Lipopolysaccharide inhibition of glucose production through the Toll-like receptor-4, myeloid differentiation factor 88, and nuclear factor kappa b pathway.

UNLABELLED: Acute exposure to lipopolysaccharide (LPS) can cause hypoglycemia and insulin resistance; the underlying mechanisms, however, are unclear. We set out to determine whether insulin resistance is linked to hypoglycemia through Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappaB (NFkappaB), a cell signaling pathway that mediates LPS induction of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). LPS induction of hypoglycemia was blocked in TLR4(-/-) and MyD88(-/-) mice but not in TNFalpha(-/-) mice. Both glucose production and glucose utilization were decreased during hypoglycemia. Hypoglycemia was associated with the activation of NFkappaB in the liver. LPS inhibition of glucose production was blocked in hepatocytes isolated from TLR4(-/-) and MyD88(-/-) mice and hepatoma cells expressing an inhibitor of NFkappaB (IkappaB) mutant that interferes with NFkappaB activation. Thus, LPS-induced hypoglycemia was mediated by the inhibition of glucose production from the liver through the TLR4, MyD88, and NFkappaB pathway, independent of LPS-induced TNFalpha. LPS suppression of glucose production was not blocked by pharmacologic inhibition of the insulin signaling intermediate phosphatidylinositol 3-kinase in hepatoma cells. Insulin injection caused a similar reduction of circulating glucose in TLR4(-/-) and TLR4(+/+) mice. These two results suggest that LPS and insulin inhibit glucose production by separate pathways. Recovery from LPS-induced hypoglycemia was linked to glucose intolerance and hyperinsulinemia in TLR4(+/+) mice, but not in TLR4(-/-) mice. CONCLUSION: Insulin resistance is linked to the inhibition of glucose production by the TLR4, MyD88, and NFkappaB pathway.

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro.

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.

Characterization of N-glycosylation consensus sequences in the Kv3.1 channel.

N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

Neuroendocrine inhibition of glucose production and resistance to cancer in dwarf mice.

Pit1 null (Snell dwarf) and Proph1 null (Ames dwarf) mutant mice lack GH, PRL and TSH. Snell and Ames dwarf mice also exhibit reduced IGF-I, resistance to cancer and a longer lifespan than control mice. Endogenous glucose production during fasting is reduced in Snell dwarf mice compared to fasting control mice. In view of cancer cell dependence on glucose for energy, low endogenous glucose production may provide Snell dwarf mice with resistance to cancer. We investigated whether endogenous glucose production is lower in Snell dwarf mice during feeding. Inhibition of endogenous glucose production by glucose injection was enhanced in 12 to 14 month-old female Snell dwarf mice. Thus, we hypothesize that lower endogenous glucose production during feeding and fasting reduces cancer cell glucose utilization providing Snell dwarf mice with resistance to cancer. The elevation of circulating adiponectin, a hormone produced by adipose tissue, may contribute to the suppression of endogenous glucose production in 12 to 14 month-old Snell dwarf mice. We compared the incidence of cancer at time of death between old Snell dwarf and control mice. Only 18% of old Snell dwarf mice had malignant lesions at the time of death compared to 82% of control mice. The median ages at death for old Snell dwarf and control mice were 33 and 26 months, respectively. By contrast, previous studies showed a high incidence of cancer in old Ames dwarf mice at the time of death. Hence, resistance to cancer in old Snell dwarf mice may be mediated by neuroendocrine factors that reduce glucose utilization besides elevated adiponectin, reduced IGF-I and a lack of GH, PRL and TSH, seen in both Snell and Ames dwarf mice. Proteomics analysis of pituitary secretions from Snell dwarf mice confirmed the absence of GH and PRL, the secretion of ACTH and elevated secretion of Chromogranin B and Secretogranin II. Radioimmune assays confirmed that circulating Chromogranin B and Secretogranin II were elevated in 12 to 14 month-old Snell dwarf mice. In summary, our results in Snell dwarf mice suggest that the pituitary gland and adipose tissue are part of a neuroendocrine loop that lowers the risk of cancer during aging by reducing the availability of glucose.

Do low levels of circulating adiponectin represent a biomarker or just another risk factor for the metabolic syndrome?

The metabolic syndrome is currently defined by various combinations of insulin resistance, obesity, dyslipidaemia and hypertension. The tendency for these risk factors to appear simultaneously suggests a single aetiologic basis. A low level of circulating adiponectin is associated with the appearance of each metabolic syndrome risk factor. The following review summarizes a large body of evidence that suggests a low level of circulating adiponectin represents an independent risk factor and a possible biomarker for the metabolic syndrome. An association between the metabolic syndrome and low adiponectin supports the view that the development of the metabolic syndrome may be triggered by a single underlying mechanism. Clinical studies in the future may show that a low level of circulating adiponectin is a primary biomarker for a specific cluster of metabolic syndrome risk factors rather than all the possible combinations of risk factors currently used to identify the metabolic syndrome. The significance of low circulating adiponectin in risk assessment models should ultimately be compared against insulin resistance, obesity, dyslipidaemia, hypertension and other metabolic syndrome risk factors presently under consideration. Adiponectin can be measured reliably in a clinical setting; circulating values of adiponectin do not fluctuate on a diurnal basis as much as insulin, glucose, triglycerides or cholesterol and only 2-4 microl of blood are currently needed for its measurement.

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice.

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.