Phosphorylation status of fetuin-A is critical for inhibition of insulin action and is correlated with obesity and insulin resistance.

Fetuin-A (Fet-A), a hepatokine associated with insulin resistance, obesity, and incident type 2 diabetes, is shown to exist in both phosphorylated and dephosphorylated forms in circulation. However, studies on fetuin-A phosphorylation status in insulin-resistant conditions and its functional significance are limited. We demonstrate that serum phosphofetuin-A (Ser312) levels were significantly elevated in high-fat diet-induced obese mice, insulin-resistant Zucker diabetic fatty rats, and in individuals with obesity who are insulin resistant. Unlike serum total fetuin-A, serum phosphofetuin-A was associated with body weight, insulin, and markers of insulin resistance. To characterize potential mechanisms, fetuin-A was purified from Hep3B human hepatoma cells. Hep3B Fet-A was phosphorylated (Ser312) and inhibited insulin-stimulated glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Furthermore, single (Ser312Ala) and double (Ser312Ala + Ser120Ala) phosphorylation-defective Fet-A mutants were without effect on glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Together, our studies demonstrate that phosphorylation status of Fet-A (Ser312) is associated with obesity and insulin resistance and raise the possibility that Fet-A phosphorylation may play a role in regulation of insulin action.

Management of severe blunt liver injuries by applying the damage control strategies with packing-oriented surgery: experiences at a single institution in Korea.

BACKGROUND/AIMS: This study was conducted to investigate effective management strategies for patients with severe blunt liver injuries. METHODOLOGY: Treatment methods and outcomes of 77 patients with grade IV-V damage among patients with liver injury managed between 2009 and 2013 were investigated. RESULTS: Of the 77 patients, 32 were managed surgically. Packing was performed in 29 of these patients, while 26 also underwent liver surgery to maximize the hemostatic effect of packing. All 32 underwent temporary abdominal closure, and the mean amount of blood products used in the first 24 hours after admission included packed red blood cell, 13.3 units; fresh frozen plasma, 12.4 units; and platelets, 12.2 units, very close to 1:1:1. A total of 9 of 77 (11.7%) patients and 8 of 32 who underwent the operation died (operative mortality rate, 25%). Liver-related uncontrolled hemorrhage contributing to death occurred in four patients (12.5%). CONCLUSIONS: Although nonoperative management can first be pursued if the patient's condition allows for it, hemodynamic instability and evidence of peritonitis requires surgical management. Surgical management should abide by the damage control surgery principles that focus on packing to minimize surgical time, followed by aggressive critical care according to damage control resuscitation.

Carnitine palmitoyltransferase-1b deficiency aggravates pressure overload-induced cardiac hypertrophy caused by lipotoxicity.

BACKGROUND: Carnitine palmitoyltransferase-1 (CPT1) is a rate-limiting step of mitochondrial ß-oxidation by controlling the mitochondrial uptake of long-chain acyl-CoAs. The muscle isoform, CPT1b, is the predominant isoform expressed in the heart. It has been suggested that inhibiting CPT1 activity by specific CPT1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure. However, clinical and animal studies have shown mixed results, thereby creating concerns about the safety of this class of drugs. Preclinical studies using genetically modified animal models should provide a better understanding of targeting CPT1 to evaluate it as a safe and effective therapeutic approach. METHODS AND RESULTS: Heterozygous CPT1b knockout (CPT1b(+/-)) mice were subjected to transverse aorta constriction-induced pressure overload. These mice showed overtly normal cardiac structure/function under the basal condition. Under a severe pressure-overload condition induced by 2 weeks of transverse aorta constriction, CPT1b(+/-) mice were susceptible to premature death with congestive heart failure. Under a milder pressure-overload condition, CPT1b(+/-) mice exhibited exacerbated cardiac hypertrophy and remodeling compared with wild-type littermates. There were more pronounced impairments of cardiac contraction with greater eccentric cardiac hypertrophy in CPT1b(+/-) mice than in control mice. Moreover, the CPT1b(+/-) heart exhibited exacerbated mitochondrial abnormalities and myocardial lipid accumulation with elevated triglycerides and ceramide content, leading to greater cardiomyocyte apoptosis. CONCLUSIONS: CPT1b deficiency can cause lipotoxicity in the heart under pathological stress, leading to exacerbation of cardiac pathology. Therefore, caution should be exercised in the clinical use of CPT1 inhibitors.

