Featured ArticlesVolume 10 | April 2018
|NAc inflammation mediates anxiodepressive behavior elicited by saturated dietary fatObesity considerably elevates the odds of developing depression. Obesity in humans and murine models is characterized by a state of chronic low-grade inflammation. Several lines of evidence implicate a neuroimmune etiology in a subset of depressed individuals; thus, persistent immune activation in obesity may give rise to mood impairments. Décarie-Spain, Sharma et al. sought to identify neuroinflammatory responses in the nucleus accumbens (NAc) and determine if they underlie anxiodepressive behavior provoked by diet-induced obesity. They found that a saturated, high fat diet (HFD) enhances systemic inflammation and anxiety-like and despair responses in behavioral tests. Concurrently, the saturated HFD was found to increase the expression of several immune markers in the NAc.
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Objective: The incidence of depression is significantly compounded by obesity. Obesity arising from excessive intake of high-fat food provokes anxiodepressive behavior and elicits molecular adaptations in the nucleus accumbens (NAc), a region well-implicated in the hedonic deficits associated with depression and in the control of food-motivated behavior. To determine the etiology of diet-induced depression, we studied the impact of different dietary lipids on anxiodepressive behavior and metabolic and immune outcomes and the contribution of NAc immune activity.
Methods: Adult C57Bl/6 mice were subjected to isocaloric high-fat/high-sucrose diets (HFD), enriched in either saturated or monounsaturated fat, or a control low-fat diet (LFD). Metabolic responses, anxiodepressive behavior, and plasma and NAc inflammatory markers were assessed after 12 weeks. In separate experiments, an adenoviral construct inhibiting IKKβ, an upstream component of the nuclear factor kappa-b (NFkB) pathway, was a priori injected into the NAc.
Results: Both HFDs resulted in obesity and hyperleptinemia; however, the saturated HFD uniquely triggered anxiety-like behavior, behavioral despair, hyperinsulinemia, glucose intolerance, peripheral inflammation, and multiple pro-inflammatory signs in the NAc, including reactive gliosis, increased expression of cytokines, antigen-presenting markers and NFкB transcriptional activity. Selective NAc IKKβ inhibition reversed the upregulated expression of inflammatory markers, prevented anxiodepressive behavior and blunted compulsive sucrose-seeking in mice fed the saturated HFD.
Conclusions: Metabolic inflammation and NFкB-mediated neuroinflammatory responses in the NAc contribute to the expression of anxiodepressive behavior and heightened food cravings caused by a diet high in saturated fat and sugar.[Hide abstract]
|Rapid sensing of leucine by hypothalamic neuronsDietary proteins strongly influence metabolic health via their effect on appetite, weight gain, and adiposity. Levels of the branched-chain amino acid leucine are likely to represent a physiological signal of protein availability in the control of appetite and metabolism. Appetite-regulating neurons in the arcuate nucleus of the hypothalamus (ARH) are well positioned to sense changes in leucine levels. However, the molecular, neurophysiological, and neurochemical mechanisms involved remain poorly understood. Heeley et al. show that a specialized group of mediobasal hypothalamus (MBH) neurons rapidly respond to physiological changes in extracellular leucine concentrations. This requires the modulation of plasma membrane calcium channels.|
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Objective: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine.
Methods: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration.
Results: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current.
Conclusions: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity.[Hide abstract]
|Prolyl carboxypeptidase in AgRP neurons modulates food intake and body weightThe anorectic peptide α-melanocyte stimulating hormone (α-MSH) is a product of the proopiomelanocortin (POMC) gene. Peripheral administration of α-MSH does not reduce food intake, suggesting that α-MSH is rapidly degraded. Prolyl carboxypeptidase (PRCP) is a key enzyme responsible for the degradation of α-MSH. Bruschetta and colleagues provide evidence that PRCP in Neuropeptide Y/Agouti-related peptide neurons modulate melanocortin signaling in the hypothalamic paraventricular nucleus via control of α-MSH degradation.
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Objective: Prolyl carboxypeptidase (PRCP) plays a role in the regulation of energy metabolism by inactivating hypothalamic α-melanocyte stimulating hormone (α-MSH) levels. Although detected in the arcuate nucleus, limited PRCP expression has been observed in the arcuate POMC neurons, and its site of action in regulating metabolism is still ill-defined.
