Francis Kim

Toll-Like Receptor-4 Mediates Vascular Inflammation and Insulin Resistance in Diet-Induced Obesity

Vascular dysfunction is a major complication of metabolic disorders such as diabetes and obesity. The current studies were undertaken to determine whether inflammatory responses are activated in the vasculature of mice with diet-induced obesity, and if so, whether Toll-Like Receptor-4 (TLR4), a key mediator of innate immunity, contributes to these responses. Mice lacking TLR4 (TLR4−/−) and wild-type (WT) controls were fed either a low fat (LF) control diet or a diet high in saturated fat (HF) for 8 weeks. In response to HF feeding, both genotypes displayed similar increases of body weight, body fat content, and serum insulin and free fatty acid (FFA) levels compared with mice on a LF diet. In lysates of thoracic aorta from WT mice maintained on a HF diet, markers of vascular inflammation both upstream (IKK&bgr; activity) and downstream of the transcriptional regulator, NF-&kgr;B (ICAM protein and IL-6 mRNA expression), were increased and this effect was associated with cellular insulin resistance and impaired insulin stimulation of eNOS. In contrast, vascular inflammation and impaired insulin responsiveness were not evident in aortic samples taken from TLR4−/− mice fed the same HF diet, despite comparable increases of body fat mass. Incubation of either aortic explants from WT mice or cultured human microvascular endothelial cells with the saturated FFA, palmitate (100 &mgr;mol/L), similarly activated IKK&bgr;, inhibited insulin signal transduction and blocked insulin-stimulated NO production. Each of these effects was subsequently shown to be dependent on both TLR4 and NF-&kgr;B activation. These findings identify the TLR4 signaling pathway as a key mediator of the deleterious effects of palmitate on endothelial NO signaling, and are the first to document a key role for TLR4 in the mechanism whereby diet-induced obesity induces vascular inflammation and insulin resistance.

Pilot Randomized Clinical Trial of Prehospital Induction of Mild Hypothermia in Out-of-Hospital Cardiac Arrest Patients With a Rapid Infusion of 4°C Normal Saline

Background— Although delayed hospital cooling has been demonstrated to improve outcome after cardiac arrest, in-field cooling started immediately after the return of spontaneous circulation may be more beneficial. The aims of the present pilot study were to assess the feasibility, safety, and effectiveness of in-field cooling. Methods and Results— We determined the effect on esophageal temperature, before hospital arrival, of infusing up to 2 L of 4°C normal saline as soon as possible after resuscitation from out-of-hospital cardiac arrest. A total of 125 such patients were randomized to receive standard care with or without intravenous cooling. Of the 63 patients randomized to cooling, 49 (78%) received an infusion of 500 to 2000 mL of 4°C normal saline before hospital arrival. These 63 patients experienced a mean temperature decrease of 1.24±1°C with a hospital arrival temperature of 34.7°C, whereas the 62 patients not randomized to cooling experienced a mean temperature increase of 0.10±0.94°C (P<0.0001) with a hospital arrival temperature of 35.7°C. In-field cooling was not associated with adverse consequences in terms of blood pressure, heart rate, arterial oxygenation, evidence for pulmonary edema on initial chest x-ray, or rearrest. Secondary end points of awakening and discharged alive from hospital trended toward improvement in ventricular fibrillation patients randomized to in-field cooling. Conclusions— These pilot data suggest that infusion of up to 2 L of 4°C normal saline in the field is feasible, safe, and effective in lowering temperature. We propose that the effect of this cooling method on neurological outcome after cardiac arrest be studied in larger numbers of patients, especially those whose initial rhythm is ventricular fibrillation.

Effect of prehospital induction of mild hypothermia on survival and neurological status among adults with cardiac arrest: a randomized clinical trial.