An early, reversible cholesterolgenic etiology of diet-induced insulin resistance.

OBJECTIVE: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. METHODS: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. RESULTS: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. CONCLUSIONS: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.

Novel Roles for the Transcriptional Repressor E4BP4 in Both Cardiac Physiology and Pathophysiology.

Circadian clocks temporally orchestrate biological processes critical for cellular/organ function. For example, the cardiomyocyte circadian clock modulates cardiac metabolism, signaling, and electrophysiology over the course of the day, such that, disruption of the clock leads to age-onset cardiomyopathy (through unknown mechanisms). Here, we report that genetic disruption of the cardiomyocyte clock results in chronic induction of the transcriptional repressor E4BP4. Importantly, E4BP4 deletion prevents age-onset cardiomyopathy following clock disruption. These studies also indicate that E4BP4 regulates both cardiac metabolism (eg, fatty acid oxidation) and electrophysiology (eg, QT interval). Collectively, these studies reveal that E4BP4 is a novel regulator of both cardiac physiology and pathophysiology.

Hepatic mTORC2 Signaling Facilitates Acute Glucagon Receptor Enhancement of Insulin-Stimulated Glucose Homeostasis in Mice.

Long-term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We used a liver-specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin receptor (INSR) and GCGR cross talk. RictorΔLiver mice were characterized by impaired AKT signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/ßSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell-autonomous cross talk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action.

Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58.

Chlamydia pecorum is an obligate intracellular bacterial pathogen that causes diverse disease in a wide variety of economically important mammals. We report the finished complete genome sequence of C. pecorum E58, the type strain for the species.

Serum fetuin-A and Ser312 phosphorylated fetuin-A responses and markers of insulin sensitivity after a single bout of moderate intensity exercise.

Fetuin-A (Fet-A), secreted by the liver and adipose tissue, inhibits insulin receptor tyrosine kinase activity and modulates insulin action. Numerous studies have shown association of elevated serum Fet-A concentrations with obesity, non-alcoholic fatty liver disease, and type 2 diabetes. Both moderate body weight loss (5%-10%) and significant body weight loss have been shown to decrease serum Fet-A and improve insulin sensitivity. Currently, there are no studies examining the effects of a single bout of exercise on serum Fet-A or Ser312-pFet-A (pFet-A) responses. We hypothesized that a single bout of moderate-intensity exercise will lower serum Fet-A and that these changes will be associated with an improvement in insulin sensitivity. Thirty-one individuals with obesity and 11 individuals with normal body weight were recruited. Participants underwent a single bout of treadmill walking, expending 500 kcal at 60%-70% VO2max . Oral glucose tolerance tests (OGTT) were administered before the single bout of exercise (Pre Ex) and 24 h after exercise (24h Post Ex). In individuals with obesity, we observed a transient elevation of serum Fet-A concentrations, but not pFet-A, immediately after exercise (Post Ex). Further, a single bout of exercise decreased glucoseAUC , insulinAUC , and insulin resistance index in individuals with obesity. Consistent with this improvement in insulin sensitivity, we observed that Fet-AAUC , pFet-AAUC , 2 h pFet-A, and 2 h pFet-A/Fet-A were significantly lower following a single bout of exercise. Further, reductions in serum Fet-AAUC 24h Post Ex were correlated with a reduction in insulin resistance index. Together, this suggests that alterations in serum Fet-A following a single bout of moderate-intensity endurance exercise may play a role in the improvement of insulin sensitivity. CLINICAL TRIAL REGISTRATION: NCT03478046; https://clinicaltrials.gov/ct2/show/NCT03478046.

