Synergistic Inhibitory Effects of Hypoxia and Iron Deficiency on Hepatic Glucose Response in Mouse Liver.

Hypoxia and iron both regulate metabolism through multiple mechanisms, including hypoxia-inducible transcription factors. The hypoxic effects on glucose disposal and glycolysis are well established, but less is known about the effects of hypoxia and iron deficiency on hepatic gluconeogenesis. We therefore assessed their effects on hepatic glucose production in mice. Weanling C57BL/6 male mice were fed an iron-deficient (4 ppm) or iron-adequate (35 ppm) diet for 14 weeks and were continued in normoxia or exposed to hypoxia (8% O2) for the last 4 weeks of that period. Hypoxic mice became hypoglycemic and displayed impaired hepatic glucose production after a pyruvate challenge, an effect accentuated by an iron-deficient diet. Stabilization of hypoxia-inducible factors under hypoxia resulted in most glucose being converted into lactate and not oxidized. Hepatic pyruvate concentrations were lower in hypoxic mice. The decreased hepatic pyruvate levels were not caused by increased utilization but rather were contributed to by decreased metabolism from gluconeogenic amino acids. Pyruvate carboxylase, which catalyzes the first step of gluconeogenesis, was also downregulated by hypoxia with iron deficiency. Hypoxia, and more so hypoxia with iron deficiency, results in hypoglycemia due to decreased levels of hepatic pyruvate and decreased pyruvate utilization for gluconeogenesis. These data highlight the role of iron levels as an important determinant of glucose metabolism in hypoxia.

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice.

Excess iron is associated with hepatic damage and diabetes in humans, although the detailed molecular mechanisms are not known. To investigate how iron regulates glucose homeostasis, we fed C57BL/6J male mice with high-iron (HI) diets (2 or 20 g Fe/kg chow). Mice fed an HI diet exhibited elevated AMP-activated protein kinase (AMPK) activity and impaired insulin signaling in skeletal muscle and liver. Consistent with the increased AMPK activity, glucose uptake was enhanced in mice fed an HI diet. The effects of improved glucose tolerance induced by HI feeding were abolished in transgenic mice with expression of muscle specific dominant-negative AMPK. Glucose output was suppressed in the liver of wild-type mice fed an HI diet, due to decreased expression of gluconeogenic genes and decreased substrate (lactate) from peripheral glycolysis. Iron activated AMPK by increasing deacetylase and decreasing LKB1 acetylation, in turn stimulating the phosphorylation of LKB1 and AMPK. The effects of HI diet were abrogated by treatment of the mice with N-acetyl cysteine, suggesting a redox-dependent mechanism for increasing deacetylase activity. In addition, tissue from iron-fed mice exhibited an elevated AMP/ATP ratio, further contributing to AMPK activation. In summary, a diet high in iron improves glucose tolerance by activating AMPK through mechanisms that include deacetylation.

Manganese supplementation protects against diet-induced diabetes in wild type mice by enhancing insulin secretion.

Mitochondrial dysfunction is both a contributing mechanism and complication of diabetes, and oxidative stress contributes to that dysfunction. Mitochondrial manganese-superoxide dismutase (MnSOD) is a metalloenzyme that provides antioxidant protection. We have previously shown in a mouse model of hereditary iron overload that cytosolic iron levels affected mitochondrial manganese availability, MnSOD activity, and insulin secretion. We therefore sought to determine the metallation status of MnSOD in wild-type mice and whether altering that status affected ß-cell function. 129/SvEVTac mice given supplemental manganese exhibited a 73% increase in hepatic MnSOD activity and increased metallation of MnSOD. To determine whether manganese supplementation offered glucose homeostasis under a situation of ß-cell stress, we challenged C57BL/6J mice, which are more susceptible to diet-induced diabetes, with a high-fat diet for 12 weeks. Manganese was supplemented or not for the final 8 weeks on that diet, after which we examined glucose tolerance and the function of isolated islets. Liver mitochondria from manganese-injected C57BL/6J mice had similar increases in MnSOD activity (81%) and metallation as were seen in 129/SvEVTac mice. The manganese-treated group fed high fat had improved glucose tolerance (24% decrease in fasting glucose and 41% decrease in area under the glucose curve), comparable with mice on normal chow and increased serum insulin levels. Isolated islets from the manganese-treated group exhibited improved insulin secretion, decreased lipid peroxidation, and improved mitochondrial function. In conclusion, MnSOD metallation and activity can be augmented with manganese supplementation in normal mice on normal chow, and manganese treatment can increase insulin secretion to improve glucose tolerance under conditions of dietary stress.

