Featured ArticlesVolume 6 | No. 8 | August 2017
|Lipid synthesis coupled to control of thermogenic programmingEarly work on adipocyte metabolism in obesity revealed that the biosynthesis of palmitate from acetyl CoA, denoted de novo lipogenesis (DNL), was markedly reduced in the insulin resistant state. Guilherme, Pedersen et al. set out to clarify the physiological role of adipocyte DNL by generating mouse models in which adipocyte fatty acid synthase (FASN) could be depleted after the animals reach maturity. Analysis of these mice following FASN deletion revealed improved glucose tolerance and showed strong inguinal white adipose tissue (iWAT) browning. Also, a markedly increased sympathetic innervation of iWAT was observed in response to induced FASN depletion in adipocytes of mature mice.|
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Background: The de novo biosynthesis of fatty acids (DNL) through fatty acid synthase (FASN) in adipocytes is exquisitely regulated by nutrients, hormones, fasting, and obesity in mice and humans. However, the functions of DNL in adipocyte biology and in the regulation of systemic glucose homeostasis are not fully understood.
Methods & results: Here we show adipocyte DNL controls crosstalk to localized sympathetic neurons that mediate expansion of beige/brite adipocytes within inguinal white adipose tissue (iWAT). Induced deletion of FASN in white and brown adipocytes of mature mice (iAdFASNKO mice) enhanced glucose tolerance, UCP1 expression, and cAMP signaling in iWAT. Consistent with induction of adipose sympathetic nerve activity, iAdFASNKO mice displayed markedly increased neuronal tyrosine hydroxylase (TH) and neuropeptide Y (NPY) content in iWAT. In contrast, brown adipose tissue (BAT) of iAdFASNKO mice showed no increase in TH or NPY, nor did FASN deletion selectively in brown adipocytes (UCP1-FASNKO mice) cause these effects in iWAT.
Conclusions: These results demonstrate that downregulation of fatty acid synthesis via FASN depletion in white adipocytes of mature mice can stimulate neuronal signaling to control thermogenic programming in iWAT.[Hide abstract]
|pH changes in mitochondria during brown adipocyte activationHow mitochondrial pH is regulated in stimulated brown adipocytes, which have high metabolic capacity, is unknown. To understand the dynamic regulation of mitochondrial pH in response to adrenergic stimulation, Hou and colleagues used the pH sensitive protein mito-pHluorin. The kinetics of mitochondrial pH changes were compared with the thermogenic process. The authors show that mitochondrial pH regulation is associated with changes in mitochondrial Ca2+ levels, which are further governed by the intracellular Ca2+ store that is the endoplasmic reticulum (ER).|
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Objective: Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes.
Methods: A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca2+ profiles in mitochondria and the intracellular Ca2+ store, the endoplasmic reticulum (ER).
Results: A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca2+. This Ca2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation.
Conclusions: We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis.[Hide abstract]
|Angiopoietin-like 4 directs uptake of dietary fat away from adipose during fastingAngiopoietin-like 4 (ANGPTL4), is a fasting induced inhibitor of lipoprotein lipase (LPL) and a regulator of triglyceride metabolism. In this study, Cushing and colleagues investigate the physiological mechanisms by which ANGPTL4 regulates plasma triglycerides. Their results support a model in which ANGPTL4 primarily acts locally in adipose tissue to inhibit LPL, and thus triglyceride uptake, during fasting. This inhibition would redirect triglyceride-derived fatty acids to tissues such as heart and muscle.|
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Objective: Angiopoietin-like 4 (ANGPTL4) is a fasting-induced inhibitor of lipoprotein lipase (LPL) and a regulator of plasma triglyceride metabolism. Here, we examined the kinetics of Angptl4 induction and tested the hypothesis that ANGPTL4 functions physiologically to reduce triglyceride delivery to adipose tissue during nutrient deprivation.
Methods: Gene expression, LPL activity, and triglyceride uptake were examined in fasted and fed wild-type and Angptl4−/− mice.
Results: Angptl4 was strongly induced early in fasting, and this induction was suppressed in mice with access to food during the light cycle. Fasted Angptl4−/− mice manifested increased LPL activity and triglyceride uptake in adipose tissue compared to wild-type mice.
