Reactive oxygen species are produced at low glucose and contribute to the activation of AMPK in insulin-secreting cells.

Excess reactive oxygen species (ROS) production is thought to play a key role in the loss of pancreatic ß-cell number and/or function, in response to high glucose and/or fatty acids. However, contradictory findings have been reported showing that in pancreatic ß cells or insulin-secreting cell lines, ROS are produced under conditions of either high or low glucose. Superoxide production was measured in attached INS1E cells as a function of glucose concentration, by following in real time the oxidation of dihydroethidine. Minimal values of superoxide production were measured at glucose concentrations of 5-20 mM, whereas superoxide generation was maximal at 0-1 mM glucose. Superoxide generation started rapidly (15-30 min) after exposure to low glucose and was suppressed by its addition within minutes. Superoxide was totally suppressed by rotenone, but not myxothiazol, suggesting a role for complex I in this process. Indirect evidence for mitochondrial ROS generation was also provided by a decrease in aconitase activity. Activation of AMPK, a cellular metabolic sensor, and its downstream target ACC by low glucose concentration was largely inhibited by addition of MnTBAP, a MnSOD and catalase mimetic that also totally suppressed superoxide production. Taken together, the data show that low glucose activates AMPK in a superoxide-dependent, AMP-independent way.

Adipose tissue plasticity during catch-up fat driven by thrifty metabolism: relevance for muscle-adipose glucose redistribution during catch-up growth.

OBJECTIVE: Catch-up growth, a risk factor for later type 2 diabetes, is characterized by hyperinsulinemia, accelerated body-fat recovery (catch-up fat), and enhanced glucose utilization in adipose tissue. Our objective was to characterize the determinants of enhanced glucose utilization in adipose tissue during catch-up fat. RESEARCH DESIGN AND METHODS: White adipose tissue morphometry, lipogenic capacity, fatty acid composition, insulin signaling, in vivo glucose homeostasis, and insulinemic response to glucose were assessed in a rat model of semistarvation-refeeding. This model is characterized by glucose redistribution from skeletal muscle to adipose tissue during catch-up fat that results solely from suppressed thermogenesis (i.e., without hyperphagia). RESULTS: Adipose tissue recovery during the dynamic phase of catch-up fat is accompanied by increased adipocyte number with smaller diameter, increased expression of genes for adipogenesis and de novo lipogenesis, increased fatty acid synthase activity, increased proportion of saturated fatty acids in triglyceride (storage) fraction but not in phospholipid (membrane) fraction, and no impairment in insulin signaling. Furthermore, it is shown that hyperinsulinemia and enhanced adipose tissue de novo lipogenesis occur concomitantly and are very early events in catch-up fat. CONCLUSIONS: These findings suggest that increased adipose tissue insulin stimulation and consequential increase in intracellular glucose flux play an important role in initiating catch-up fat. Once activated, the machinery for lipogenesis and adipogenesis contribute to sustain an increased insulin-stimulated glucose flux toward fat storage. Such adipose tissue plasticity could play an active role in the thrifty metabolism that underlies glucose redistribution from skeletal muscle to adipose tissue.

Fatty acids do not activate UCP2 in pancreatic beta cells: comparison with UCP1.

UCP2 is expressed in pancreatic beta cells where its postulated uncoupling activity will modulate glucose-induced changes in ATP/ADP ratio and insulin secretion. The consequences of UCP2 over/underexpression on beta-cell function has mainly been studied in the basal state; however, a UCP has no uncoupling activity unless stimulated by fatty acids and/or reactive oxygen species. Here, UCP2 was overexpressed in INS-1 cells and parameters reflecting mitochondrial coupling measured in the basal state and after stimulation by fatty acids. For comparison, UCP1 was expressed to similar levels and the same parameters measured. Neither UCP1 expression nor UCP2 overexpression modified basal or glucose-stimulated metabolic changes. Upon addition of fatty acids, UCP1-expressing cells displayed the expected mitochondrial uncoupling effect, while UCP2 did not elicit any measurable change in mitochondrial function. Taken together, our data demonstrate that, in pancreatic beta-cells, UCP2 has no uncoupling activity in the basal state or after fatty acid stimulation.

Independent evolution of heart autonomic function and insulin sensitivity during weight loss.