Adipocyte iron regulates adiponectin and insulin sensitivity.

Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores.

Metabolic insight into mechanisms of high-altitude adaptation in Tibetans.

Recent studies have identified genes involved in high-altitude adaptation in Tibetans. Genetic variants/haplotypes within regions containing three of these genes (EPAS1, EGLN1, and PPARA) are associated with relatively decreased hemoglobin levels observed in Tibetans at high altitude, providing corroborative evidence for genetic adaptation to this extreme environment. The mechanisms that afford adaptation to high-altitude hypoxia, however, remain unclear. Considering the strong metabolic demands imposed by hypoxia, we hypothesized that a shift in fuel preference to glucose oxidation and glycolysis at the expense of fatty acid oxidation would improve adaptation to decreased oxygen availability. Correlations between serum free fatty acid and lactate concentrations in Tibetan groups living at high altitude and putatively selected haplotypes provide insight into this hypothesis. An EPAS1 haplotype that exhibits a signal of positive selection is significantly associated with increased lactate concentration, the product of anaerobic glycolysis. Furthermore, the putatively advantageous PPARA haplotype is correlated with serum free fatty acid concentrations, suggesting a possible decrease in the activity of fatty acid oxidation. Although further studies are required to assess the molecular mechanisms underlying these patterns, these associations suggest that genetic adaptation to high altitude involves alteration in energy utilization pathways.

Early mitochondrial adaptations in skeletal muscle to diet-induced obesity are strain dependent and determine oxidative stress and energy expenditure but not insulin sensitivity.

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake.

Pleiotropic and age-dependent effects of decreased protein modification by O-linked N-acetylglucosamine on pancreatic ß-cell function and vascularization.

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of ß-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal ß-cell function, however, is not well understood. We studied transgenic mice that over express ß-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic ß-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in ß-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

Increased hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance: evidence for a shared pathway.

Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.

Iron overload and diabetes risk: a shift from glucose to Fatty Acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis.

OBJECTIVE: Excess tissue iron levels are a risk factor for diabetes, but the mechanisms underlying the association are incompletely understood. We previously published that mice and humans with a form of hereditary iron overload, hemochromatosis, exhibit loss of ß-cell mass. This effect by itself is not sufficient, however, to fully explain the diabetes risk phenotype associated with all forms of iron overload. RESEARCH DESIGN AND METHODS: We therefore examined glucose and fatty acid metabolism and hepatic glucose production in vivo and in vitro in a mouse model of hemochromatosis in which the gene most often mutated in the human disease, HFE, has been deleted (Hfe⁻(/)⁻). RESULTS: Although Hfe⁻(/)⁻ mice exhibit increased glucose uptake in skeletal muscle, glucose oxidation is decreased and the ratio of fatty acid to glucose oxidation is increased. On a high-fat diet, the Hfe⁻(/)⁻ mice exhibit increased fatty acid oxidation and are hypermetabolic. The decreased glucose oxidation in skeletal muscle is due to decreased pyruvate dehydrogenase (PDH) enzyme activity related, in turn, to increased expression of PDH kinase 4 (pdk4). Increased substrate recycling to liver contributes to elevated hepatic glucose production in the Hfe⁻(/)⁻ mice. CONCLUSIONS: Increased hepatic glucose production and metabolic inflexibility, both of which are characteristics of type 2 diabetes, may contribute to the risk of diabetes with excessive tissue iron.

Dietary iron restriction or iron chelation protects from diabetes and loss of beta-cell function in the obese (ob/ob lep-/-) mouse.

Iron overload can cause insulin deficiency, but in some cases this may be insufficient to result in diabetes. We hypothesized that the protective effects of decreased iron would be more significant with increased beta-cell demand and stress. Therefore, we treated the ob/ob mouse model of type 2 diabetes with an iron-restricted diet (35 mg/kg iron) or with an oral iron chelator. Control mice were fed normal chow containing 500 mg/kg iron. Neither treatment resulted in iron deficiency or anemia. The low-iron diet significantly ameliorated diabetes in the mice. The effect was long lasting and reversible. Ob/ob mice on the low-iron diet exhibited significant increases in insulin sensitivity and beta-cell function, consistent with the phenotype in mouse models of hereditary iron overload. The effects were not accounted for by changes in weight or feeding behavior. Treatment with iron chelation had a more dramatic effect, allowing the ob/ob mice to maintain normal glucose tolerance for at least 10.5 wk despite no effect on weight. Although dietary iron restriction preserved beta-cell function in ob/ob mice fed a high-fat diet, the effects on overall glucose levels were less apparent due to a loss of the beneficial effects of iron on insulin sensitivity. Beneficial effects of iron restriction were minimal in wild-type mice on normal chow but were apparent in mice on high-fat diets. We conclude that, even at "normal" levels, iron exerts detrimental effects on beta-cell function that are reversible with dietary restriction or pharmacotherapy.

Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure.

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR(-/-)) or mice with genetic ablation of Akt1 (Akt1-/-). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR(-/-) mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1-/- mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.

Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria.

Mycobacterium avium complex (MAC) and rapidly growing mycobacteria (RGM) such as M. abscessus, M. mucogenicum, M. chelonae, and M. fortuitum, implicated in health care-associated infections, are often isolated from potable water supplies as part of the microbial flora. To understand factors that influence growth in their environmental source, clinical RGM and slowly growing MAC isolates were grown as biofilm in a laboratory batch system. High and low nutrient levels were compared, as well as stainless steel and polycarbonate surfaces. Biofilm growth was measured after 72 h of incubation by enumeration of bacteria from disrupted biofilms and by direct quantitative image analysis of biofilm microcolony structure. RGM biofilm development was influenced more by nutrient level than by substrate material, though both affected biofilm growth for most of the isolates tested. Microcolony structure revealed that RGM develop several different biofilm structures under high-nutrient growth conditions, including pillars of various shapes (M. abscessus and M. fortuitum) and extensive cording (M. abscessus and M. chelonae). Although it is a slowly growing species in the laboratory, a clinical isolate of M. avium developed more culturable biofilm in potable water in 72 h than any of the 10 RGM examined. This indicates that M. avium is better adapted for growth in potable water systems than in laboratory incubation conditions and suggests some advantage that MAC has over RGM in low-nutrient environments.

Mechanisms for increased myocardial fatty acid utilization following short-term high-fat feeding.

AIMS: Diet-induced obesity is associated with increased myocardial fatty acid (FA) utilization, insulin resistance, and cardiac dysfunction. The study was designed to test the hypothesis that impaired glucose utilization accounts for initial changes in FA metabolism. METHODS AND RESULTS: Ten-week-old C57BL6J mice were fed a high-fat diet (HFD, 45% calories from fat) or normal chow (4% calories from fat). Cardiac function and substrate metabolism in isolated working hearts, glucose uptake in isolated cardiomyocytes, mitochondrial function, insulin-stimulated protein kinase B (Akt/PKB) and Akt substrate (AS-160) phosphorylation, glucose transporter 4 (GLUT4) translocation, pyruvate dehydrogenase (PDH) activity, and mRNA levels for metabolic genes were determined after 2 or 5 weeks of HFD. Two weeks of HFD reduced basal rates of glycolysis and glucose oxidation and prevented insulin stimulation of glycolysis in hearts and reduced insulin-stimulated glucose uptake in cardiomyocytes. Insulin-stimulated Akt/PKB and AS-160 phosphorylation were preserved, and PDH activity was unchanged. GLUT4 content was reduced by 55% and GLUT4 translocation was significantly attenuated. HFD increased FA oxidation rates and myocardial oxygen consumption (MVO2), which could not be accounted for by mitochondrial uncoupling or by increased expression of peroxisome proliferator activated receptor-alpha (PPAR-alpha) target genes, which increased only after 5 weeks of HFD. CONCLUSION: Rates of myocardial glucose utilization are altered early in the course of HFD because of reduced GLUT4 content and GLUT4 translocation despite normal insulin signalling to Akt/PKB and AS-160. The reciprocal increase in FA utilization is not due to PPAR-alpha-mediated signalling or mitochondrial uncoupling. Thus, the initial increase in myocardial FA utilization in response to HFD likely results from impaired glucose transport that precedes impaired insulin signalling.

The role of mPer2 clock gene in glucocorticoid and feeding rhythms.

