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Iron is an essential cofactor for many proteins that function in electron transport or oxygen transport as heme or iron-sulfur cluster. On the contrary, iron also has the potential to cause oxidative damage if not carefully regulated and when in labial iron excess. Clinical studies show that elevated serum ferritin levels are observed in most patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this context, p53 is shown to induces some mitochondrial iron regulatory genes. The role of crosstalk between p53 and iron metabolism has not been sufficiently examined in the pathogenesis of diabetes and NAFLD. Here, we examined the role of ferredoxin reductase (FDXR), a key mitochondrial regulator for iron metabolism, as p53-inducible gene with focusing on the hepatocyte and liver. We confirmed that p53 induced FDXR expression in HepG2 cells and SKEHP1 cells. Biochemical analysis demonstrated that FDXR regulated ROS levels via iron metabolism. In vivo analysis, high-fat diet activated the p53-FDXR pathway in mice liver. We generated transgene expression in mice liver using adenovirus infection carrying shRNA or CRISPR Cas9 system. Treatment with the FDXR knockdown increased hepatic iron content and aggravated glucose intolerance. Besides, forkhead box protein O1 (FOXO1), a key transcriptional factor that induces phosphoenolpyruvate carboxylase and glucose-6-phosphatase increased ratio of nuclear localization, indicating hepatic gluconeogenesis activation. Consistently, biochemical analysis in HepG2 cells demonstrated that FDXR regulated insulin-dependent FOXO1 nuclear exclusion through oxidative stress. In conclusion, p53-inducible FDXR regulates iron metabolism and oxidative stress. FDXR inhibits iron accumulation and oxidative stress in liver and links to suppression of hepatic gluconeogenesis via insulin-dependent FOXO1 nuclear exclusion. The results of this study provide important new insights into relationship between iron metabolism and glucose metabolism as well as potentially identify novel therapeutic targets for the treatment of diabetes and NAFLD.

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow– and high-fat diet (HFD)–fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow– and HFD-fed mice. This improvement in insulin responsiveness in FGF21-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cϵ activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

Context: Lipotoxicity (increased tissue fat content) has been implicated in the development of muscle insulin resistance and type 2 diabetes mellitus (T2DM). Objective: The aim was to study the effect of pioglitazone on intramyocellular fat metabolism. Research Design: Twenty-four T2DM subjects (glycosylated hemoglobin = 8.3 ± 0.4%) participated in three similar study protocols before and after 4 months of 45 mg/d pioglitazone treatment: 1) 3-h euglycemic insulin (80 mU/m2 · min) clamp with measurement of intramyocellular fat with proton nuclear magnetic resonance; 2) vastus lateralis muscle biopsy for measurement of LC-FACoAs 60 min before start of the insulin clamp; and 3) muscle biopsy for measurement of diacylglycerol 60 min before start of the insulin clamp. Results: In all three protocols, pioglitazone similarly reduced (all P < 0.05) the glycosylated hemoglobin (Δ = 0.8–1.2%), fasting plasma glucose (39–76 mg/dl), fasting free fatty acid (132–236 μmol/liter), and increased insulin-stimulated glucose disposal (by 25–56%). Intramyocellular fat (protocol I) declined from 1.5 to 0.9% (P < 0.05) and correlated with the increase in glucose disposal rate (r = 0.65; P < 0.05). Long chain-fatty acyl-coenzyme A decreased from 12.5 to 8.1 nmol/g (P < 0.05) and correlated with the increase in disposal rate (r = 0.76; P < 0.05). Pioglitazone therapy had no effect on muscle diacylglycerol content. Conclusions: Pioglitazone improves insulin resistance in T2DM in association with mobilization of fat and toxic lipid metabolites out of muscle.

The exact mechanisms through which ghrelin promotes lipogenesis are unknown. Uncoupling protein (UCP)-2 is a mitochondrial protein important in regulating reactive oxygen species; however, recent research shows that it may play an important role fat metabolism. Given that ghrelin increases UCP2 mRNA in white adipose tissue, we examined whether the lipogenic actions of ghrelin are modulated by UCP2 using ucp2(+/+) and ucp2(-/-) mice. Chronic ghrelin treatment either via osmotic minipumps or daily ip injections induced body weight gain in both ucp2(+/+) and ucp2(-/-) mice; however, body weight gain was potentiated in ucp2(-/-) mice. Increased body weight gain was completely due to increased body fat as a result of decreased fat oxidation in ucp2(-/-) mice. Ghrelin treatment of ucp2(-/-) mice resulted in a gene expression profile favoring lipogenesis. In a calorie-restriction model of negative energy balance, ghrelin to ucp2(+/+) mice did not increase body weight; however, ghrelin to ucp2(-/-) mice still induced body weight. These results show that UCP2 plays an important role in fat metabolism by promoting fat oxidation and restricts ghrelin-induced lipogenesis.

