A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle.

AIMS/HYPOTHESIS: Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals. METHODS: Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp. RESULTS: Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of inhibitor of kappa Bα (p = 0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation. CONCLUSIONS/INTERPRETATION: A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT01740817.

Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control.

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine diminution was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete beta-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.

A low dose lipid infusion is sufficient to induce insulin resistance and a pro-inflammatory response in human subjects.

OBJECTIVE: The root cause behind the low-grade inflammatory state seen in insulin resistant (obesity and type 2 diabetes) states is unclear. Insulin resistant subjects have elevations in plasma free fatty acids (FFA), which are ligands for the pro-inflammatory toll-like receptor (TLR)4 pathway. We tested the hypothesis that an experimental elevation in plasma FFA (within physiological levels) in lean individuals would upregulate TLR4 and activate downstream pathways (e.g., MAPK) in circulating monocytes. RESEARCH DESIGN AND METHODS: Twelve lean, normal glucose-tolerant subjects received a low dose (30 ml/h) 48 h lipid or saline infusion on two different occasions. Monocyte TLR4 protein level, MAPK phosphorylation, and expression of genes in the TLR pathway were determined before and after each infusion. RESULTS: The lipid infusion significantly increased monocyte TLR4 protein and phosphorylation of JNK and p38 MAPK. Lipid-mediated increases in TLR4 and p38 phosphorylation directly correlated with reduced peripheral insulin sensitivity (M value). Lipid increased levels of multiple genes linked to inflammation, including several TLRs, CD180, MAP3K7, and CXCL10. Monocytes exposed in vivo to lipid infusion exhibited enhanced in vitro basal and LPS-stimulated IL-1ß secretion. CONCLUSIONS: In lean subjects, a small increase in plasma FFA (as seen in insulin resistant subjects) is sufficient to upregulate TLR4 and stimulate inflammatory pathways (MAPK) in monocytes. Moreover, lipids prime monocytes to endotoxin. We provide proof-of-concept data in humans indicating that the low-grade inflammatory state characteristic of obesity and type 2 diabetes could be caused (at least partially) by pro-inflammatory monocytes activated by excess lipids present in these individuals.

Isolation of the B3 transcription factor of the Xenopus TFIIIA gene.

The selective expression of the Xenopus TFIIIA gene in immature oocytes is principally regulated by a single 5'-flanking DNA sequence element, termed element 3 (i.e. E3). We describe the isolation and characterization of a cDNA for a protein present in immature Xenopus ooctyes, termed B3.65, which appears to bind to and activate E3-mediated expression. The approximate molecular weight of the E3 binding protein(s) was determined by ultraviolet light cross-linking analysis. B3.65, a protein of the appropriate molecular weight, was purified biochemically from immature Xenopus ooctye extracts by affinity chromatography. Antiserum to purified B3.65 super-shifted the E3 activator complex. In addition, B3.65 mRNA was found to be highly enriched in immature oocytes. All of these data are consistent with B3.65 either being the E3 activator, or antigenically related to the specific activator required for XenopusTFIIIA gene transcription. B3.65 is a member of the K-homologous (KH) domain family of proteins, with almost absolute identity to Xenopus Vg1 RBP/VERA (97%) and significant similarity to human koc (82%). The koc mRNA is over-expressed in human pancreatic cancer tissues, and B3.65 mRNA was detected in Xenopus pancreas and kidney. Interestingly, KH proteins, like Vg1RBP/VERA, are most commonly associated with RNA metabolism, in their capacity to regulate RNA localization, stability, and translation. Our results suggest that B3.65 is a key regulator of both RNA- and DNA metabolism.

Enhanced fitness: a randomized controlled trial of the effects of home-based physical activity counseling on glycemic control in older adults with prediabetes mellitus.