Methods: We performed immunostaining to assess the localization of PRCP in arcuate Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons. Hypothalamic explants were then used to assess the intracellular localization of PRCP and its release at the synaptic levels. Finally, we generated a mouse model to assess the role of PRCP in NPY/AgRP neurons of the arcuate nucleus in the regulation of metabolism.
Results: Here we show that PRCP is expressed in NPY/AgRP-expressing neurons of the arcuate nucleus. In hypothalamic explants, stimulation by ghrelin increased PRCP concentration in the medium and decreased PRCP content in synaptic extract, suggesting that PRCP is released at the synaptic level. In support of this, hypothalamic explants from mice with selective deletion of PRCP in AgRP neurons (PrcpAgRPKO) showed reduced ghrelin-induced PRCP concentration in the medium compared to controls mice. Furthermore, male PrcpAgRPKO mice had decreased body weight and fat mass compared to controls. However, this phenotype was sex-specific as female PrcpAgRPKO mice show metabolic differences only when challenged by high fat diet feeding. The improved metabolism of PrcpAgRPKO mice was associated with reduced food intake and increased energy expenditure, locomotor activity, and hypothalamic α-MSH levels. Administration of SHU9119, a potent melanocortin receptor antagonist, selectively in the PVN of PrcpAgRPKO male mice increased food intake to a level similar to that of control mice.
Conclusions: Altogether, our data indicate that PRCP is released at the synaptic levels and that PRCP in AgRP neurons contributes to the modulation of α-MSH degradation and related metabolic control in mice.[Hide abstract]
|PPARα is dispensable for cold-induced adipose tissue browningCold exposure in mice causes specific white adipose tissue (WAT) depots to adopt features of brown adipose tissue (BAT), a process known as browning. One of the transcription factors that has been implicated in browning is Peroxisome Proliferator-Activated Receptor α (PPARα), mainly known as the master regulator of lipid metabolism in the liver during fasting. Defour and colleagues find that cold-induced changes in gene expression in inguinal WAT are unaltered in mice lacking PPARα, indicating that PPARα is dispensable for cold-induced browning.|
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Objective: Chronic cold exposure causes white adipose tissue (WAT) to adopt features of brown adipose tissue (BAT), a process known as browning. Previous studies have hinted at a possible role for the transcription factor Peroxisome Proliferator-Activated Receptor alpha (PPARα) in cold-induced browning. Here we aimed to investigate the importance of PPARα in driving transcriptional changes during cold-induced browning in mice.
Methods: Male wildtype and PPARα−/− mice were housed at thermoneutrality (28 °C) or cold (5 °C) for 10 days. Whole genome expression analysis was performed on inguinal WAT. In addition, other analyses were carried out. Whole genome expression data of livers of wildtype and PPARα−/− mice fasted for 24 h served as positive control for PPARα-dependent gene regulation.
Results: Cold exposure increased food intake and decreased weight of BAT and WAT to a similar extent in wildtype and PPARα−/− mice. Except for plasma non-esterified fatty acids, none of the cold-induced changes in plasma metabolites were dependent on PPARα genotype. Histological analysis of inguinal WAT showed clear browning upon cold exposure but did not reveal any morphological differences between wildtype and PPARα−/− mice. Transcriptomics analysis of inguinal WAT showed a marked effect of cold on overall gene expression, as revealed by principle component analysis and hierarchical clustering. However, wildtype and PPARα−/− mice clustered together, even after cold exposure, indicating a similar overall gene expression profile in the two genotypes. Pathway analysis revealed that cold upregulated pathways involved in energy usage, oxidative phosphorylation, and fatty acid β-oxidation to a similar extent in wildtype and PPARα−/− mice. Furthermore, cold-mediated induction of genes related to thermogenesis such as Ucp1, Elovl3, Cox7a1, Cox8, and Cidea, as well as many PPAR target genes, was similar in wildtype and PPARα−/− mice. Finally, pharmacological PPARα activation had a minimal effect on expression of cold-induced genes in murine WAT.