IMPORTANCE Hospital cooling improves outcome after cardiac arrest, but prehospital cooling immediately after return of spontaneous circulation may result in better outcomes. OBJECTIVE To determine whether prehospital cooling improves outcomes after resuscitation from cardiac arrest in patients with ventricular fibrillation (VF) and without VF. DESIGN, SETTING, AND PARTICIPANTS A randomized clinical trial that assigned adults with prehospital cardiac arrest to standard care with or without prehospital cooling, accomplished by infusing up to 2 L of 4°C normal saline as soon as possible following return of spontaneous circulation. Adults in King County, Washington, with prehospital cardiac arrest and resuscitated by paramedics were eligible and 1359 patients (583 with VF and 776 without VF) were randomized between December 15, 2007, and December 7, 2012. Patient follow-up was completed by May 1, 2013. Nearly all of the patients resuscitated from VF and admitted to the hospital received hospital cooling regardless of their randomization. MAIN OUTCOMES AND MEASURES The primary outcomes were survival to hospital discharge and neurological status at discharge. RESULTS The intervention decreased mean core temperature by 1.20°C (95% CI, -1.33°C to -1.07°C) in patients with VF and by 1.30°C (95% CI, -1.40°C to -1.20°C) in patients without VF by hospital arrival and reduced the time to achieve a temperature of less than 34°C by about 1 hour compared with the control group. However, survival to hospital discharge was similar among the intervention and control groups among patients with VF (62.7% [95% CI, 57.0%-68.0%] vs 64.3% [95% CI, 58.6%-69.5%], respectively; P = .69) and among patients without VF (19.2% [95% CI, 15.6%-23.4%] vs 16.3% [95% CI, 12.9%-20.4%], respectively; P = .30). The intervention was also not associated with improved neurological status of full recovery or mild impairment at discharge for either patients with VF (57.5% [95% CI, 51.8%-63.1%] of cases had full recovery or mild impairment vs 61.9% [95% CI, 56.2%-67.2%] of controls; P = .69) or those without VF (14.4% [95% CI, 11.3%-18.2%] of cases vs 13.4% [95% CI,10.4%-17.2%] of controls; P = .30). Overall, the intervention group experienced rearrest in the field more than the control group (26% [95% CI, 22%-29%] vs 21% [95% CI, 18%-24%], respectively; P = .008), as well as increased diuretic use and pulmonary edema on first chest x-ray, which resolved within 24 hours after admission. CONCLUSION AND RELEVANCE Although use of prehospital cooling reduced core temperature by hospital arrival and reduced the time to reach a temperature of 34°C, it did not improve survival or neurological status among patients resuscitated from prehospital VF or those without VF. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00391469.

Free Fatty Acid Impairment of Nitric Oxide Production in Endothelial Cells Is Mediated by IKKβ

Objective—Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. Methods and Results—We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 &mgr;mol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKKβ activity, which regulates activation of NF- &kgr;B, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKKβ activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKKβ blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKKβ recapitulates the effect of FFA on insulin-dependent NO production. Conclusions—Elevated levels of common FFA found in human serum activate IKKβ in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction.

Vascular Inflammation, Insulin Resistance, and Reduced Nitric Oxide Production Precede the Onset of Peripheral Insulin Resistance

Objectives—Obesity causes inflammation and insulin resistance in the vasculature as well as in tissues involved in glucose metabolism such as liver, muscle, and adipose tissue. To investigate the relative susceptibility of vascular tissue to these effects, we determined the time course over which inflammation and insulin resistance develops in various tissues of mice with diet-induced obesity (DIO) and compared these tissue-based responses to changes in circulating inflammatory markers. Methods and Results—Adult male C57BL/6 mice were fed either a control low-fat diet (LF; 10% saturated fat) or a high-fat diet (HF, 60% saturated fat) for durations ranging between 1 to 14 weeks. Cellular inflammation and insulin resistance were assessed by measuring phospho-I&kgr;Bα and insulin-induced phosphorylation of Akt, respectively, in extracts of thoracic aorta, liver, skeletal muscle, and visceral fat. As expected, HF feeding induced rapid increases of body weight, fat mass, and fasting insulin levels compared to controls, each of which achieved statistical significance within 4 weeks. Whereas plasma markers of inflammation became elevated relatively late in the course of DIO (eg, serum amyloid A [SAA], by Week 14), levels of phospho-I&kgr;Bα in aortic lysates were elevated by 2-fold within the first week. The early onset of vascular inflammation was accompanied by biochemical evidence of both endothelial dysfunction (reduced nitric oxide production; induction of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and insulin resistance (impaired insulin-induced phosphorylation of Akt and eNOS). Although inflammation and insulin resistance were also detected in skeletal muscle and liver of HF-fed animals, these responses were observed much later (between 4 and 8 weeks of HF feeding), and they were not detected in visceral adipose tissue until 14 weeks. Conclusions—During obesity induced by HF feeding, inflammation and insulin resistance develop in the vasculature well before these responses are detected in muscle, liver, or adipose tissue. This observation suggests that the vasculature is more susceptible than other tissues to the deleterious effects of nutrient overload.