The transcriptional co-regulator LDB1 is required for brown adipose function.

OBJECTIVE: Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. METHODS: LDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the ß3-adrenergic receptor (ß3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. RESULTS: Reducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. CONCLUSIONS: Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

Glucagon receptor signaling regulates weight loss via central KLB receptor complexes.

Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon's weight loss effects. FGF21 signaling requires an obligate coreceptor (ß-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.

Sam68 promotes hepatic gluconeogenesis via CRTC2.

Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.

Metformin reduces saturated fatty acid-induced lipid accumulation and inflammatory response by restoration of autophagic flux in endothelial cells.

Autophagy, an integral part of the waste recycling process, plays an important role in cellular physiology and pathophysiology. Impaired autophagic flux causes ectopic lipid deposition, which is defined as the accumulation of lipids in non-adipose tissue. Ectopic lipid accumulation is observed in patients with cardiometabolic syndrome, including obesity, diabetes, insulin resistance, and cardiovascular complications. Metformin is the first line of treatment for type 2 diabetes, and one of the underlying mechanisms for the anti-diabetic effect of metformin is mediated by the stimulation of AMP-activated protein kinase (AMPK). Because the activation of AMPK is crucial for the initiation of autophagy, we hypothesize that metformin reduces the accumulation of lipid droplets by increasing autophagic flux in vascular endothelial cells. Incubation of vascular endothelial cells with saturated fatty acid (SFA) increased the accumulation of lipid droplets and impaired autophagic flux. We observed that the accumulation of lipid droplets was reduced, and the autophagic flux was enhanced by treatment with metformin. The knock-down of AMPKα by using siRNA blunted the effect of metformin. Furthermore, treatment with SFA or inhibition of autophagy increased leukocyte adhesion, whereas treatment with metformin decreased the SFA-induced leukocyte adhesion. The results suggest a novel mechanism by which metformin protects vascular endothelium from SFA-induced ectopic lipid accumulation and pro-inflammatory responses. In conclusion, improving autophagic flux may be a therapeutic strategy to protect endothelial function from dyslipidemia and diabetic complications.

Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression.

Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.

Alterations of Serum Ser312-Phosphorylated Fetuin-A from Exercise-Induced Moderate Body Weight Loss in Individuals with Obesity.

OBJECTIVE: Phosphorylated fetuin-A (pFet-A) inhibits insulin action and has been shown to be associated with obesity and insulin resistance. The objective of this cohort study was to assess the effect of incremental body weight loss on alterations in serum pFet-A and indexes of insulin sensitivity. METHODS: A total of 16 men with obesity attained a targeted weight loss of 8% to 10% of their initial body weight by achieving an energy expenditure/deficit of 2,000 to 2,500 kcal/wk. Anthropometric assessments and blood samples were obtained every 4 weeks. Weight loss was calculated and partitioned as 2% to 4%, 4% to 6%, 6% to 8%, and 8% to 10% compared with initial body weight. RESULTS: Targeted body weight loss of 8% to 10% decreased serum pFet-A, pFet-A:Fet-A ratio, fasting insulin, log(homeostasis model assessment of insulin resistance), quantitative insulin sensitivity check index, adipose insulin resistance, and insulin resistance index significantly. Percent changes in serum pFet-A were associated with percent changes in indexes of insulin sensitivity. Unlike insulin sensitivity indexes, which were altered starting with 6% to 8% weight loss, serum pFet-A levels were significantly decreased by 19.6% starting with 2% to 4% weight loss and decreased by 25.6%, 36.8%, and 42.3% with 4% to 6%, 6% to 8%, and 8% to 10% weight loss, respectively. CONCLUSIONS: This study reports for the first time that the insulin-sensitizing effects of moderate weight loss are associated with a reduction in serum pFet-A levels.