Obesity and osteoporosis are major health issues affecting millions of individuals. Transgenic mice overexpressing ΔFosB, an activator protein-1 transcription factor, under the control of the enolase 2 (ENO2) promoter exhibit both an increase in bone density and a decrease in adipose mass. Here we demonstrate that ΔFosB overexpression increases fatty-acid oxidation and energy expenditure, leading to a decrease in adipocyte size and adipose mass. In addition, the ENO2-ΔFosB mice exhibit increased insulin sensitivity and glucose tolerance. Targeted overexpression of ΔFosB in adipocytes using the adipocyte protein 2 promoter failed to induce changes in fat or in bone, showing that the effect on metabolic activity is not due to cell-autonomous effects of ΔFosB within adipocytes. Detailed analysis of the ENO2-ΔFosB mice demonstrated that energy expenditure was increased in muscle, independent of locomotor activity. These findings provide evidence that signaling downstream of ΔFosB is a potential target for not only osteoporosis but also obesity and diabetes. Overexpression of ΔFosB under the control of the enolase 2 promoter increases bone mass and energy expenditure by increasing fatty-acid oxidation within muscle, reducing adiposity.

Endocrine defensive mechanisms provide for energy supply during hypoglycemia. Intramyocellular lipids (IMCL) were recently shown to contribute to energy supply during exercise.The objective of this study was to assess the contribution of IMCL compared with lipolysis and endogenous glucose production (EGP) to insulin-mediated hypoglycemia counterregulation in patients with type 1 diabetes mellitus (T1DM).This was a prospective explorative study performed in a university research facility.Six well-controlled T1DM (age, 29 +/- 4 yr; body mass index, 23.4 +/- 1.0 kg/m2; hemoglobin A1c, 6.3 +/- 0.1%) and six nondiabetic humans (controls; age, 28 +/- 2 yr; body mass index, 23.4 +/- 1.0 kg/m2; hemoglobin A1c, 5.1 +/- 0.1%) were studied.We performed 240-min hypoglycemic (approximately 3 mM)-hyperinsulinemic (0.8 mU/kg x min) clamps on separate days to measure: 1) systemic lipolysis ([2H5]glycerol turnover), EGP ([6,6-(2)H2]glucose), and local lipolysis in abdominal s.c. adipose tissue and gastrocnemius muscle (microdialysis); and 2) IMCL (by 1H nuclear magnetic resonance spectroscopy) in soleus and tibialis anterior muscle.The main outcome measures were changes in IMCL during prolonged hypoglycemia.At baseline, EGP, glycerol turnover, and IMCL were not different between the groups. During hypoglycemia, hormonal counterregulation was blunted in T1DM (peak: glucagon, 68 +/- 4 vs. 170 +/- 37 pg/ml; cortisol, 16 +/- 2 vs. 24 +/- 2 microg/dl; epinephrine, 274 +/- 84 vs. 597 +/- 212 pg/ml; all P0.05 vs. control). T1DM had approximately 50% lower EGP (4.6 +/- 0.6 vs. 10.9 +/- 0.5 micromol/kg x min; P0.005), but approximately 40% higher glycerol turnover (374 +/- 21 vs. 272 +/- 19 micromol/kg x min; P0.01). Glycerol concentrations in muscle (T1DM, 302 +/- 22 control, 346 +/- 17 micromol/liter) and adipose tissue (264 +/- 25 vs. 318 +/- 25 micromol/liter) did not differ between groups. IMCL in soleus and tibialis anterior muscle did not change from baseline during hypoglycemia.In well-controlled T1DM, impaired hypoglycemia counterregulation is associated with decreased glucose production and augmented whole body lipolysis, which cannot be explained by either hydrolysis of muscle triglycerides or increased abdominal s.c. adipose tissue lipolysis.