Pilot Study of Rapid Infusion of 2 L of 4°C Normal Saline for Induction of Mild Hypothermia in Hospitalized, Comatose Survivors of Out-of-Hospital Cardiac Arrest

Background—Recent clinical studies have demonstrated that mild hypothermia (32°C to 34°C) induced by surface cooling improves neurological outcome after resuscitation from out-of-hospital cardiac arrest. Results from animal models suggest that the effectiveness of mild hypothermia could be improved if initiated as soon as possible after return of spontaneous circulation. Infusion of cold, intravenous fluid has been proposed as a safe, effective, and inexpensive technique to induce mild hypothermia after cardiac arrest. Methods and Results—In 17 hospitalized survivors of out-of-hospital cardiac arrest, we determined the effect on temperature and hemodynamics of infusing 2 L of 4°C cold, normal saline during 20 to 30 minutes into a peripheral vein with a high-pressure bag. Data on vital signs, electrolytes, arterial blood gases, and coagulation were collected before and after fluid infusion. Cardiac function was assessed by transthoracic echocardiography before fluid administration and 1 hour after infusion. Passive (fans, leaving patient uncovered) or active (cooling blankets, neuromuscular blockade) cooling measures were used to maintain mild hypothermia for 24 hours. Infusion of 2 L of 4°C cold, normal saline resulted in a mean temperature drop of 1.4°C 30 minutes after the initiation of infusion. Rapid infusion of fluid was not associated with clinically important changes in vital signs, electrolytes, arterial blood gases, or coagulation parameters. The initial mean ejection fraction was 34%, and fluid infusion did not affect ejection fraction or increase central venous pressure, pulmonary pressures, or left atrial filling pressures as assessed by echocardiography. Passive measures were ineffective in maintaining hypothermia compared with active measures. Conclusions—Infusion of 2 L of 4°C cold, normal saline is safe and effective in rapidly lowering body temperature in survivors of out-of-hospital cardiac arrest.

Hypothalamic leptin signaling regulates hepatic insulin sensitivity via a neurocircuit involving the vagus nerve.

Recent evidence suggests that hormones such as insulin and leptin act in the hypothalamus to regulate energy balance and glucose metabolism. Here we show that in leptin receptor-deficient Koletsky (fa(k)/fa(k)) rats, adenovirally induced expression of leptin receptors in the area of the hypothalamic arcuate nucleus improved peripheral insulin sensitivity via enhanced suppression of hepatic glucose production, with no change of insulin-stimulated glucose uptake or disposal. This effect was associated with increased insulin signal transduction via phosphatidylinositol-3-OH kinase (as measured by pY-insulin receptor substrate-1 and pS-PKB/Akt) in liver, but not skeletal muscle, and with reduced hepatic expression of the gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate kinase. Moreover, the beneficial effects of hypothalamic leptin signaling on hepatic insulin sensitivity were blocked by selective hepatic vagotomy. We conclude that hypothalamic leptin action increases peripheral insulin sensitivity primarily via effects on the liver and that the mechanism underlying this effect is dependent on the hepatic branch of the vagus nerve.

Active surface cooling protocol to induce mild therapeutic hypothermia after out-of-hospital cardiac arrest: a retrospective before-and-after comparison in a single hospital.

Objective: To evaluate whether implementation of a therapeutic hypothermia protocol on arrival in a community hospital improved survival and neurologic outcomes in patients initially found to have ventricular fibrillation, pulseless electrical activity, or asystole, and then successfully resuscitated from out-of-hospital cardiac arrest. Design: A retrospective study of patients who presented after implementation of a therapeutic hypothermia protocol compared with those who presented before the protocol was implemented. Setting: Harborview Medical Center, Seattle, WA. Patients: A total of 491 consecutive adults with out-of-hospital, nontraumatic cardiac arrest who presented between January 1, 2000 and December 31, 2004. Interventions: An active cooling therapeutic hypothermia protocol, using ice packs, cooling blankets, or cooling pads to achieve a temperature of 32°C to 34°C was initiated on November 18, 2002 for unconscious patients resuscitated from cardiac arrest. Measurements and Main Results: Demographics and outcomes were obtained from medical records and an emergency medical database. The primary outcomes were survival and favorable neurologic outcome at discharge associated with the therapeutic hypothermia protocol. An adjusted analysis was performed, using a multivariate regression. During the therapeutic hypothermia period, 204 patients were brought to the emergency department; of these 204 patients, 132 (65%) ultimately achieved temperatures of <34°C. Of the 72 patients who did not achieve goal temperatures: 40 (20%) died in the emergency department or shortly after being admitted to the hospital, 15 (7%) regained consciousness, four (2%) had contraindications, 13 (6%) had temperature increase or did not have documented use of the therapeutic hypothermia protocol. In the prior period, none of the 287 patients received active cooling. Patients admitted in the therapeutic hypothermia period had a mean esophageal temperature of 34.1°C during the first 12 hrs compared with 35.2°C in the pretherapeutic hypothermia period (p < .01). Survival to hospital discharge improved in the therapeutic hypothermia period in patients with an initial rhythm of ventricular fibrillation (odds ratio, 1.88, 95% confidence interval, 1.03–3.45), however not in patients with nonventricular fibrillation (odds ratio, 1.17, 95% confidence interval, 0.66–2.05). In adjusted analysis, ventricular fibrillation patients during the therapeutic hypothermia period trended toward improved survival (odds ratio, 1.71, 95% confidence interval, 0.85–3.46) and had favorable neurologic outcome (odds ratio, 2.62, 95% confidence interval, 1.1–6.27) compared with the earlier period. This benefit was not observed in patients whose initial rhythm was pulseless electrical activity or asystole. Conclusions: The therapeutic hypothermia period was associated with a significant improvement in neurologic outcomes in patients whose initial rhythm was ventricular fibrillation, but not in patients with other rhythms.