In order to investigate the improvement of insulin resistance and cardiac autonomic function along massive weight loss, 12 obese women were evaluated before, and 3 and 12 months after Roux-en-Y gastric bypass. The 12-month values were compared to those of BMI-matched controls. Insulin sensitivity was assessed by euglycemic clamp and the cardiac autonomic function by the analysis of the Heart Rate Variability (HRV). After surgery, glucose uptake progressively increased from 4.3 +/- 0.5 mg/kg lean body mass (LBM)/min preoperative (pre-op) to 4.9 +/- 0.5 and 7.0 +/- 0.5, 3- and 12-month postoperative (post-op) (P = 0.04 and P = 0.006 vs. pre-op), whereas the cardiac autonomic function showed a biphasic pattern. HRV values increased 3 months post-op, and decreased at 12 months, thus indicating an early sympathetic withdrawal followed by a later reactivation (e.g., the standard deviation of the normal-to-normal intervals was 116 +/- 7 ms in pre-op, 161 +/- 10 at 3 months, P = 0.008 vs. pre-op, and 146 +/- 15 at 12 months, P = 0.03 vs. pre-op and P = 0.02 vs. 3 m). Insulin sensitivity was significantly related to body weight (P = 0.02), whereas the cardiac indexes were significantly linked to the profile of energy intake (e.g., HRV triangular index vs. energy intake P = 0.003). No significant relationship linked insulin sensitivity to the cardiac autonomic indexes. Insulin sensitivity and cardiac parameters of the 12-month post-op patients were similar to their matched controls. During massive weight loss, the cardiac autonomic deregulation and insulin resistance improved concomitantly but independently from each other. Our results suggest that the extent of the improvement is associated with the final body weight.

Characterization and localization of cocaine- and amphetamine-regulated transcript (CART) binding sites.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the brain and various endocrine tissues. CART is implicated in many physiological functions including food intake, drug reward, stress and nociception. No CART receptor has been identified yet. We fused CART(55-102) to the green fluorescent protein (GFP) and found that the ligand suppresses significantly food intake after intracerebroventricular (i.c.v.) injection in mice. Using this ligand, we show specific CART binding sites on HepG2 cells and hypothalamic dissociated cells. In brain sections, CART displaceable binding sites were observed on cell bodies mainly localized in hypothalamic periventricular areas.

Cloning, ontogenesis, and localization of an atypical uncoupling protein 4 in Xenopus laevis.

Uncoupling protein 1 (UCP1) is the first UCP described. It belongs to the family of mitochondrial carrier proteins and is expressed mainly in brown adipose tissue. Recently, the family of the UCPs has rapidly been growing due to the successive cloning of UCP2, UCP3, UCP4, and UCP5, also called brain mitochondrial carrier protein 1. Phylogenetic studies suggest that UCP1/UCP2/UCP3 on one hand and UCP4/UCP5 on the other hand belong to separate subfamilies. In this study, we report the cloning from a frog Xenopus laevis (Xl) oocyte cDNA library of a novel UCP that was shown, by sequence homology, to belong to the family of ancestral UCP4. This cloning provides a milestone in the gap between Drosophila melanogaster or Caenorhabditis elegans on one hand and mammalian UCP4 on the other. Xl UCP4 is already expressed in the oocyte, being the first UCP described in germ cell lineage. During development, it segregates in the neural cord, and, in the adult, in situ hybridization shows its expression in the neurons and also in the choroid plexus of the brain. By RT-PCR analysis, it was found that Xl UCP4 is present in all the subdivisions of the brain and also that it differs from mammalian UCP4 by a very high relative level of expression in peripheral tissues such as the liver and kidney. The peripheral tissue distribution of Xl UCP4 reinforces the hypothesis that UCP4 might be the ancestral UCP from which other UCPs diverged from.

Intracerebroventricular infusion of a triglyceride emulsion leads to both altered insulin secretion and hepatic glucose production in rats.

