6 fatty acid-enriched diet (corn oil) on the following: a) lung microvascular protein permeability, arterial blood pressure, and platelet and white blood cells in a model of endotoxin-induced acute lung injury; b) alveolar macrophage prostaglandin and leukotriene synthesis; and c) liver and alveolar macrophage phospholipid fatty acid composition. DESIGN Prospective, randomized, controlled, double-blind study. SETTING Research laboratory at a university medical center. SUBJECTS Male Long-Evans rats, weighing 250 g. INTERVENTIONS Rats were randomized into four dietary treatment groups and fed nutritionally complete diets (300 kcal/kg/day), containing 55.2% of the total calories from fat with either 97% corn oil, 20% fish oil, 20% fish and 5% borage oil, or 20% fish and 20% borage oil for 21 days. On day 22, lung microvascular protein permeability, mean arterial pressure, and platelet and white blood cell counts were determined for 2 hrs after an intravenous injection of Salmonella enteritidis endotoxin (10 mg/kg). In a second group of prefed rats, the phospholipid fatty acid composition was determined in liver and alveolar macrophages. Alveolar macrophages were harvested by bronchoalveolar lavage and stimulated in vitro with a calcium ionophore (A23187), and the concentrations of leukotrienes B4 and B5, thromboxane A2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in a third group of prefed rats. MEASUREMENT AND MAIN RESULTS Lung permeability was greatest with corn oil and was significantly attenuated with 20% fish oil and 20% fish and 5% borage oil, and this effect approached significance with 20% fish and 20% borage oil (p = .06). The early and late hypotensive effects of endotoxin were attenuated with 20% fish oil, 20% fish and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Concentrations of leukotriene B4, prostaglandin E2, and thromboxane B2 released from A23187-stimulated alveolar macrophages were significantly lower with 20% fish oil and 20% fish and 20% borage oil, as compared with corn oil. The increase in lung microvascular protein permeability with 20% fish and 20% borage oil was not significantly different than the lung microvascular protein permeability that was found in animals receiving 20% fish oil (p = .20) and 20% fish and 5% borage oil (p = .31). Alveolar macrophage and liver phospholipid concentrations of arachidonic acid were lower, and the concentrations of eicosapentaenoic acid and docosahexaenic acid were higher, with 20% fish oil, and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Dihomo-gamma-linolenic acid, the desaturated and elongated intermediate of gamma-linolenic acid, was increased with 20% fish and 20% borage oil, as compared with 20% fish oil and 20% fish and 5% borage oil. CONCLUSIONS The severity of pulmonary microvascular protein permeability and the degree of hypotension were reduced with fish or fish and borage oil diets, as compared with corn oil, in endotoxic rats. The reduced synthesis of the proinflammatory arachidonic acid-derived mediators, leukotriene B4, thromboxane B2, and prostaglandin E2 from stimulated alveolar macrophages was indicative of a decrease in arachidonic acid and an increase in eicosapentaenoic acid and docosahexaenoic acid in cell membrane phospholipids.

DIETARY FISH OIL AND FISH AND BORAGE OIL SUPPRESS INTRAPULMONARY PROINFLAMMATORY EICOSANOID BIOSYNTHESIS AND ATTENUATE PULMONARY NEUTROPHIL ACCUMULATION IN ENDOTOXIC RATS