Conclusions: Angptl4 is induced early in fasting to divert uptake of fatty acids and triglycerides away from adipose tissues.[Hide abstract]
|Nicotinamide riboside kinases display redundancy Because of the activity of Nicotinamide adenine dinucleotide (NAD+) consuming enzymes, replenishment of NAD+ through biosynthesis and salvage pathways is vital. Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have emerged as NAD+ precursors with the potential to circumvent the adverse side effects associated with high dose niacin and augment NAD+ synthesis. Fletcher et al. have investigated NR kinase (NRK) expression in skeletal muscle and define the influence of NRKs on NR and NMN metabolism to NAD+. They show that the NRKs have overlapping and redundant activity in muscle cells critical to the conversion of exogenous NR and NMN to NAD+.|
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Objective: Augmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.
Methods: We exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization.
Results: Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability.
Conclusions: These results identify skeletal muscle cells as requiring NAMPT to maintain NAD+ availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD+ availability.[Hide abstract]
|Amyloid formation disrupts the balance between interleukin-1β and interleukin-1 receptor antagonistA key regulator of islet inflammation is the pro-inflammatory cytokine interleukin-1β (IL-1β). Islet amyloid is a pathological characteristic of the pancreas in type 2 diabetes (T2D). IL-1 receptor antagonist (IL-1Ra) is a natural inhibitor of IL-1β. Hui and colleagues show that amyloid formation impairs the balance between islet IL-1β and IL-1Ra both by increasing IL-1β and reducing IL-1Ra production, thereby promoting β-cell dysfunction and death. Pharmacological strategies to reduce the IL-1β/IL-1Ra ratio may effectively protect β-cells from amyloid toxicity in conditions associated with islet amyloid formation such as T2D and human islet grafts in T1D.|
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Objective: β-cell dysfunction and apoptosis associated with islet inflammation play a key role in the pathogenesis of type 2 diabetes (T2D). Growing evidence suggests that islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to islet inflammation and β-cell death in T2D. We recently showed the role of interleukin-1β (IL-1β)/Fas/caspase-8 apoptotic pathway in amyloid-induced β-cell death. In this study, we used human islets in culture as an ex vivo model of amyloid formation to: (1) investigate the effects of amyloid on islet levels of the natural IL-1 receptor antagonist (IL-1Ra); (2) examine if modulating the IL-1β/IL-1Ra balance can prevent amyloid-induced β-cell Fas upregulation and apoptosis.
Methods: Isolated human islets (n = 10 donors) were cultured in elevated glucose (to form amyloid) with or without a neutralizing human IL-1β antibody for up to 7 days. Parallel studies were performed with human islets in which amyloid formation was prevented by adeno-siRNA-mediated suppression of hIAPP expression (as control). β-cell levels of IL-1Ra, Fas, apoptosis as well as islet function, insulin- and amyloid-positive areas, and IL-1Ra release were assessed.
Results: Progressive amyloid formation in human islets during culture was associated with alterations in IL-1Ra. Islet IL-1Ra levels were higher at early stages but were markedly reduced at later stages of amyloid formation. Furthermore, IL-1Ra release from human islets was reduced during 7-day culture in a time-dependent manner. These changes in IL-1Ra production and release from human islets during amyloid formation adversely correlated with islet IL-1β levels, β-cell Fas expression and apoptosis. Treatment with IL-1β neutralizing antibody markedly reduced amyloid-induced β-cell Fas expression and apoptosis, thereby improving islet β-cell survival and function during culture.
