Eva Rother

Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity

Leptin and insulin have been identified as fuel sensors acting in part through their hypothalamic receptors to inhibit food intake and stimulate energy expenditure. As their intracellular signaling converges at the PI3K pathway, we directly addressed the role of phosphatidylinositol3,4,5-trisphosphate-mediated (PIP3-mediated) signals in hypothalamic proopiomelanocortin (POMC) neurons by inactivating the gene for the PIP3 phosphatase Pten specifically in this cell type. Here we show that POMC-specific disruption of Pten resulted in hyperphagia and sexually dimorphic diet-sensitive obesity. Although leptin potently stimulated Stat3 phosphorylation in POMC neurons of POMC cell-restricted Pten knockout (PPKO) mice, it failed to significantly inhibit food intake in vivo. POMC neurons of PPKO mice showed a marked hyperpolarization and a reduction in basal firing rate due to increased ATP-sensitive potassium (KATP) channel activity. Leptin was not able to elicit electrical activity in PPKO POMC neurons, but application of the PI3K inhibitor LY294002 and the KATP blocker tolbutamide restored electrical activity and leptin-evoked firing of POMC neurons in these mice. Moreover, icv administration of tolbutamide abolished hyperphagia in PPKO mice. These data indicate that PIP3-mediated signals are critical regulators of the melanocortin system via modulation of KATP channels.

PDK1 Deficiency in POMC-Expressing Cells Reveals FOXO1-Dependent and -Independent Pathways in Control of Energy Homeostasis and Stress Response

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.

Activation of Stat3 Signaling in AgRP Neurons Promotes Locomotor Activity

Leptin, an adipocyte-derived hormone, acts on hypothalamic neurons located in the arcuate nucleus (ARC) of the hypothalamus to regulate energy homeostasis. One of the leptin-regulated neuronal subtypes in the ARC are agouti-related peptide (AgRP)-expressing neurons, which are involved in the regulation of food intake and are directly inhibited by leptin. Leptin activates the signal transducer and activator of transcription 3 (Stat3), but the role of Stat3 in the regulation of AgRP neurons is unclear. Here we show that mice expressing a constitutively active version of Stat3 selectively in AgRP neurons are lean and exhibit relative resistance to diet-induced obesity. Surprisingly, this phenotype arises from increased locomotor activity in the presence of unaltered AgRP expression. These data demonstrate that Stat3-dependent signaling in AgRP neurons in the ARC controls locomotor activity independently of AgRP regulation.

Secondary Consequences of β Cell Inexcitability: Identification and Prevention in a Murine Model of KATP-Induced Neonatal Diabetes Mellitus

ATP-insensitive K(ATP) channel mutations cause neonatal diabetes mellitus (NDM). To explore the mechanistic etiology, we generated transgenic mice carrying an ATP-insensitive mutant K(ATP) channel subunit. Constitutive expression in pancreatic beta cells caused neonatal hyperglycemia and progression to severe diabetes and growth retardation, with loss of islet insulin content and beta cell architecture. Tamoxifen-induced expression in adult beta cells led to diabetes within 2 weeks, with similar secondary consequences. Diabetes was prevented by transplantation of normal islets under the kidney capsule. Moreover, the endogenous islets maintained normal insulin content and secretion in response to sulfonylureas, but not glucose, consistent with reduced ATP sensitivity of beta cell K(ATP) channels. In NDM, transfer to sulfonylurea therapy is less effective in older patients. This may stem from poor glycemic control or lack of insulin because glibenclamide treatment prior to tamoxifen induction prevented diabetes and secondary complications in mice but failed to halt disease progression after diabetes had developed.

Hypothalamic JNK1 and IKKβ activation and impaired early postnatal glucose metabolism after maternal perinatal high-fat feeding.