We investigated here whether non-esterified fatty acids (NEFA) influence insulin secretion and action through a direct effect on central nervous system sites involved in the control of glucose homeostasis. Normal Wistar rats received a 48-h intracerebroventricular infusion of either a 10% triglyceride (Intralipid, IL)/heparin emulsion (IL/h) or saline/heparin solution (control). At 48 h, insulin secretion as measured by an intravenous glucose tolerance test, was more elevated in IL/h than in control rats. Pancreatic noradrenaline turnover was decreased by 57% in IL/h rats, suggesting low pancreatic sympathetic output that could account partly for the elevated insulin secretion. The time course of glycaemia was similar in both groups, suggesting insulin resistance. Euglycaemic-hyperinsulinaemic clamps were imposed to assess peripheral and hepatic insulin sensitivity. At each insulin concentration glucose utilization was increased to a similar extent in both groups, whereas hepatic glucose production decreased much less in IL/h than in control rats. Hepatic insulin insensitivity could be related partly to activation of the hypothalamic-pituitary-adrenocortical axis, since plasma corticosterone concentration was significantly increased in IL/h rats compared with controls. Our data indicate that lipids may alter both insulin secretion and hepatic sensitivity to insulin through their effect on central nervous system.

Progressive defect of insulin action on glycogen synthase in obesity and diabetes.

The purpose of the present work was to have a closer view on the changes in the regulation of glycogen synthase (GS) activity by insulin in relationship with the impairment of nonoxidative glucose disposal in human obesity. Obese patients with normal glucose tolerance (12), impaired glucose tolerance (11), diabetes (10), and lean control subjects (15) participated to the study. A euglycemic, hyperinsulinemic clamp was performed and associated with indirect calorimetry. Muscle needle biopsies were taken before and at the end of the 2-hour clamp for measurements of glycogen synthase fractional velocity and total activity. Total GS activity was significantly decreased (P <.05), while its percent activation by insulin was still normal in the obese glucose-tolerant group, and nonoxidative glucose disposal was decreased by 56% (P <.001) and glucose oxidation still normal. Total GS activity was decreased by about 50% (P <.01) and GS was unresponsive to insulin in the glucose-intolerant and diabetic groups. In conclusion, our data show that insulin-stimulated nonoxidative glucose disposal and total glycogen synthase are very early defects observed in obese patients.

IL-1 receptor antagonist serum levels are increased in human obesity: a possible link to the resistance to leptin?

We have recently shown that human monocytic cells express functional leptin receptors and that leptin is capable of inducing the expression and secretion of the IL-1 receptor antagonist (IL-1Ra). Although IL-1Ra has anti-inflammatory and possibly anti-atherogenic properties, it has also been shown to antagonize the action of leptin at the hypothalamic level in rodents, thereby inducing leptin resistance. We have therefore examined whether IL-1Ra levels are increased in human hyperleptinemic conditions, such as obesity. To this end, we measured serum IL-1Ra levels in 20 morbidly obese nondiabetic subjects [body mass index (BMI), 45 +/- 6 kg/m(2); serum leptin, 52 +/- 20 ng/ml] as well as in 10 age- and sex-matched lean controls (BMI, 22 +/- 2 kg/m(2); serum leptin, 7 +/- 4 ng/ml). Serum IL-1Ra concentrations proved to be elevated 6.5-fold in the obese subjects, and they were positively correlated in a linear manner with the leptin levels (r(2) = 0.34; P = 0.01), although lean body mass (LBM) and the insulin resistance index were even better predictors of IL-1Ra levels (r(2) = 0.45 and 0.58, respectively; P < 0.01). Six months after 15 of the 20 obese subjects had undergone bypass surgery for their morbid obesity, their mean BMI and leptin levels decreased to 33 +/- 7 kg/m(2) and 18 plus minus 12 ng/ml, respectively. This change in leptin concentrations was associated with a significant reduction in IL-1Ra levels (P < 0.02). However, there was a better correlation between the decrease in IL-1Ra level and the change in LBM than with the reduction in leptin levels, indicating that leptin is not the sole determinant of circulating IL-1Ra in obesity. In summary, we demonstrate that IL-1Ra levels are highly elevated in human obesity and that its concentrations decrease after weight loss from bypass surgery. However, LBM and insulin resistance are better predictors of serum IL-1Ra concentrations than are leptin levels, suggesting that additional metabolic factors control the secretion of this cytokine antagonist. Although the immunological consequences of this alteration remain unknown, it is tempting to speculate that the obesity-related increase in IL-1Ra might contribute to the central resistance to leptin in obese patients, similar to the inhibition of the hypothalamic signaling of leptin by IL-1Ra in rodents.