Conclusions: These data suggest that amyloid formation impairs the balance between IL-1β and IL-1Ra in islets by increasing IL-1β production and reducing IL-1Ra levels thereby promoting β-cell dysfunction and death. Restoring the IL-1β/IL-1Ra ratio may provide an effective strategy to protect islet β-cells from amyloid toxicity in T2D.[Hide abstract]
|Myeloid protein tyrosine phosphatase 1B (PTP1B) deficiency protects against atherosclerotic plaque formation Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with Type 1 or Type 2 diabetes, due to accelerated atherosclerosis. Thompson and colleagues demonstrate here that myeloid-specific genetic deletion of protein tyrosine phosphatase (PTP) 1B in the ApoE-/- mouse model of atherosclerosis leads to protection against atherosclerosis development, suggesting beneficial effects of PTP1B inhibition for the reduction of cardiovascular risk and treatment of CVDs.|
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Objective: Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with Type 1 or Type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance due to impaired insulin receptor (IR) signaling. Moreover, inflammatory cells, in particular macrophages, play a key role in pathogenesis of atherosclerosis and insulin resistance in humans. We hypothesized that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR, specifically in macrophages, would have beneficial anti-inflammatory effects and lead to protection against atherosclerosis and CVD.
Methods: We generated novel macrophage-specific PTP1B knockout mice on atherogenic background (ApoE−/−/LysM-PTP1B). Mice were fed standard or pro-atherogenic diet, and body weight, adiposity (echoMRI), glucose homeostasis, atherosclerotic plaque development, and molecular, biochemical and targeted lipidomic eicosanoid analyses were performed.
Results: Myeloid-PTP1B knockout mice on atherogenic background (ApoE−/−/LysM-PTP1B) exhibited a striking improvement in glucose homeostasis, decreased circulating lipids and decreased atherosclerotic plaque lesions, in the absence of body weight/adiposity differences. This was associated with enhanced phosphorylation of aortic Akt, AMPKα and increased secretion of circulating anti-inflammatory cytokine interleukin-10 (IL-10) and prostaglandin E2 (PGE2), without measurable alterations in IR phosphorylation, suggesting a direct beneficial effect of myeloid-PTP1B targeting.
Conclusions: Here we demonstrate that inhibiting the activity of PTP1B specifically in myeloid lineage cells protects against atherosclerotic plaque formation, under atherogenic conditions, in an ApoE−/− mouse model of atherosclerosis. Our findings suggest for the first time that macrophage PTP1B targeting could be a therapeutic target for atherosclerosis treatment and reduction of CVD risk.[Hide abstract]
|Beta-adrenergic receptors are critical for weight loss but not for other metabolic adaptations to a ketogenic diet The consumption of a low-carbohydrate ketogenic diet (KD) by mice leads to a distinct physiologic state associated with weight loss, increased metabolic rate, and improved insulin sensitivity. Douris, Desai et al. hypothesized that the sympathetic nervous system (SNS) may play a role in the adaptation to ketogenic diets. Their findings confirm that SNS activity, mediated through β-adrenergic receptors, is required for the physiologic response and adaptation to the ketogenic diet that ultimately results in weight loss.|
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Objective: We have previously shown that the consumption of a low-carbohydrate ketogenic diet (KD) by mice leads to a distinct physiologic state associated with weight loss, increased metabolic rate, and improved insulin sensitivity . Furthermore, we identified fibroblast growth factor 21 (FGF21) as a necessary mediator of the changes, as mice lacking FGF21 fed KD gain rather than lose weight . FGF21 activates the sympathetic nervous system (SNS) , which is a key regulator of metabolic rate. Thus, we considered that the SNS may play a role in mediating the metabolic adaption to ketosis.
Methods: To test this hypothesis, we measured the response of mice lacking all three β-adrenergic receptors (β-less mice) to KD feeding.
Results: In contrast to wild-type (WT) controls, β-less mice gained weight, increased adipose tissue depots mass, and did not increase energy expenditure when consuming KD. Remarkably, despite weight-gain, β-less mice were insulin sensitive. KD-induced changes in hepatic gene expression of β-less mice were similar to those seen in WT controls eating KD. Expression of FGF21 mRNA rose over 60-fold in both WT and β-less mice fed KD, and corresponding circulating FGF21 levels were 12.5 ng/ml in KD-fed wild type controls and 35.5 ng/ml in KD-fed β-less mice.