Hypothalamic inflammation has been demonstrated to be an important mechanism in the pathogenesis of obesity-induced type 2 diabetes mellitus. Feeding pregnant and lactating rodents a diet rich in saturated fatty acids has consistently been shown to predispose the offspring for the development of obesity and impaired glucose metabolism. However, hypothalamic inflammation in the offspring has not been addressed as a potential underlying mechanism. In this study, virgin female C57BL/6 mice received high-fat feeding starting at conception until weaning of the offspring at postnatal d 21. The offspring developed increased body weight, body fat content, and serum leptin concentrations during the nursing period. Analysis of hypothalamic tissue of the offspring at postnatal d 21 showed up-regulation of several members of the toll-like receptor 4 signaling cascade and subsequent activation of c-Jun N-terminal kinase 1 and IκB kinase-β inflammatory pathways. Interestingly, glucose tolerance testing in the offspring revealed signs of impaired glucose tolerance along with increased hepatic expression of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase. In addition, significantly increased hepatic and pancreatic PGC1α expression suggests a role for sympathetic innervation in mediating the effects of hypothalamic inflammation to the periphery. Taken together, our data indicate an important role for hypothalamic inflammation in the early pathogenesis of glucose intolerance after maternal perinatal high-fat feeding.

Neurocircuits integrating hormone and nutrient signaling in control of glucose metabolism

As obesity, diabetes, and associated comorbidities are on a constant rise, large efforts have been put into better understanding the cellular and molecular mechanisms by which nutrients and metabolic signals influence central and peripheral energy regulation. For decades, peripheral organs as a source and a target of such cues have been the focus of study. Their ability to integrate metabolic signals is essential for balanced energy and glucose metabolism. Only recently has the pivotal role of the central nervous system in the control of fuel partitioning been recognized. The rapidly expanding knowledge on the elucidation of molecular mechanisms and neuronal circuits involved is the focus of this review.

Acute selective ablation of rat insulin promoter-expressing (RIPHER) neurons defines their orexigenic nature

Rat insulin promoter (RIP)-expressing neurons in the hypothalamus control body weight and energy homeostasis. However, genetic approaches to study the role of these neurons have been limited by the fact that RIP expression is predominantly found in pancreatic β-cells, which impedes selective targeting of neurons. To define the function of hypothalamic RIP-expressing neurons, we set out to acutely and selectively eliminate them via diphtheria toxin-mediated ablation. Therefore, the diphtheria toxin receptor transgene was specifically expressed upon RIP-specific Cre recombination using a RIP-Cre line first described by Herrera (RIPHER-Cre) [Herrera PL (2000) Development 127:2317–2322]. Using proopiomelanocortin–expressing cells located in the arcuate nucleus of the hypothalamus and in the pituitary gland as a model, we established a unique protocol of intracerebroventricular application of diphtheria toxin to efficiently ablate hypothalamic cells with no concomitant effect on pituitary proopiomelanocortin–expressing corticotrophs in the mouse. Using this approach to ablate RIPHER neurons in the brain, but not in the pancreas, resulted in decreased food intake and loss of body weight and fat mass. In addition, ablation of RIPHER neurons caused increased c-Fos immunoreactivity of neurons in the paraventricular nucleus (PVN) of the hypothalamus. Moreover, transsynaptic tracing of RIPHER neurons revealed labeling of neurons located in the PVN and dorsomedial hypothalamic nucleus. Thus, our experiments indicate that RIPHER neurons inhibit anorexigenic neurons in the PVN, revealing a basic orexigenic nature of these cells.

Early Postnatal Hyperalimentation Impairs Renal Function via SOCS-3 Mediated Renal Postreceptor Leptin Resistance

Early postnatal hyperalimentation has long-term implications for obesity and developing renal disease. Suppressor of cytokine signaling (SOCS) 3 inhibits phosphorylation of signal transducer and activator of transcription (STAT) 3 and ERK1/2 and thereby plays a pivotal role in mediating leptin resistance. In addition, SOCS-3 is induced by both leptin and inflammatory cytokines. However, little is known about the intrinsic-renal leptin synthesis and function. Therefore, this study aimed to elucidate the implications of early postnatal hyperalimentation on renal function and on the intrinsic-renal leptin signaling. Early postnatal hyperalimentation in Wistar rats during lactation was induced by litter size reduction at birth (LSR) either to LSR10 or LSR6, compared with home cage control male rats. Assessment of renal function at postnatal day 70 revealed decreased glomerular filtration rate and proteinuria after LSR6. In line with this impairment of renal function, renal inflammation and expression as well as deposition of extracellular matrix molecules, such as collagen I, were increased. Furthermore, renal expression of leptin and IL-6 was up-regulated subsequent to LSR6. Interestingly, the phosphorylation of Stat3 and ERK1/2 in the kidney, however, was decreased after LSR6, indicating postreceptor leptin resistance. In accordance, neuropeptide Y (NPY) gene expression was down-regulated; moreover, SOCS-3 protein expression, a mediator of postreceptor leptin resistance, was strongly elevated and colocalized with NPY. Thus, our findings not only demonstrate impaired renal function and profibrotic processes but also provide compelling evidence of a SOCS-3-mediated intrinsic renal leptin resistance and concomitant up-regulated NPY expression as an underlying mechanism.