OBJECTIVE Proinflammatory eicosanoids and cytokines are important mediators of local inflammation in acute lung injury. We determined if enteral nutrition with anti-inflammatory fatty acids, eicosapentaenoic acid, and gamma-linolenic acid would reduce the intrapulmonary synthesis of proinflammatory eicosanoids and cytokines and pulmonary neutrophil accumulation in a rat model of acute lung injury. DESIGN Prospective, randomized, controlled, double-blind study. SETTING Research laboratory at a university medical center. SUBJECTS Male Long-Evans rats (250 g). INTERVENTIONS Rats were randomly assigned to three dietary treatment groups and fed nutritionally complete diets (300 kcal/kg/day) containing 55.2% of the total calories from fat with either 97% corn oil, 20% fish oil, or 20% fish and 20% borage oil for 21 days. On day 22, bronchoalveolar lavage was performed 2 hrs after an intravenous injection of Salmonella enteritidis endotoxin (10 mg/kg) or saline. Bronchoalveolar lavage fluid was analyzed for leukotriene B4, leukotriene C4/D4, thromboxane B2, prostaglandin E2, 6 keto-prostaglandin F1alpha, tumor necrosis factor (TNF)-alpha, and macrophage inflammatory protein-2 (MIP-2). Lung myeloperoxidase activity (a marker for neutrophil accumulation) and phospholipid fatty acid composition were also determined. MEASUREMENTS AND MAIN RESULTS Lung phospholipid concentrations of arachidonic acid were lower and the concentrations of eicosapentaenoic acid and docosahexaenoic acid were higher with fish oil and fish and borage oil as compared with corn oil. Dihomo-gamma-linolenic acid, the desaturated and elongated intermediate of gamma-linolenic acid, increased with fish and borage oil as compared with fish oil and corn oil. The levels of leukotriene B4, leukotriene C4/D4, 6-keto-prostaglandin F1alpha, and thromboxane B2 with corn oil were significantly increased with endotoxin as compared with saline. In contrast to the corn oil group, endotoxin did not significantly increase bronchoalveolar lavage levels of leukotriene B4, leukotriene C4/D4, and thromboxane B2 above those of saline-treated rats with fish oil and fish and borage oil. Lung myeloperoxidase activity was significantly increased in endotoxin-treated rats compared with those rats given saline in all dietary treatment groups. However, lung myeloperoxidase activity was significantly lower with either fish oil or fish and borage oil as compared with corn oil after endotoxin. Although endotoxin increased the levels of TNF-alpha and MIP-2 with all dietary treatment groups as compared with saline-treated rats, there were no significant differences in the levels of either cytokine between the dietary treatment groups. CONCLUSIONS These results indicate that dietary fish oil and fish and borage oil as compared with corn oil may ameliorate endotoxin-induced acute lung injury by suppressing the levels of proinflammatory eicosanoids (but not TNF-alpha or MIP-2) in bronchoalveolar lavage fluid and reducing pulmonary neutrophil accumulation.

Enhanced skeletal muscle and liver protein synthesis with structured lipid in enterally fed burned rats

We assessed the effects of total enteral nutrition with long-chain triacylglycerides (LCT), medium-chain triacylglycerides (MCT), or two structured lipids, modified dairy fat (MDF) and modified MCT (Captex 810B, Capital City Products, Columbus, OH), on protein and energy metabolism in hypermetabolic burned rats (25% to 30% body surface area). Male Sprague-Dawley rats (200 +/- 10 g) were continuously gastrostomy-fed isovolemic diets that provided 50 kcal/d, 2 g amino acids/d and 40% nonprotein calories as lipid for three days. Changes in body weight, nitrogen balance, serum albumin, indirect calorimetry, whole body leucine kinetics, and rectus muscle and liver protein kinetics were determined. Whole body leucine kinetics and tissue fractional protein synthetic rates (FSR, percent per day) were estimated using a four-hour constant intravenous infusion of L-[1-14C]leucine on day 3. The group of rats enterally fed MDF lost less body weight than the other groups (P less than or equal to .05). MDF and Captex 810B produced a positive and significantly greater (P less than or equal to .05) daily and cumulative nitrogen balance than either LCT or MCT. Oxygen consumption (P less than or equal to .05) and total energy expenditure (P less than or equal to .05) were elevated approximately 22% with MDF as compared with LCT or MCT. Rectus muscle FSR and absolute rate of protein synthesis were increased 19% with MDF (P less than or equal to .05) as compared with LCT or MCT.(ABSTRACT TRUNCATED AT 250 WORDS)

STUDIES IN SMALL BOWEL TRANSPLANTATION: Prevention of Graft-Versus-Host Disease With Preservation of Allograft Function by Donor Pretreatment With Antilymphocyte Serum