There is a wide interindividual variation in peripheral insulin sensitivity at any given body mass index or percent body fat among obese adolescents with normal glucose tolerance. The goals of this study were to determine whether variability in insulin sensitivity is associated with differences in patterns of lipid partitioning or substrate use under fasting and hyperinsulinemic conditions. We compared 14 obese insulin-resistant adolescents with 14 obese insulin-sensitive controls, pair matched for age, gender, pubertal stage and body composition. Insulin sensitivity was assessed by the hyperinsulinemic-euglycemic clamp, intramyocellular lipid content by (1)H-nuclear magnetic resonance and visceral fat by magnetic resonance imaging. Obese insulin-sensitive subjects had lower intramyocellular (1.64 +/- 0.68 vs.2.26 +/- 0.62% of water peak, P = 0.017) and visceral lipid deposition (45 +/- 23 vs. 77 +/- 52 cm(2), P = 0.04) and a higher level of adiponectin, compared with their obese-resistant counterparts (8.8 +/- 3.6 vs. 6.5 +/- 1.8 mug/dl, P = 0.015). Glycerol fluxes were similar between the two obese groups yet occurred in the face of different concentrations of insulin. Intramyocellular lipid and visceral fat were negatively related to insulin sensitivity. Obese insulin-sensitive adolescents are characterized by lower lipid deposition in the intramyocellular and visceral compartments and greater levels of adiponectin, despite similar degree of adiposity.

We examined the impact of adolescent obesity on circulating adiponectin levels and the relationship between adiponectin and insulin sensitivity, intramyocellular (IMCL) lipid content, plasma triglycerides, and free fatty acids. Plasma adiponectin levels were measured in 8 nonobese (percentage fat, 18 +/- 1.8) and 14 obese adolescents (percentage fat, 41 +/- 1.6). Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp. Intramuscular lipid content was quantified using (1)H-nuclear magnetic resonance spectroscopy, and abdominal fat distribution by magnetic resonance imaging. Adiponectin levels were lower in obese adolescents (9.2 +/- 1 microg/ml, P < 0.001) and were positively related to insulin sensitivity in all subjects (r = 0.531, P < 0.02). Strong inverse relationships were found between adiponectin and triglyceride levels (r = -0.80, P < 0.001) and IMCL (r = -0.73, P < 0.001). Triglycerides (partial r(2) = 0.52; P < 0.0002) and IMCL (partial r(2) = 0.10; P < 0.05) were the most significant predictors of adiponectin levels, explaining 62% of the variation. In conclusion, plasma adiponectin levels are reduced in adolescent obesity and related to insulin resistance, independent of total body fat and central adiposity. There is a strong relationship between adiponectin and IMCL lipid content in this pediatric population. The putative modulatory effects of adiponectin on insulin sensitivity may, in part, be mediated via its effects on IMCL lipid content.

The increased risk of hypoglycemia during intensified treatment of type 1 diabetes mellitus (T1DM) patients, who have a deficient glucagon secretory response, is largely attributed to the development of suppressed adrenomedullary responses. A consequence of this impairment of catecholamine secretion might be reduced lipolysis in major target tissues (muscle and adipose) and, in turn, increased glucose metabolism. To test this hypothesis, we used microdialysis to monitor glycerol (index of lipolysis) in the extracellular fluid of skeletal muscle and adipose tissue and assessed whole-body glucose use by measuring [6,6-2H2]glucose enrichment in plasma in seven intensively treated T1DM patients and eight nondiabetic subjects who received a 3-h insulin infusion (0.8 mU/kg·min) on two occasions: during mild-moderate hypoglycemia or euglycemia. In the hypoglycemic study, the rise in plasma epinephrine was approximately 50% less in the T1DM patients despite a greater fall in plasma glucose (to 3.0 vs. 3.5 mm in controls; P < 0.05). Moreover, the rate of glucose flux and the plasma-extracellular fluid glucose gradient in muscle was increased during hypoglycemia in T1DM subjects compared with controls. Glycerol levels in muscle, adipose, and plasma fell similarly in both groups in the first hour. Thereafter, tissue glycerol remained suppressed in the T1DM patients but rebounded significantly (P < 0.01) in the control subjects. The glycerol response in muscle and adipose tissue was significantly correlated with plasma epinephrine concentration (r = 0.73, P = 0.002; and r = 0.52, P = 0.04, respectively), and inversely correlated with whole-body glucose disposal (r = −0.51, P = 0.05; and r = −0.50, P = 0.05). To determine whether the absence of the lipolytic response is limited to deficient catecholamine release, we perfused muscle and adipose tissue in situ with the selective β2-agonist terbutaline during hyperinsulinemic euglycemia. Local addition of agonist increased glycerol and blood flow in both muscle and adipose (P < 0.01 and P < 0.05, respectively) similarly in T1DM and control subjects. We conclude that deficient release of (rather than impaired responsiveness to) catecholamines in T1DM prevents the local fat breakdown within muscle and adipose tissue that normally occurs during mild-moderate hypoglycemia. This defect within peripheral tissues may lead to a delayed increase in glucose disposal that could contribute to the severity of hypoglycemia when it is prolonged.