Recent Advances in Obesity-Induced Inflammation and Insulin Resistance

It has been demonstrated in rodents and humans that chronic inflammation characterized by macrophage infiltration occurs mainly in adipose tissue or liver during obesity, in which activation of immune cells is closely associated with insulin sensitivity. Macrophages can be classified as classically activated (M1) macrophages that support microbicidal activity or alternatively activated (M2) macrophages that support allergic and antiparasitic responses. In the context of insulin action, M2 macrophages sustain insulin sensitivity by secreting IL-4 and IL-10, while M1 macrophages induce insulin resistance through the secretion of proinflammatory cytokines, such as TNFα. Polarization of M1/M2 is controlled by various dynamic functions of other immune cells. It has been demonstrated that, in a lean state, TH2 cells, Treg cells, natural killer T cells, or eosinophils contribute to the M2 activation of macrophages by secreting IL-4 or IL-10. In contrast, obesity causes alteration of the constituent immune cells, in which TH1 cells, B cells, neutrophils, or mast cells induce M1 activation of macrophages by the elevated secretion of TNFα and IFNγ. Increased secretion of TNFα and free fatty acids from hypertrophied adipocytes also contributes to the M1 activation of macrophages. Since obesity-induced insulin resistance is established by macrophage infiltration and the activation of immune cells inside tissues, identification of the factors that regulate accumulation and the intracellular signaling cascades that define polarization of M1/M2 would be indispensable. Regulation of these factors would lead to the pharmacological inhibition of obesity-induced insulin resistance. In this review, we introduce molecular mechanisms relevant to the pathophysiology and review the most recent studies of clinical applications targeting chronic inflammation.

Distribution of insulin receptor substrate-2 in brain areas involved in energy homeostasis.

Body weight regulation depends on neuronal signaling by adiposity-related hormones such as insulin and leptin. Activation of receptors for these hormones induces cell signaling via the insulin receptor substrate/phosphatidylinositol 3-kinase (IRS-PI3K) pathway, and growing evidence from knockout models implicates IRS-2 as a key component of this signal transduction mechanism. As a first step towards the identification of brain areas that utilize IRS-PI3K signaling in the control of energy homeostasis, we used immunohistochemical techniques to investigate the neuronal distribution of IRS-2 protein in rat brain. In the hypothalamus, strong IRS-2 staining was detected chiefly in the arcuate (ARC), ventromedial (VMN) nucleus and parvocellular paraventricular nucleus (PVN). Within the ARC, IRS-2 was co-localized with alpha melanocyte stimulating hormone (alpha-MSH) as well as neuropeptide Y (NPY). In the hindbrain, IRS-2 staining was detected in the area postrema (AP), medial nucleus of the solitary tract (mNTS), dorsal motor nucleus of the vagus nerve (DMV) and the hypoglossal nucleus (HN). Co-localization studies in the mNTS demonstrated the presence of IRS-2 in catecholamine neurons. IRS-2 protein was also found in the ventral tegmental area (VTA), an important area for reward perception, and was detected in dopamine neurons in this brain area. In summary, neurons containing IRS-2 immunoreactivity were identified in forebrain, midbrain and hindbrain areas and in cell types that are crucial for the control of food intake and autonomic function. An improved understanding of mechanisms underlying normal and abnormal energy homeostasis may be gained by analysis of the role played by signaling through IRS-2 in these brain areas.