Donor pretreatment with antilymphocyte serum (ALS) effectively prevents graft-versus-host disease (GVHD) in a unidirectional (parent—to—F1 hybrid) rat small bowel transplantation model. ALS must be administered prior to or at the time of transplantation, and the intraperitoneal route is more effective than subcutaneous administration. Donor pretreatment with ALS uniformly prevents GVHD without impairing subsequent allograft function as measured by absorption of dietary energy and nitrogen, weight gain, and bowel morphology. These rodent studies suggest that ALS treatment of donors as well as recipients in small bowel transplantation may be a highly effective, simple, and easily applicable method to prevent or ameliorate GVHD in human small bowel transplantation.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing β-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1β markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and β-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1β to promote transcription. In addition, IL-1β induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1β induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1β-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic β-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Synergistic Expression of the CXCL10 Gene in Response to IL-1β and IFN-γ Involves NF-κB, Phosphorylation of STAT1 at Tyr701, and Acetylation of Histones H3 and H4

The CXCL10 gene encodes a peptide that chemoattracts a variety of leukocytes associated with type 1 and type 2 diabetes. The present study was undertaken to determine the molecular mechanisms required for expression of the CXCL10 gene in response to IL-1β and IFN-γ using rat islets and β cell lines. IL-1β induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1β plus IFN-γ was synergistic. Small interfering RNA–mediated suppression of NF-κB p65 markedly inhibited the ability of cytokines to induce the expression of the CXCL10 gene, whereas targeting STAT1 only diminished the synergy provided by IFN-γ. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-γ–controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr701. We further discovered that, although the Tyr701 phosphorylation site is inducible (within 15 min of IFN-γ exposure), the Ser727 site within STAT1 is constitutively phosphorylated. Thus, we generated single-mutant STAT1 Y701F and double-mutant STAT1 Y701F/S727A adenoviruses. Using these recombinant adenoviruses, we determined that overexpression of either the single- or double-mutant STAT1 decreased the IFN-γ–mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser727 phosphorylation was neither contingent on a functional Y701 site in β cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1β and IFN-γ to induce expression of the CXCL10 gene requires NF-κB, STAT1 phosphorylated at Tyr701, recruitment of coactivators, and acetylation of histones H3 and H4.

Methylprednisolone does not influence endotoxin translocation during cardiopulmonary bypass.

This study investigated whether the prophylactic administration of methylprednisolone sodium succinate (MPSS) could prevent an increase in plasma endotoxin levels during cardiac surgery with cardiopulmonary bypass. MPSS (1 g/patient) or saline was given intravenously with induction in the steroid (n = 6) and control (n = 7) groups, respectively. Blood samples were collected preinduction and postinduction, during and after cardiopulmonary bypass, and 1 and 24 hours postoperatively. Plasma endotoxin was determined by a chromogenic Limulus amebocyte lysate assay. There was an intraoperative increase in the level of plasma endotoxin that occurred primarily after initiation of cardiopulmonary bypass and removal of the aortic cross-clamp. Endotoxin at 1 and 24 hours postoperatively was lower than the peak intraoperative levels and approached the preinduction level in both groups. The pump prime and other administered fluids contained low levels of endotoxin that were at or below the preinduction or postinduction level of the patients. MPSS did not prevent or attenuate the degree of endotoxemia during cardiopulmonary bypass. The loss of normal gut mucosal barrier function during cardiopulmonary bypass may result in endotoxemia and/or bacterial translocation, either of which could initiate or contribute to postoperative complications.