Conclusions: The response of β-less mice distinguishes at least two distinct categories of physiologic effects in mice consuming KD. In the liver, KD regulates peroxisome proliferator-activated receptor alpha (PPARα)-dependent pathways through an action of FGF21 independent of the SNS and beta-adrenergic receptors. In sharp contrast, induction of interscapular brown adipose tissue (BAT) and increased energy expenditure absolutely require SNS signals involving action on one or more β-adrenergic receptors. In this way, the key metabolic actions of FGF21 in response to KD have diverse effector mechanisms.[Hide abstract]
|Nrg4 promotes fuel oxidation and a healthy adipokine profile Neuregulin 4 (Nrg4) has emerged as a brown adipose tissue (BAT) -enriched endocrine factor that exerts robust effects on hepatic lipid metabolism and systemic homeostasis. Chen and colleagues revealed a surprisingly pleiotropic effect of Nrg4 signaling on key aspects of systemic energy and glucose metabolism. They identified hepatic fatty acid oxidation and ketogenesis as a new metabolic target of endocrine signaling by Nrg4. Also, Nrg4 promotes a beneficial adipokine profile during obesity. Together, this work provides important insights into the physiological actions of Nrg4 that contribute to its beneficial effects on metabolic homeostasis.|
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Objective: Brown and white adipose tissue exerts pleiotropic effects on systemic energy metabolism in part by releasing endocrine factors. Neuregulin 4 (Nrg4) was recently identified as a brown fat-enriched secreted factor that ameliorates diet-induced metabolic disorders, including insulin resistance and hepatic steatosis. However, the physiological mechanisms through which Nrg4 regulates energy balance and glucose and lipid metabolism remain incompletely understood. The aims of the current study were: i) to investigate the regulation of adipose Nrg4 expression during obesity and the physiological signals involved, ii) to elucidate the mechanisms underlying Nrg4 regulation of energy balance and glucose and lipid metabolism, and iii) to explore whether Nrg4 regulates adipose tissue secretome gene expression and adipokine secretion.
Methods: We examined the correlation of adipose Nrg4 expression with obesity in a cohort of diet-induced obese mice and investigated the upstream signals that regulate Nrg4 expression. We performed metabolic cage and hyperinsulinemic-euglycemic clamp studies in Nrg4 transgenic mice to dissect the metabolic pathways regulated by Nrg4. We investigated how Nrg4 regulates hepatic lipid metabolism in the fasting state and explored the effects of Nrg4 on adipose tissue gene expression, particularly those encoding secreted factors.
Results: Adipose Nrg4 expression is inversely correlated with adiposity and regulated by pro-inflammatory and anti-inflammatory signaling. Transgenic expression of Nrg4 increases energy expenditure and augments whole body glucose metabolism. Nrg4 protects mice from diet-induced hepatic steatosis in part through activation of hepatic fatty acid oxidation and ketogenesis. Finally, Nrg4 promotes a healthy adipokine profile during obesity.
Conclusions: Nrg4 exerts pleiotropic beneficial effects on energy balance and glucose and lipid metabolism to ameliorate obesity-associated metabolic disorders. Biologic therapeutics based on Nrg4 may improve both type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) in patients.[Hide abstract]
|Repletion of branched chain amino acids reverses mTORC1 signalling but not improved metabolism Dietary protein dilution (PD), in which dietary protein intake is reduced and replaced by calories from carbohydrate and/or fat, has emerged as an influential environmental variable affecting aging and age-related disease such as type 2 diabetes (T2D). DPD can retard the development of insulin resistance and T2D in mouse models, via the induction of the liver-derived hormone fibroblast growth factor 21 (FGF21). Maida et al. show that branched chain amino acids (BCAAs) are consistently affected by dietary PD. Despite normalization of circulating BCAAs and mTORC1 signaling, they demonstrate that dietary BCAA-replacement to an AA-diluted (AAD) diet did not reverse FGF21 induction or restore hyperglycaemia in experimental T2D.|
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Objective: Dietary protein dilution (PD) has been associated with metabolic advantages such as improved glucose homeostasis and increased energy expenditure. This phenotype involves liver-induced release of FGF21 in response to amino acid insufficiency; however, it has remained unclear whether dietary dilution of specific amino acids (AAs) is also required. Circulating branched chain amino acids (BCAAs) are sensitive to protein intake, elevated in the serum of obese humans and mice and thought to promote insulin resistance. We tested whether replenishment of dietary BCAAs to an AA-diluted (AAD) diet is sufficient to reverse the glucoregulatory benefits of dietary PD.