Conclusions: Cold-induced changes in gene expression in inguinal WAT are unaltered in mice lacking PPARα, indicating that PPARα is dispensable for cold-induced browning.[Hide abstract]
|Disruption of seipin causes early-onset lipodystrophy and altered fuel utilisation Congenital generalized lipodystrophy (CGL) is a rare, autosomal recessive, genetic disorder characterized by dramatically reduced adipose tissue mass from birth. Individuals with CGL develop hepatic steatosis, hyperlipidaemia, and severe insulin resistance. One possible cause for CGL is a mutation of the gene BSCL2, which codes for the protein seipin. Mcilroy et al. used the non-inducible Adipoq-Cre model to investigate the consequences of ablating Bscl2 in developing and mature adipocytes. The data reveal that seipin deficiency in developing adipocytes is sufficient to cause severe generalized lipodystrophy early in life but that this does not induce all of the metabolic consequences of congenital seipin deficiency.|
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Objective: Mutations to the BSCL2 gene disrupt the protein seipin and cause the most severe form of congenital generalised lipodystrophy (CGL). Affected individuals exhibit a near complete loss of white adipose tissue (WAT) and suffer from metabolic disease. Seipin is critical for adipocyte development in culture and mice with germline disruption to Bscl2 recapitulate the effects of BSCL2 disruption in humans. Here we examined whether loss of Bscl2 specifically in developing adipocytes in vivo is sufficient to prevent adipose tissue development and cause all features observed with congenital BSCL2 disruption.
Methods: We generated and characterised a novel mouse model of Bscl2 deficiency in developing adipocytes (Ad-B2(−/−)) using the adipose-specific Adiponectin-Cre line.
Results: We demonstrate that Ad-B2(−/−) mice display early onset lipodystrophy, in common with congenital Bscl2 null mice and CGL2 patients. However, glucose intolerance, insulin resistance, and severe hepatic steatosis are not apparent. Food intake and energy expenditure are unchanged, but Ad-B2(−/−) mice exhibit significantly altered substrate utilisation. We also find differential effects of seipin loss between specific adipose depots revealing new insights regarding their varied characteristics. When fed a high-fat diet, Ad-B2(−/−) mice entirely fail to expand adipose mass but remain glucose tolerant.
Conclusions: Our findings demonstrate that disruption of Bscl2 specifically in developing adipocytes is sufficient to cause the early-onset generalised lipodystrophy observed in patients with mutations in BSCL2. However, this significant reduction in adipose mass does not cause the overt metabolic dysfunction seen in Bscl2 knockout mice, even following a high-fat diet challenge.[Hide abstract]
|Evidence against a role for NLRP3-driven islet inflammation in db/db miceType 2 diabetes (T2D) is now recognized as a chronic inflammatory disease. In particular, the pro-inflammatory cytokine interleukin-1β (IL-1β) has been highlighted as a potent driver of beta cell dysfunction. Mature IL-1β is mainly produced through the multi-protein inflammasome complexes such as NOD-like receptor pyrin domain containing protein 3 (NLRP3) inflammasome. The orally available small molecule MCC950 potently and specifically inhibits NLRP3 activation and should prevent the decay of pancreatic islets. However, Kammoun et al. found that that MCC950 has no effect in db/db mice. Also, neither NLRP3 nor IL-1β mRNA expression were elevated in the islets from db/db mice. Their data caution the use of db/db mice as an appropriate pre-clinical model of T2D, particularly when testing the efficacy of drugs that target insulin secretory pathways and islet inflammation.|
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Objective: Type 2 diabetes (T2D) is associated with chronic, low grade inflammation. Activation of the NLRP3 inflammasome and secretion of its target interleukin-1β (IL-1β) have been implicated in pancreatic β cell failure in T2D. Specific targeting of the NLRP3 inflammasome to prevent pancreatic β cell death could allow for selective T2D treatment without compromising all IL-1β-associated immune responses. We hypothesized that treating a mouse model of T2D with MCC950, a compound that specifically inhibits NLRP3, would prevent pancreatic β cell death, thereby preventing the onset of T2D.
Methods: Diabetic db/db mice were treated with MCC950 via drinking water for 8 weeks from 6 to 14 weeks of age, a period over which they developed pancreatic β cell failure. We assessed metabolic parameters such as body composition, glucose tolerance, or insulin secretion over the course of the intervention.
Results: MCC950 was a potent inhibitor of NLRP3-induced IL-1β in vitro and was detected at high levels in the plasma of treated db/db mice. Treatment of pre-diabetic db/db mice with MCC950, however, did not prevent pancreatic dysfunction and full onset of the T2D pathology. When examining the NLRP3 pathway in the pancreas of db/db mice, we could not detect an activation of this pathway nor increased levels of its target IL-1β.