Developmental regulation of inflammatory cytokine-mediated Stat3 signaling: the missing link between intrauterine growth restriction and pulmonary dysfunction?

Intrauterine growth restriction (IUGR) is a risk factor for impairment of lung function in adolescence and adulthood. Inflammatory and proliferative processes linking IUGR and perturbed extracellular matrix (ECM) as an underlying mechanism have not been addressed so far. Therefore, in this study, we aimed to investigate the developmental regulation of inflammatory and profibrotic processes in the lung subsequent to IUGR. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 28 and P70. Lungs were obtained at P1, P42, and P70 for assessment of mRNA, protein expression, immunohistochemistry, and gelatinolytic activity. Both respiratory system resistance and compliance were impaired subsequent to IUGR at P28 and this impairment was even more pronounced at P70. In line with these results, the expression of ECM components and metabolizing enzymes was deregulated. The deposition of collagen was increased at P70. In addition, the expression of inflammatory cytokines and both the activity and the expression of target genes of Stat3 signaling were dynamically regulated, with unaltered or decreased expression at P1 and significantly increased expression art P70. Taken together, these data give evidence for an age-dependent impairment of lung function as a result of a developmentally regulated increase in inflammatory and profibrotic processes subsequent to IUGR.

Prevention of early postnatal hyperalimentation protects against activation of transforming growth factor-β/bone morphogenetic protein and interleukin-6 signaling in rat lungs after intrauterine growth restriction.

BACKGROUND Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date. OBJECTIVE The aim of this study was to investigate whether prevention of HA subsequent to IUGR protects the lung from 1) deregulation of the transforming growth factor-β(TGF-β)/bone morphogenetic protein (BMP) pathway, 2) activation of interleukin (IL)-6 signaling, and 3) profibrotic processes. METHODS IUGR was induced in Wistar rats by isocaloric protein restriction during gestation by feeding a control (Co) or a low-protein diet with 17% or 8% casein, respectively. On postnatal day 1 (P1), litters from both groups were randomly reduced to 6 pups per dam to induce HA or adjusted to 10 pups and fed with standard diet: Co, Co with HA (Co-HA), IUGR, and IUGR with HA (IUGR-HA). RESULTS Birth weights in rats after IUGR were lower than in Co rats (P < 0.05). HA during lactation led to accelerated body weight gain from P1 to P23 (Co vs. Co-HA, IUGR vs. IUGR-HA; P < 0.05). At P70, prevention of HA after IUGR protected against the following: 1) activation of both TGF-β [phosphorylated SMAD (pSMAD) 2; plasminogen activator inhibitor 1 (Pai1)] and BMP signaling [pSMAD1; inhibitor of differentiation (Id1)] compared with Co (P < 0.05) and Co or IUGR (P < 0.05) rats, respectively; 2) greater mRNA expression of interleukin (Il) 6 and Il13 (P < 0.05) as well as activation of signal transducer and activator of transcription 3 (STAT3) signaling (P < 0.05) after IUGR-HA; and 3) greater gene expression of collagen Iα1 and osteopontin (P < 0.05) and increased deposition of bronchial subepithelial connective tissue in IUGR-HA compared with Co and IUGR rats. Moreover, HA had a significant additive effect (P < 0.05) on the increased enhanced pause (indicator of airway resistance) in the IUGR group (P < 0.05) at P70. CONCLUSIONS This study demonstrates a dual mechanism in IUGR-associated lung disease that is 1) IUGR-dependent and 2) HA-mediated and thereby offers new avenues to develop innovative preventive strategies for perinatal programming of adult lung diseases.