Nuclear magnetic resonance (NMR) spectroscopy has made noninvasive and repetitive measurements of human hepatic glycogen concentrations possible. Monitoring of liver glycogen in real-time mode has demonstrated that glycogen concentrations decrease linearly and that net hepatic glycogenolysis contributes only about 50 percent to glucose production during the early period of a fast. Following a mixed meal, hepatic glycogen represents approximately 20 percent of the ingested carbohydrates, while only about 10 percent of an intravenous glucose load is retained by the liver as glycogen. During mixed-meal ingestion, poorly controlled type 1 diabetic patients synthesize only about 30 percent of the glycogen stored in livers of nondiabetic humans studied under similar conditions. Reduced net glycogen synthesis can be improved but not normalized by short-term, intensified insulin treatment. A decreased increment in liver glycogen content following meals was also found in patients with maturity-onset diabetes of the young due to glucokinase mutations (MODY-2). In patients with poorly controlled type 2 diabetes, fasting hyperglycemia can be attributed mainly to increased rates of endogenous glucose production, which was found by 13C NMR to be due to increased rates of gluconeogenesis. Metformin treatment improved fasting hyperglycemia in these patients through a reduction in hepatic glucose production, which could be attributed to a decrease in gluconeogenesis. In conclusion, NMR spectroscopy has provided new insights into the pathogenesis of hyperglycemia in type 1, type 2, and MODY diabetes and offers the potential of providing new insights into the mechanism of action of novel antidabetic therapies.

To examine the effect of caffeine ingestion on muscle glycogen utilization and the neuroendocrine axis during exercise, we studied 20 muscle glycogen-loaded subjects who were given placebo or caffeine (6 mg/kg) in a double blinded fashion 90 min before cycling for 2 h at 65% of their maximal oxygen consumption. Exercise-induced glycogen depletion in the thigh muscle was noninvasively measured by means of 13C nuclear magnetic resonance spectroscopy (NMR) spectroscopy, and plasma concentrations of substrates and neuroendocrine hormones, including beta-endorphins, were also assessed. Muscle glycogen content was increased 140% above normal values on the caffeine trial day (P < 0.001). After cycling for 2 h, caffeine ingestion was associated with a greater increase in plasma lactate (caffeine: +1.0 +/- 0.2 mmol/L; placebo, +0.1 +/- 0.2 mmol/L; P < 0.005), epinephrine (caffeine, +223 +/- 82 pg/mL; placebo, +56 +/- 26 pg/mL; P < 0.05), and cortisol (caffeine, +12 +/- 3 mg/mL; placebo, +2 +/- 2 mg/mL; P < 0.001) levels. However, plasma free fatty acid concentrations increased (caffeine, +814 +/- 133 mmol/L; placebo, +785 +/- 85 mmol/L; P = NS), and muscle glycogen content decreased (caffeine, -57 +/- 6 mmol/L muscle; placebo, -53 +/- 5 mmol/L muscle; P = NS) to the same extent in both groups. At the same time, plasma beta-endorphin levels almost doubled (from 30 +/- 5 to 53 +/- 13 pg/mL; P < 0.05) in the caffeine-treated group, whereas no change occurred in the placebo group. We conclude that caffeine ingestion 90 min before prolonged exercise does not exert a muscle glycogen-sparing effect in athletes with high muscle glycogen content. However, these data suggest that caffeine lowers the threshold for exercise-induced beta-endorphin and cortisol release, which may contribute to the reported benefits of caffeine on exercise endurance.