A Small Molecule, UAB126, Reverses Diet-Induced Obesity and its Associated Metabolic Disorders.

Targeting retinoid X receptor (RXR) has been proposed as one of the therapeutic strategies to treat individuals with metabolic syndrome, as RXR heterodimerizes with multiple nuclear receptors that regulate genes involved in metabolism. Despite numerous efforts, RXR ligands (rexinoids) have not been approved for clinical trials to treat metabolic syndrome due to the serious side effects such as hypertriglyceridemia and altered thyroid hormone axis. In this study, we demonstrate a novel rexinoid-like small molecule, UAB126, which has positive effects on metabolic syndrome without the known side effects of potent rexinoids. Oral administration of UAB126 ameliorated obesity, insulin resistance, hepatic steatosis, and hyperlipidemia without changes in food intake, physical activity, and thyroid hormone levels. RNA-sequencing analysis revealed that UAB126 regulates the expression of genes in the liver that are modulated by several nuclear receptors, including peroxisome proliferator-activated receptor α and/or liver X receptor in conjunction with RXR. Furthermore, UAB126 not only prevented but also reversed obesity-associated metabolic disorders. The results suggest that optimized modulation of RXR may be a promising strategy to treat metabolic disorders without side effects. Thus, the current study reveals that UAB126 could be an attractive therapy to treat individuals with obesity and its comorbidities.

Curcumin activates AMPK and suppresses gluconeogenic gene expression in hepatoma cells.

Curcumin, the bioactive component of curry spice turmeric, and its related structures possess potent anti-oxidant and anti-inflammatory properties. Several lines of evidence suggest that curcumin may play a beneficial role in animal models of diabetes, both by lowering blood glucose levels and by ameliorating the long-term complications of diabetes. However, current understanding of the mechanism of curcumin action is rudimentary and is limited to its anti-oxidant and anti-inflammatory effects. In this study we examine potential anti-diabetic mechanisms of curcumin, curcumin C3 complex), and tetrahydrocurcuminoids (THC). Curcuminoids did not exert a direct effect on receptor tyrosine kinase activity, 2-deoxy glucose uptake in L6-GLUT4myc cells, or intestinal glucose metabolism measured by DPP4/alpha-glucosidase inhibitory activity. We demonstrate that curcuminoids effectively suppressed dexamethasone-induced phosphoenol pyruvate carboxy kinase (PEPCK) and glucose6-phosphatase (G6Pase) in H4IIE rat hepatoma and Hep3B human hepatoma cells. Furthermore, curcuminoids increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase (ACC) in H4IIE and Hep3B cells with 400 times (curcumin) to 100,000 times (THC) the potency of metformin. These results suggest that AMPK mediated suppression of hepatic gluconeogenesis may be a potential mechanism mediating glucose-lowering effects of curcuminoids.

Extended-release niacin decreases serum fetuin-A concentrations in individuals with metabolic syndrome.

BACKGROUND: Fetuin-A, a liver-secreted phosphoprotein and physiological inhibitor of insulin receptor tyrosine kinase, is associated with insulin resistance, metabolic syndrome (MetS), and an increased risk for type 2 diabetes. However, studies on the modulation of circulating levels of fetuin-A are limited. The goal of this study was to determine the effect of niacin administration on serum total- and phosphorylated fetuin-A (phosphofetuin-A) concentrations in individuals with MetS and correlate with changes in serum lipids, insulin sensitivity, and markers of inflammation. METHODS: Fifteen sedentary, obese, male participants, who met the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria for MetS, were treated with extended-release niacin (Niaspan) for 6 weeks. Blood samples were obtained before and after treatment with niacin. RESULTS: Serum fetuin-A and phosphofetuin-A concentrations were decreased following niacin administration (p < 0.005). Changes in fetuin-A concentrations were correlated with changes in triglyceride (r = 0.62, p = 0.01) and C-reactive protein (CRP) concentrations (r = 0.58, p < 0.05) after niacin treatment. Changes in high-density lipoproteins (HDL)-cholesterol following niacin intervention were negatively correlated with changes in serum fetuin-A (p < 0.05) and phosphofetuin-A concentrations (p < 0.05). Serum cortisol levels were significantly elevated after niacin administration. CONCLUSIONS: Niacin treatment lowers serum total- and phosphofetuin-A concentrations in individuals with MetS, and these changes correlate with the beneficial changes in serum lipids. Because niacin is known to induce insulin resistance, these findings suggest that fetuin-A may not be a mediator of niacin-induced insulin resistance but it may blunt the insulin resistance induced by niacin by decreasing its circulating concentrations.