Conclusions: NLRP3 driven-pancreatic IL-1β inflammation does not play a key role in the pathogenesis of the db/db murine model of T2D.[Hide abstract]
|Modeling pancreatic beta cell dedifferentiationType 2 diabetes develops as a consequence of a combination of insulin resistance and insufficient β cell mass. The decline in β cell mass has been attributed to a decrease in both β cell number and function. It has been suggested recently that β cell dedifferentiation represents a mechanism to explain loss of functioning β cells. Diedisheim et al. used EndoC-βH1, a recently developed functional human β cell line, to model β cell dedifferentiation in a human context. Their model implicates fibroblast growth factor signaling in the dedifferentiation process.|
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Objective: Dedifferentiation could explain reduced functional pancreatic β-cell mass in type 2 diabetes (T2D).
Methods: Here we model human β-cell dedifferentiation using growth factor stimulation in the human β-cell line, EndoC-βH1, and human pancreatic islets.
Results: Fibroblast growth factor 2 (FGF2) treatment reduced expression of β-cell markers, (INS, MAFB, SLC2A2, SLC30A8, and GCK) and activated ectopic expression of MYC, HES1, SOX9, and NEUROG3. FGF2-induced dedifferentiation was time- and dose-dependent and reversible upon wash-out. Furthermore, FGF2 treatment induced expression of TNFRSF11B, a decoy receptor for RANKL and protected β-cells against RANKL signaling. Finally, analyses of transcriptomic data revealed increased FGF2 expression in ductal, endothelial, and stellate cells in pancreas from T2D patients, whereas FGFR1, SOX,9 and HES1 expression increased in islets from T2D patients.
Conclusions: We thus developed an FGF2-induced model of human β-cell dedifferentiation, identified new markers of dedifferentiation, and found evidence for increased pancreatic FGF2, FGFR1, and β-cell dedifferentiation in T2D.[Hide abstract]
|Unique pharmacology of a novel insulin receptor antibodyA ubiquitous feature of type 2 diabetes (T2D) is decreased sensitivity to insulin. Insulin sensitizers such as thiazolidinediones can impact insulin resistance but have been associated with weight gain and cardiac events. There is a need for novel insulin-sensitizing agents that provide safer and more effective means to treat T2D. Hinke and colleagues characterize a novel monoclonal antibody against insulin receptor, IRAB-A. In vitro testing with IRAB-A indicates that it binds allosterically to the insulin receptor (IR) and has both sensitizer and agonist properties distinct from those of insulin.|
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Objective: Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D.
Methods: A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice.
Results: In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge.
Conclusions: Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.[Hide abstract]
|Galanin enhances glucose metabolism through enteric NOS neuronsGalanin is a neuropeptide largely expressed in the brain but also in enteric nervous system (ENS) neurons. Galanin is known to have beneficial effects on glucose metabolism. Aberrant duodenal hyper-contractility leads to an increase of glucose absorption that contributes to the chronic hyperglycemia observed in type 2 diabetes (T2D). Abot et al. show that oral galanin decreases duodenal contraction in mice by stimulating nitric oxide release from enteric neurons. This increases insulin sensitivity, which is associated with an improvement of several metabolic parameters.|
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Objective: Decreasing duodenal contraction is now considered as a major focus for the treatment of type 2 diabetes. Therefore, identifying bioactive molecules able to target the enteric nervous system, which controls the motility of intestinal smooth muscle cells, represents a new therapeutic avenue. For this reason, we chose to study the impact of oral galanin on this system in diabetic mice.
Methods: Enteric neurotransmission, duodenal contraction, glucose absorption, modification of gut–brain axis, and glucose metabolism (glucose tolerance, insulinemia, glucose entry in tissue, hepatic glucose metabolism) were assessed.
Results: We show that galanin, a neuropeptide expressed in the small intestine, decreases duodenal contraction by stimulating nitric oxide release from enteric neurons. This is associated with modification of hypothalamic nitric oxide release that favors glucose uptake in metabolic tissues such as skeletal muscle, liver, and adipose tissue. Oral chronic gavage with galanin in diabetic mice increases insulin sensitivity, which is associated with an improvement of several metabolic parameters such as glucose tolerance, fasting blood glucose, and insulin.
Conclusions: Here, we demonstrate that oral galanin administration improves glucose homeostasis via the enteric nervous system and could be considered a therapeutic potential for the treatment of T2D.[Hide abstract]