OBJECTIVES: To determine whether a home-based multicomponent physical activity counseling (PAC) intervention is effective in reducing glycemic measures in older outpatients with prediabetes mellitus. DESIGN: Controlled clinical trial. SETTING: Primary care clinics of the Durham Veterans Affairs (VA) Medical Center between September 29, 2008, and March 25, 2010. PARTICIPANTS: Three hundred two overweight (body mass index 25-45 kg/m(2) ), older (60-89) outpatients with impaired glucose tolerance (fasting blood glucose 100-125 mg/dL, glycosylated hemoglobin (HbA1c) <7%) randomly assigned to a PAC intervention group (n = 180) or a usual care control group (n = 122). INTERVENTION: A 12-month, home-based multicomponent PAC program including one in-person baseline counseling session, regular telephone counseling, physician endorsement in clinic with monthly automated encouragement, and customized mailed materials. All study participants, including controls, received a consultation in a VA weight management program. MEASUREMENTS: The primary outcome was a homeostasis model assessment of insulin resistance (HOMA-IR), calculated from fasting insulin and glucose levels at baseline and 3 and 12 months. HbA1c was the secondary indicator of glycemic control. Other secondary outcomes were anthropometric measures and self-reported physical activity, health-related quality of life, and physical function. RESULTS: There were no significant differences between the PAC and control groups over time for any of the glycemic indicators. Both groups had small declines over time of approximately 6% in fasting blood glucose (P < .001), and other glycemic indicators remained stable. The declines in glucose were not sufficient to affect the change in HOMA-IR scores due to fluctuations in insulin over time. Endurance physical activity increased significantly in the PAC group (P < .001) and not in the usual care group. CONCLUSION: Home-based telephone counseling increased physical activity levels but was insufficient to improve glycemic indicators in older outpatients with prediabetes mellitus.

Plasma acylcarnitines are associated with physical performance in elderly men.

BACKGROUND: Metabolic profiling might provide insight into the biologic underpinnings of disability in older adults. METHODS: A targeted mass spectrometry-based platform was used to identify and quantify 45 plasma acylcarnitines in 77 older men with a mean age of 79 years and average body mass index of 28.4 kg/m(2). To control for type I error inherent in a test of multiple analytes, principal components analysis was employed to reduce the acylcarnitines from 45 separate metabolites, into a single "acylcarnitine factor." We then tested for an association between this acylcarnitine factor and multiple indices of physical performance and self-reported function. RESULTS: The acylcarnitine factor accounted for 40% of the total variance in 45 acylcarnitines. Of the metabolites analyzed, those that contributed most to our one-factor solution were even-numbered medium and long-chain species with side chains containing 10-18 carbons (factor loadings ≥0.70). Odd-numbered chain species, in contrast, had factor loadings 0.50 or less. Acylcarnitine factor scores were inversely related to physical performance as measured by the Short Physical Performance Battery total score, two of its three component scores (gait and chair stands Short Physical Performance Battery), and usual and maximal gait speeds (ρ = -0.324, -0.348, -0.309, -0.241, and -0.254, respectively; p < .05). CONCLUSIONS: Higher acylcarnitine factor scores were associated with lower levels of objectively measured physical performance in this group of older, largely overweight men. Metabolic profiles of rodents exhibiting lipid-induced mitochondrial dysfunction show a similar phenotypic predominance of medium- and long-chain acylcarnitines.

Lessons learned when innovations go awry: a baseline description of a behavioral trial-the Enhancing Fitness in Older Overweight Veterans with Impaired Fasting Glucose study.

Individuals diagnosed with impaired glucose tolerance (i.e., prediabetes) are at increased risk for developing diabetes. We proposed a clinical trial with a novel adaptive randomization designed to examine the impact of a home-based physical activity (PA) counseling intervention on metabolic risk in prediabetic elders. This manuscript details the lessons learned relative to recruitment, study design, and implementation of a 12-month randomized controlled PA counseling trial. A detailed discussion on how we responded to unforeseen challenges is provided. A total of 302 older patients with prediabetes were randomly assigned to either PA counseling or usual care. A novel adaptive design that reallocated counseling intensity based on self-report of adherence to PA was initiated but revised when rates of non-response were lower than projected. This study presents baseline participant characteristics and discusses unwelcome adaptations to a highly innovative study design to increase PA and enhance glucose metabolism when the best-laid plans went awry.