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 B (NF B), a cell signaling pathway that mediates LPS induction of the proinflammatory cytokine tumor necrosis factor alpha (TNF␣). LPS induction of hypoglycemia was blocked in TLR4 ؊/؊ and MyD88 ؊/؊ mice but not in TNF␣ ؊/؊ mice. Both glucose production and glucose utilization were decreased during hypoglycemia. Hypoglycemia was associated with the activation of NF B 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 NF B (I B) mutant that interferes with NF B activation. Thus, LPS-induced hypoglycemia was mediated by the inhibition of glucose production from the liver through the TLR4, MyD88, and NF B pathway, independent of LPS-induced TNF␣. 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 NF B pathway. (HEPATOLOGY 2009;50:592-600.) L ipopolysaccharide (LPS) from Gram-negative bacterial infection can cause hypoglycemia and insulin resistance in both humans and mice; however, the underlying mechanisms are unclear. 1-5 Low levels of LPS exposure through gastrointestinal tract and airborne particles can also lead to insulin resistance. 6-8 Thus, insulin resistance is decreased in mice by antibiotic treatment or housing in germ-free facilities. 9,10 Insulin resistance is also decreased by gene deletion of Toll-like receptor-4 (TLR4) or cluster of differentiation 14 (CD14), a glycoprotein that binds to the extracellular portion of TLR4. [11][12][13] TLR4 is a plasma membrane protein that mediates LPS induction of inflammatory cytokines such as tumor necrosis factor alpha (TNF␣) and interleukin-1 beta (IL-1␤). 14 The main goals of this study were to (1) determine whether TLR4 and its downstream signaling targets mediate the induction of hypoglycemia by LPS and, if so, (2) determine whether the induction of hypoglycemia by TLR4 is linked to the development of insulin resistance.LPS was postulated to cause hypoglycemia through the induction of the cytokines TNF␣ and IL-1␤. 15 Given that LPS does not induce hypoglycemia in IL-1␣ Ϫ/Ϫ and IL-1␤ Ϫ/Ϫ "double-knockout" mice, LPS induction of IL-1␤ is essential. 16 Whether LPS induction of TNF␣

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.

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.Glucose metabolism is altered in centenarians and long-lived mice (1-8). Our current hypothesis is that a decrease in fasting glucose utilization may increase longevity by lowering oxygen radical production, a potential cause of aging. Changes in glucose metabolism have been indicated in long-lived rodents by (a) oral glucose tolerance test, (b) insulin tolerance test, (c) inhibition of glucose production and stimulation of glucose disposal under euglycemic clamp conditions, and (d) the levels of circulating glucose and insulin under physiologic conditions (9 -13). These methods suggest that glucose utilization is elevated in long-lived mice during feeding. However, they do not reveal whether glucose utilization is lower in long-lived mice during fasting, a period of the day when glucose utilization is relatively low and fatty acid utilization is high.During fasting (a) the appearance in the circulation of glucose from the gastrointestinal tract is low, (b) the concentrations of circulating glucose and insulin are low, (c) circulating glucose is used for energy rather than storage, and (d) the circulating glucose used is replenished largely by liver glucose production, via gluconeogenesis and glycogenolysis. Thus, whole body glucose utilization may be lowered during fasting by decreasing glucose production. Whether fasting glucose pro...

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.

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.