Diazoxide enhances basal metabolic rate and fat oxidation in obese Zucker rats

Persistent suppression of hyperinsulinemia in genetically obese (fa/fa) Zucker rats by diazoxide (DZ) reduces food intake and weight gain; improves insulin sensitivity, glycemic control, and lipid profile; and enhances beta(3)-adrenergic function and lipolysis in adipose tissue. The aim of this study was to elucidate the effects of DZ on basal metabolic rate (BMR), fat oxidation, and adrenergic function of lean and obese Zucker rats. Diazoxide (150 mg/kg/d) or vehicle (control) was administered for 4 weeks in 7-week-old obese and lean Zucker rats (n = 8-9 per subgroup). Animals underwent indirect calorimetry, body composition analysis, and determination of uncoupling proteins (UCPs) messenger RNA (mRNA) in brown and white adipose tissues (BAT and WAT) and skeletal muscle (SM), beta(3)-adrenergic receptor (AR) mRNA in BAT and WAT, beta(2)-AR in SM as well as WAT, and SM adenylate cyclase (AC) activity at the completion of study. Diazoxide treatment decreased food intake, weight gain, and body fat in obese rats (P < .01). Although DZ treatment lowered fasting plasma glucose, insulin, leptin, adiponectin, and lipids in obese rats (P < .01), it increased adiponectin-leptin ratio (P < .01). Plasma adiponectin-leptin ratio was inversely correlated with fat mass in obese and lean rats (r = -0.86, P < .0001). Diazoxide treatment resulted in higher BMR and fat oxidation rate in obese compared with control animals (P < .01), without any effect in lean animals. Furthermore, plasma adiponectin was inversely correlated with BMR (-0.56, P < .001) and lipid oxidation rate (-0.61, P < .0005) and was positively correlated with nonprotein respiratory quotient (r = 0.41, P < .01) in obese and lean rats. This was associated with increased beta(3)-AR mRNA expression in BAT and WAT (P < .01), UCP-1 and UCP-3 in BAT and AC activity in WAT (P < .02), and AC activity in SM of DZ obese rats compared with controls (P < .01), without significant change in SM beta(2)-AR mRNA expressions. Diazoxide attenuation of hyperinsulinemia decreased the rate of weight gain but enhanced insulin sensitivity, BMR, and fat oxidation in obese rats. This was associated with increased receptor- and non-receptor-mediated adrenergic function in adipose and muscle tissues in obese rats, respectively. These metabolic changes in obese Zucker rats suggest that antiobesity effects of DZ appear to be not only through its anorectic effect, modification of disturbed insulin metabolism, and inhibition of lipogenesis, but also due to augmentation of adrenergic function, energy expenditure, and fat utilization.

Regulation of the CCL2 Gene in Pancreatic β-Cells by IL-1β and Glucocorticoids: Role of MKP-1

Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic β-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived β-cell lines. We discovered that activation of the CCL2 gene by IL-1β required the p65 subunit of NF-κB and was dependent on genomic response elements located in the −3.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1β was blocked by pharmacological inhibition of the IKKβ and p38 MAPK pathways. The IL-1β-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1β-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1β-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of β-cell function.

Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism

The proinflammatory cytokines IL-1β and IFN-γ decrease functional islet β-cell mass in part through the increased expression of specific genes, such as inducible nitric oxide synthase (iNOS). Dysregulated iNOS protein accumulation leads to overproduction of nitric oxide, which induces DNA damage, impairs β-cell function, and ultimately diminishes cellular viability. However, the transcriptional mechanisms underlying cytokine-mediated expression of the iNOS gene are not completely understood. Herein, we demonstrated that individual mutations within the proximal and distal nuclear factor-κB sites impaired cytokine-mediated transcriptional activation. Surprisingly, mutating IFN-γ-activated site (GAS) elements in the iNOS gene promoter, which are classically responsive to IFN-γ, modulated transcriptional sensitivity to IL-1β. Transcriptional sensitivity to IL-1β was increased by generation of a consensus GAS element and decreased correspondingly with 1 or 2 nucleotide divergences from the consensus sequence. The nuclear factor-κB subunits p65 and p50 bound to the κB response elements in an IL-1β-dependent manner. IL-1β also promoted binding of serine-phosphorylated signal transducer and activator of transcription-1 (STAT1) (Ser727) but not tyrosine-phosphorylated STAT1 (Tyr701) to GAS elements. However, phosphorylation at Tyr701 was required for IFN-γ to potentiate the IL-1β response. Furthermore, coactivator p300 and coactivator arginine methyltransferase were recruited to the iNOS gene promoter with concomitant displacement of the coactivator CREB-binding protein in cells exposed to IL-1β. Moreover, these coordinated changes in factor recruitment were associated with alterations in acetylation, methylation, and phosphorylation of histone proteins. We conclude that p65 and STAT1 cooperate to control iNOS gene transcription in response to proinflammatory cytokines by a coactivator exchange mechanism. This increase in transcription is also associated with signal-specific chromatin remodeling that leads to RNA polymerase II recruitment and phosphorylation.