Methods: We conducted AA profiling of serum from healthy humans and lean and high fat-fed or New Zealand obese (NZO) mice following dietary PD. We fed wildtype and NZO mice one of three amino acid defined diets: control, total AAD, or the same diet with complete levels of BCAAs (AAD + BCAA). We quantified serum AAs and characterized mice in terms of metabolic efficiency, body composition, glucose homeostasis, serum FGF21, and tissue markers of the integrated stress response (ISR) and mTORC1 signaling.
Results: Serum BCAAs, while elevated in serum from hyperphagic NZO, were consistently reduced by dietary PD in humans and murine models. Repletion of dietary BCAAs modestly attenuated insulin sensitivity and metabolic efficiency in wildtype mice but did not restore hyperglycemia in NZO mice. While hepatic markers of the ISR such as P-eIF2α and FGF21 were unabated by dietary BCAA repletion, hepatic and peripheral mTORC1 signaling were fully or partially restored, independent of changes in circulating glucose or insulin.
Conclusions: Repletion of BCAAs in dietary PD is sufficient to oppose changes in somatic mTORC1 signaling but does not reverse the hepatic ISR nor induce insulin resistance in type 2 diabetes during dietary PD.[Hide abstract]
|Ghrelin-responsive mediobasal hypothalamic neurons mediate feeding responseOf all the actions mediated through ghrelin receptors (GHSRs), one of the most robust and well-characterized is the induction of an acute feeding response after peripheral or central administration of ghrelin. Mani et al. tested whether the activity of mediobasal hypothalamic (MBH) GHSR neurons is required for the normal rebound food intake following fasting. Their results suggest that the activity of MBH GHSR neurons is required for the full acute orexigenic response to administered ghrelin and the usual rebound food intake response following a 24 hour fast and is sufficient to induce spontaneous food intake.|
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Objective: Ghrelin is a stomach-derived hormone that affects food intake and regulates blood glucose. The best-characterized actions of ghrelin are mediated by its binding to and activation of the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Adequate examination of the identity, function, and relevance of specific subsets of GHSR-expressing neurons has been hampered by the absence of a suitable Cre recombinase (Cre)-expressing mouse line with which to manipulate gene expression in a targeted fashion within GHSR-expressing neurons. The present study aims to characterize the functional significance and neurocircuitry of GHSR-expressing neurons in the mediobasal hypothalamus (MBH), as they relate to ghrelin-induced food intake and fasting-associated rebound hyperphagia, using a novel mouse line in which Cre expression is controlled by the Ghsr promoter.
Methods: A Ghsr-IRES-Cre mouse line that expresses Cre directed by the Ghsr promoter was generated. The line was validated by comparing Cre activity in reporter mice to the known brain distribution pattern of GHSR. Next, the requirement of MBH GHSR-expressing neuronal activity in mediating food intake in response to administered ghrelin and in response to fasting was assessed after stereotaxic delivery of inhibitory designer receptor exclusively activated by designer drugs (DREADD) virus to the MBH. In a separate cohort of Ghsr-IRES-Cre mice, stereotaxic delivery of stimulatory DREADD virus to the MBH was performed to assess the sufficiency of MBH GHSR-expressing neuronal activity on food intake. Finally, the distribution of MBH GHSR-expressing neuronal axonal projections was assessed in the DREADD virus-injected animals.
Results: The pattern of Cre activity in the Ghsr-IRES-Cre mouse line mostly faithfully reproduced the known GHSR expression pattern. DREADD-assisted inhibition of MBH GHSR neuronal activity robustly suppressed the normal orexigenic response to ghrelin and fasting-associated rebound food intake. DREADD-assisted stimulation of MBH GHSR neuronal activity was sufficient to induce food intake. Axonal projections of GHSR-expressing MBH neurons were observed in a subset of hypothalamic and extra-hypothalamic regions.