Imaging systems for westerns: chemiluminescence vs. infrared detection.

Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging. Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum. Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal. A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.

Temporal delay of peak T-cell immunity determines Chlamydia pneumoniae pulmonary disease in mice.

Severe chlamydial disease typically occurs after previous infections and results from a hypersensitivity response that is also required for chlamydial elimination. Here, we quantitatively dissected the immune and disease responses to repeated Chlamydia pneumoniae lung infection by multivariate modeling with four dichotomous effects: mouse strain (A/J or C57BL/6), dietary protein content (14% protein and 0.3% L-cysteine-0.9% L-arginine, or 24% protein and 0.5% L-cysteine-2.0% L-arginine), dietary antioxidant content (90 IU alpha-tocopherol/kg body weight versus 450 IU alpha-tocopherol/kg and 0.1% g L-ascorbate), and time course (3 or 10 days postinfection). Following intranasal C. pneumoniae challenge, C57BL/6 mice on a low-protein/low-antioxidant diet, but not C57BL/6 mice on other diets or A/J mice, exhibited profoundly suppressed early lung inflammatory and pan-T-cell (CD3delta(+)) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severe C. pneumoniae disease as being a delayed-type hypersensitivity (DTH) response with increased lung macrophage and Th1 cell marker transcripts, increased Th1:Th2 ratios, and Th1 cytokine-driven inflammation. Results from functional analyses by DTH, enzyme-linked immunospot, and immunohistofluorescence assays were consistent with the results obtained by transcript analysis. Thus, chlamydial disease after secondary infection is a temporal dysregulation of the T-cell response characterized by a profoundly delayed T-helper cell response that results in a failure to eliminate the pathogen and provokes later pathological Th1 inflammation. This delayed T-cell response is under host genetic control and nutritional influence. The mechanism that temporally and quantitatively regulates the host T-cell population is the critical determinant in chlamydial pathogenesis.

Hepatic Glucagon Receptor Signaling Enhances Insulin-Stimulated Glucose Disposal in Rodents.

Glucagon receptor (GCGR) agonists cause hyperglycemia but also weight loss. However, GCG-like peptide 1 receptor (GLP1R)/GCGR mixed agonists do not exhibit the diabetogenic effects often attributed to GCGR activity. Thus, we sought to investigate the effect of glucagon agonism on insulin action and glucose homeostasis. Acute GCGR agonism induced immediate hyperglycemia, followed by improved glucose tolerance and enhanced glucose-stimulated insulin secretion. Moreover, acute GCGR agonism improved insulin tolerance in a dose-dependent manner in both lean and obese mice. Improved insulin tolerance was independent of GLP1R, FGF21, and hepatic glycogenolysis. Moreover, we observed increased glucose infusion rate, disposal, uptake, and suppressed endogenous glucose production during euglycemic clamps. Mice treated with insulin and GCGR agonist had enhanced phosphorylation of hepatic AKT at Ser473; this effect was reproduced in isolated mouse primary hepatocytes and resulted in increased AKT kinase activity. These data reveal that GCGR agonism enhances glucose tolerance, in part, by augmenting insulin action, with implications for the use of GCGR agonism in therapeutic strategies for diabetes.