Depletion of muscle glycogen is considered a limiting performance factor during prolonged exercise, whereas the role of the intramyocellular lipid (IMCL) pool is not yet fully understood. We examined 1) intramyocellular glycogen and lipid utilization during prolonged exercise, 2) resynthesis of muscle glycogen and lipids during recovery, and 3) changes in glycogen content between nonexercising and exercising muscles during recovery. Subjects ran on a treadmill at submaximal intensity until exhaustion. Glycogen concentrations were assessed in thigh, calf, and nonexercising forearm muscle, and IMCL content was measured in soleus muscle using magnetic resonance spectroscopy techniques. At the time of exhaustion, glycogen depletion was 2-fold greater in calf than in thigh muscles, but a significant amount of glycogen was left in both leg muscles. The glycogen concentration in nonexercising forearm muscle decreased during the initial 5 h of recovery to 73% of the baseline value. During the exercise, the IMCL content decreased to 67% and subsequently during recovery increased to 83% of the baseline value. In summary, we found during prolonged running 1) significantly greater muscle glycogen utilization in the calf muscle group than in the thigh muscle group, 2) significant utilization of IMCL in the soleus muscle, and 3) a decrease in glycogen content in nonexercising muscle and an increase in glycogen content in recovering muscles during the postexercise phase. These latter data are consistent with the hypothesis that there is transfer of glycogen by the glucose→lactate and the glucose→alanine cycle from the resting muscle (forearm) to recovering muscles (thigh and calf) after running exercise .

We examined the effect of chronic hyperglycemia on phosphoinositide hydrolysis and insulin secretion in isolated perifused rat islets. Rats were infused for 44 h with 40% dextrose in order to raise and maintain the plasma glucose concentration at 350 mg/dl. Control animals were infused with equiosmolar amounts of mannitol. In vivo insulin secretion and rats of glucose disposal were monitored throughout the study. At the end of the infusion, islets were collagenase isolated, and phosphoinositide (PI) hydrolysis (assessed by measuring the increment in [3H]inositol efflux as well as labeled inositol phosphates) and insulin output in response to a 20-mM glucose challenge were quantitated. Plasma insulin concentration and in vivo glucose disposal rates decreased significantly, by 47% and 35% respectively, after 6-8 h of hyperglycemia. In islets perifused immediately after isolation, prior in vivo hyperglycemia markedly altered the pattern of insulin output in response to 20-mM glucose challenge. Compared to mannitol infusion, 20 mM glucose stimulation resulted in an exaggerated first phase insulin secretory response (1121 +/- 88 vs. 467 +/- 75 pg/islets.min) and a blunted second phase insulin secretory response (392 +/- 90 vs. 1249 +/- 205 pg/islet.min). In islets prelabeled with myo-[2-3H]inositol for 2 h, PI hydrolysis, particularly [3H]inositol efflux in response to glucose stimulation was also reduced (0.28 +/- 0.03%/min) compared to that in mannitol-infused animals (0.53 +/- 0.08%/min). Two hours of preincubation in a low glucose medium (2.75 mM) were able to completely reverse the islet defect in both PI hydrolysis and insulin secretion. Our results demonstrate that chronic in vivo hyperglycemia impairs PI hydrolysis in perifused rat islets and suggest that this defect accounts in part for the abnormal pattern of glucose-induced insulin secretion.

We studied two obese type II diabetic patients before, during, and after 3 yr of continuous iv insulin infusion, delivered by means of totally implanted insulin infusion pumps. Tolerance of the devices was excellent, and no side-effects or episodes of significant hypoglycemia occurred. Glycosuria was eliminated, and mean 24-h plasma glucose and hemoglobin A1c levels decreased in both patients and remained in or near the normal range for 3 yr. Improvements were also noted in serum triglyceride concentrations and vitreous fluorescein concentrations after iv fluorescein injection. Euglycemic insulin clamp studies showed that no significant change in glucose disposal rate occurred after 6 and 12 months of treatment. However, some improvement in insulin secretion during hyperglycemic insulin clamp studies occurred in both patients after 6 months of insulin infusion. Evaluation of the insulin-glycerol mixture used in the pump revealed that moderate degradation of insulin occurred in the pump during the 21-day flow cycle, resulting in 6-12% increases in fasting blood glucose levels; in addition, higher mol wt species of immunoreactive insulin were present in the patients' serum. We conclude that long term continuous iv infusion of insulin using a totally implantable infusion pump is practical in type II diabetic patients, is acceptable to patients, and is capable of providing near-normal glycemic control.