Conclusions: These results suggest that 1) activation of GHSR-expressing neurons in the MBH is required for the normal feeding responses following both peripheral administration of ghrelin and fasting, 2) activation of MBH GHSR-expressing neurons is sufficient to induce feeding, and 3) axonal projections to a subset of hypothalamic and/or extra-hypothalamic regions likely mediate these responses. The Ghsr-IRES-Cre line should serve as a valuable tool to further our understanding of the functional significance of ghrelin-responsive/GHSR-expressing neurons and the neuronal circuitry within which they act.[Hide abstract]
|Dietary sugars, not lipids, drive hypothalamic inflammationThe hypothalamus of hypercalorically diet-induced obese animals is characterized by an increased inflammatory response. In hypercaloric diet-induced obesity, dietary fats, and especially saturated fatty acids, are considered to be the essential component initiating pro-inflammatory responses in the hypothalamus. Gao, Bielohuby et al. documented that the mediobasal hypothalamic inflammatory response occurs in response to high-fat diets rich in carbohydrates (high-carbohydrate high-fat (HCHF) diets) but not in response to a low-carbohydrate, high-fat (LCHF) diet.|
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Objective: The hypothalamus of hypercaloric diet-induced obese animals is featured by a significant increase of microglial reactivity and its associated cytokine production. However, the role of dietary components, in particular fat and carbohydrate, with respect to the hypothalamic inflammatory response and the consequent impact on hypothalamic control of energy homeostasis is yet not clear.
Methods: We dissected the different effects of high-carbohydrate high-fat (HCHF) diets and low-carbohydrate high-fat (LCHF) diets on hypothalamic inflammatory responses in neurons and non-neuronal cells and tested the hypothesis that HCHF diets induce hypothalamic inflammation via advanced glycation end-products (AGEs) using mice lacking advanced glycation end-products (AGEs) receptor (RAGE) and/or the activated leukocyte cell-adhesion molecule (ALCAM).
Results: We found that consumption of HCHF diets, but not of LCHF diets, increases microgliosis as well as the presence of N(ε)-(Carboxymethyl)-Lysine (CML), a major AGE, in POMC and NPY neurons of the arcuate nucleus. Neuron-secreted CML binds to both RAGE and ALCAM, which are expressed on endothelial cells, microglia, and pericytes. On a HCHF diet, mice lacking the RAGE and ALCAM genes displayed less microglial reactivity and less neovasculature formation in the hypothalamic ARC, and this was associated with significant improvements of metabolic disorders induced by the HCHF diet.
Conclusions: Combined overconsumption of fat and sugar, but not the overconsumption of fat per se, leads to excessive CML production in hypothalamic neurons, which, in turn, stimulates hypothalamic inflammatory responses such as microgliosis and eventually leads to neuronal dysfunction in the control of energy metabolism.[Hide abstract]
|Serotonergic modulation of the activity of GLP-1-producing neurons Glucagon-like peptide-1 (GLP-1) is an incretin best known for its role in glucose homeostasis and appetite regulation. Within the brain, GLP-1 is produced by preproglucagon (PPG) neurons in the lower brainstem. Serotonin (5-hydroxytryptamine; 5-HT) is another key neurotransmitter involved in both regulation of stress and anxiety and control of food intake. Holt and colleagues have investigated the cellular effects of 5-HT on PPG neurons, as well as the innervation that underlies these effects. Their data suggest a multifaceted link between 5-HT and central GLP- 1, which is tightly regulated through excitatory or inhibitory responses to 5-HT occurring within different subcellular compartments of PPG neurons.|
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Objective: Glucagon-like peptide-1 (GLP-1) and 5-HT are potent regulators of food intake within the brain. GLP-1 is expressed by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS). We have previously shown that PPG neurons innervate 5-HT neurons in the ventral brainstem. Here, we investigate whether PPG neurons receive serotonergic input and respond to 5-HT.
Methods: We employed immunohistochemistry to reveal serotonergic innervation of PPG neurons. We investigated the responsiveness of PPG neurons to 5-HT using in vitro Ca2+ imaging in brainstem slices from transgenic mice expressing the Ca2+ indicator, GCaMP3, in PPG neurons, and cell-attached patch-clamp recordings.