The circadian clock synchronizes the activity level of an organism to the light-dark cycle of the environment. Energy intake, as well as energy metabolism, also has a diurnal rhythm. Although the role of the clock genes in the sleep-wake cycle is well characterized, their role in the generation of the metabolic rhythms is poorly understood. Here, we use mice deficient in the clock protein mPer2 to study how the circadian clock regulates two critical metabolic rhythms: glucocorticoid and food intake rhythms. Our findings indicate that mPer2-/- mice do not have a glucocorticoid rhythm even though the corticosterone response to hypoglycemia, ACTH, and restraint stress is intact. In addition, the diurnal feeding rhythm is absent in mPer2-/- mice. On high-fat diet, they eat as much during the light period as they do during the dark period and develop significant obesity. The diurnal rhythm of neuroendocrine peptide alphaMSH, a major effector of appetite control, is disrupted in the hypothalamus of mPer2-/- mice even though the diurnal rhythm of ACTH, the alphaMSH precursor, is intact. Peripheral injection of alphaMSH, which has been shown to enter the brain, restored the feeding rhythm and induced weight loss in mPer2-/- mice. These findings emphasize the requirement of mPer2 in appetite control during the inactive period and the potential role of peripherally administered alphaMSH in restoring night-day eating pattern in individuals with circadian eating disorders such as night-eating syndrome, which is also associated with obesity.

RETRACTED: Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria.

This article has been retracted.

Saturated fatty acids inhibit hepatic insulin action by modulating insulin receptor expression and post-receptor signalling.

Free fatty acids (FFAs) are proposed to play a pathogenic role in both peripheral and hepatic insulin resistance. We have examined the effect of saturated FFA on insulin signalling (100 nM) in two hepatocyte cell lines. Fao hepatoma cells were treated with physiological concentrations of sodium palmitate (0.25 mM) (16:0) for 0.25-48 h. Palmitate decreased insulin receptor (IR) protein and mRNA expression in a dose- and time-dependent manner (35% decrease at 12 h). Palmitate also reduced insulin-stimulated IR and IRS-2 tyrosine phosphorylation, IRS-2-associated PI 3-kinase activity, and phosphorylation of Akt, p70 S6 kinase, GSK-3 and FOXO1A. Palmitate also inhibited insulin action in hepatocytes derived from wild-type IR (+/+) mice, but was ineffective in IR-deficient (-/-) cells. The effects of palmitate were reversed by triacsin C, an inhibitor of fatty acyl CoA synthases, indicating that palmitoyl CoA ester formation is critical. Neither the non-metabolized bromopalmitate alone nor the medium chain fatty acid octanoate (8:0) produced similar effects. However, the CPT-1 inhibitor (+/-)-etomoxir and bromopalmitate (in molar excess) reversed the effects of palmitate. Thus, the inhibition of insulin signalling by palmitate in hepatoma cells is dependent upon oxidation of fatty acyl-CoA species and requires intact insulin receptor expression.

Captopril normalizes insulin signaling and insulin-regulated substrate metabolism in obese (ob/ob) mouse hearts.

The goal of this study was to determine whether inhibiting the renin-angiotensin system would restore insulin signaling and normalize substrate use in hearts from obese ob/ob mice. Mice were treated for 4 wk with Captopril (4 mg/kg x d). Circulating levels of free fatty acids, triglycerides, and insulin were measured and glucose tolerance tests performed. Rates of palmitate oxidation and glycolysis, oxygen consumption, and cardiac power were determined in isolated working hearts in the presence and absence of insulin, along with levels of phosphorylation of Akt and AMP-activated protein kinase (AMPK). Captopril treatment did not correct the hyperinsulinemia or impaired glucose tolerance in ob/ob mice. Rates of fatty acid oxidation were increased and glycolysis decreased in ob/ob hearts, and insulin did not modulate substrate use in hearts of ob/ob mice and did not increase Akt phosphorylation. Captopril restored the ability of insulin to regulate fatty acid oxidation and glycolysis in hearts of ob/ob mice, possibly by increasing Akt phosphorylation. Moreover, AMPK phosphorylation, which was increased in hearts of ob/ob mice, was normalized by Captopril treatment, suggesting that in addition to restoring insulin sensitivity, Captopril treatment improved myocardial energetics. Thus, angiotensin-converting enzyme inhibitors restore the responsiveness of ob/ob mouse hearts to insulin and normalizes AMPK activity independently of effects on systemic metabolic homeostasis.

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital.

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.

Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis.

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe(-/-)) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe(-/-) mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe(-/-) mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe(-/-) mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe(-/-) mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.

Increased glucose disposal and AMP-dependent kinase signaling in a mouse model of hemochromatosis.

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe(-/-)). Hfe(-/-) mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe(-/-), for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe(-/-) mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-(13)C]D-glucose clearance from plasma is significantly increased in Hfe(-/-) mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe(-/-) mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe(-/-) mice (p < 0.03). Compared with controls Hfe(-/-) mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe(-/-) mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.