Results: Close appositions from 5-HT-immunoreactive axons occurred on many PPG neurons. Application of 20 μM 5-HT produced robust Ca2+ responses in NTS PPG dendrites but little change in somata. Dendritic Ca2+ spikes were concentration-dependent (2, 20, and 200 μM) and unaffected by blockade of glutamatergic transmission, suggesting 5-HT receptors on PPG neurons. Neither activation nor blockade of 5-HT3 receptors affected [Ca2+]i. In contrast, inhibition of 5-HT2 receptors attenuated increases in intracellular Ca2+ and 5-HT2C receptor activation produced Ca2+ spikes. Patch-clamp recordings revealed that 44% of cells decreased their firing rate under 5-HT, an effect blocked by 5-HT1A receptor antagonism.
Conclusions: PPG neurons respond directly to 5-HT with a 5-HT2C receptor-dependent increase in dendritic [Ca2+]i. Electrical responses to 5-HT revealed additional inhibitory effects due to somatic 5-HT1A receptors. Reciprocal innervation between 5-HT and PPG neurons suggests that the coordinated activity of these brainstem neurons may play a role in the regulation of food intake.[Hide abstract]
|Maternal obesity programs increased leptin gene expression in offspringAccording to the Developmental Origin of Health and Disease (DOHaD) concept , maternal obesity and accelerated growth in neonates predispose offspring to obesity and metabolic pathologies. Adult rat offspring from high-fat diet-fed dams (HF) exhibits hypertrophic adipocyte, hyperleptinemia, and increased leptin mRNA levels in a depot-specific manner. Lecoutre et al. show that maternal obesity differently affects epigenetic remodeling of a promoter and two enhancers linked to higher leptin gene expression during visceral perirenal and subcutaneous inguinal deposits development in HF neonates. They also report that retained active marks that are correlated with persistent increased leptin mRNA levels occur in a depot-specific manner in adult obesity-prone HF offspring. |
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Objective: According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates predispose offspring to white adipose tissue (WAT) accumulation. In rodents, adipogenesis mainly develops during lactation. The mechanisms underlying the phenomenon known as developmental programming remain elusive. We previously reported that adult rat offspring from high-fat diet-fed dams (called HF) exhibited hypertrophic adipocyte, hyperleptinemia and increased leptin mRNA levels in a depot-specific manner. We hypothesized that leptin upregulation occurs via epigenetic malprogramming, which takes place early during development of WAT.
Methods: As a first step, we identified in silico two potential enhancers located upstream and downstream of the leptin transcription start site that exhibit strong dynamic epigenomic remodeling during adipocyte differentiation. We then focused on epigenetic modifications (methylation, hydroxymethylation, and histone modifications) of the promoter and the two potential enhancers regulating leptin gene expression in perirenal (pWAT) and inguinal (iWAT) fat pads of HF offspring during lactation (postnatal days 12 (PND12) and 21 (PND21)) and in adulthood.
Results: PND12 is an active period for epigenomic remodeling in both deposits especially in the upstream enhancer, consistent with leptin gene induction during adipogenesis. Unlike iWAT, some of these epigenetic marks were still observable in pWAT of weaned HF offspring. Retained marks were only visible in pWAT of 9-month-old HF rats that showed a persistent “expandable” phenotype.
Conclusions: Consistent with the DOHaD hypothesis, persistent epigenetic remodeling occurs at regulatory regions especially within intergenic sequences, linked to higher leptin gene expression in adult HF offspring in a depot-specific manner.[Hide abstract]
|A Unique Resource for Studying Organ Crosstalk in DiabetesTo generate a unique resource for studying consequences of chronic insulin insufficiency and hyperglycemia in a multi-tissue, multi-omics approach, Blutke et al. generated a complex biobank of more than 50 different tissues and body fluids from two-year-old MIDY pigs and WT littermate controls. A comprehensive standardized protocol was established to ensure uniform high quality of representative samples for a broad spectrum of analyses, including molecular profiling as well as qualitative and quantitative morphological investigations. To characterize the quality of samples in the Munich MIDY pig biobank, the authors extracted and characterized RNA and proteins from a subset of tissues. These pilot studies revealed excellent sample quality. Future detailed omics studies will provide insights into tissue specific molecular changes induced by chronic hyperglycemia and insulin insufficiency.|
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Objective: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates.
Methods: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics.
Results: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples.
Conclusions: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.[Hide abstract]