Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes.

The mammalian respiratory chain complexes I, III2, and IV (CI, CIII2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation.

Sex-specific effects of Cre expression in Syn1Cre mice.

The Cre-loxP system has been used to generate cell-type specific mutations in mice, allowing researchers to investigate the underlying biological mechanisms of disease. However, the Cre-recombinase alone can induce phenotypes that confound comparisons among genotypes if the appropriate Cre control is not included. In this study, we characterised behavioural, morphological and metabolic phenotypes of the pan-neuronal Syn1Cre line. We found that these mice possess intact neuromuscular parameters but have reduced exploratory activity and a male-specific increase in anxiety-like behaviour. Moreover, we observed a male-specific deficit in learning and long-term memory of Syn1Cre mice that could be a result of decreased visual acuity. Furthermore, we found that over-expression of human growth hormone (hGH) from Syn1Cre results in a male-specific reduction in body weight and femur length, potentially through decreased hepatic Igf1 expression. However, metabolic characteristics of Syn1Cre mice such as glucose metabolism, energy expenditure and feeding were unaffected by the presence of Syn1Cre. In conclusion, our data demonstrate that Syn1Cre expression has effects on behavioural and morphological traits. This finding highlights the importance of including the Cre control in all comparisons, while the male-specific effects on some phenotypes highlight the importance of including both sexes.

Heterogeneous effects of individual high-fat diet compositions on phenotype, metabolic outcome, and hepatic proteome signature in BL/6 male mice.

The multitude of obesogenic diets used in rodent studies can hardly be overviewed. Since standardization is missing and assuming that individual compositions provoke individual effects, the choice of quality, quantity and combination of diet ingredients seems to be crucial for the outcome and interpretation of obesity studies. Therefore, the present study was conducted to compare the individual effects of three commonly used obesogenic diets, mainly differing in sugar and fat content. Besides basic phenotypic and metabolic characterization, one main aspect was a comparative liver proteome analysis. As expected, the obtained results picture differentiated consequences mainly depending on fat source and/or fat- and sugar quantity. By confirming the general presumption that the choice of nutritional composition is a pivotal factor, the present findings demonstrate that a conscious selection is indispensable for obtaining reliable and sound results in obesity research. In conclusion, we strongly recommend a careful selection of the appropriate diet in advance of a new experiment, taking into account the specific research question.

Reduced insulin signaling in neurons induces sex-specific health benefits.

Reduced activity of insulin/insulin-like growth factor signaling (IIS) extends health and life span in mammals. Loss of the insulin receptor substrate 1 (Irs1) gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS-mediated longevity are currently unknown. Here, we measured survival and health span in mice lacking IRS1 specifically in liver, muscle, fat, and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of IRS1 in more than one tissue is required for life-span extension. Loss of IRS1 in liver, muscle, and fat did not improve health. In contrast, loss of neuronal IRS1 increased energy expenditure, locomotion, and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4, and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of aging in response to reduced IIS associated with improved health at old age.

Micro-CT Analysis of Fat in the Killifish Nothobranchius furzeri.

Aging is associated with an increase in body fat mass and a concomitant decrease in lean mass and bone density in mammals. Body adiposity can also be redistributed with age, resulting in abdominal fat accumulation and subcutaneous fat reduction. In addition, specific variation in fat distribution is considered to be a risk factor for a number of age-related metabolic disorders. Micro computed tomography (micro-CT) is a nondestructive high-resolution imaging method that uses planar X-ray images captured at various angles around a sample of interest to yield a three-dimensional array of radiodensity values, which can then be used to computationally extract the adipose volume in situ using its innate contrast properties. This method was successfully used to study adipose tissue dynamics in rodents and more recently in zebrafish. The naturally short-lived African turquoise killifish is an emerging model organism to study the biology of aging. Like mammals, killifish also have different fat deposits (visceral and subcutaneous), making them a suitable model to study age-related changes in fat mass and distribution. However, procedures allowing precise quantification of fat content and distribution are missing in this species. Here, we provide an optimized protocol to measure and quantify fat distribution in turquoise killifish by micro-CT scan analysis and show the applicability of the method in young and old animals of both sexes.

Hexosamine pathway activation improves memory but does not extend lifespan in mice.

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.

Brain-Restricted Inhibition of IL-6 Trans-Signaling Mildly Affects Metabolic Consequences of Maternal Obesity in Male Offspring.

Maternal obesity greatly affects next generations, elevating obesity risk in the offspring through perinatal programming and flawed maternal and newborn nutrition. The exact underlying mechanisms are poorly understood. Interleukin-6 (IL-6) mediates its effects through a membrane-bound receptor or by trans-signaling (tS), which can be inhibited by the soluble form of the co-receptor gp130 (sgp130). As IL-6 tS mediates western-style diet (WSD) effects via chronic low-grade inflammation (LGI) and LGI is an important mediator in brain-adipose tissue communication, this study aims at determining the effects of maternal obesity in a transgenic mouse model of brain-restricted IL-6tS inhibition (GFAPsgp130) on offspring's short- and long-term body composition and epigonadal white adipose tissue (egWAT) metabolism. Female wild type (WT) or transgenic mice were fed either standard diet (SD) or WSD pregestationally, during gestation, and lactation. Male offspring received SD from postnatal day (P)21 to P56 and were metabolically challenged with WSD from P56 to P120. At P21, offspring from WT and transgenic dams that were fed WSD displayed increased body weight and egWAT mass, while glucose tolerance testing showed the strongest impairment in GFAPsgp130WSD offspring. Simultaneously, egWAT proteome reveals a characteristic egWAT expression pattern in offspring as a result of maternal conditions. IL-6tS inhibition in transgenic mice was in tendency associated with lower body weight in dams on SD and their respective offspring but blunted by the WSD. In conclusion, maternal nutrition affects offspring's body weight and egWAT metabolism predominantly independent of IL-6tS inhibition, emphasizing the importance of maternal and newborn nutrition for long-term offspring health.

Lithium can mildly increase health during ageing but not lifespan in mice.

Lithium is a nutritional trace element, used clinically as an anti-depressant. Preclinically, lithium has neuroprotective effects in invertebrates and mice, and it can also extend lifespan in fission yeast, C. elegans and Drosophila. An inverse correlation of human mortality with the concentration of lithium in tap water suggests a possible, evolutionarily conserved mechanism mediating longevity. Here, we assessed the effects of lithium treatment on lifespan and ageing parameters in mice. Lithium has a narrow therapeutic dose range, and overdosing can severely affect organ health. Within the tolerable dosing range, we saw some mildly positive effects of lithium on health span but not on lifespan.

High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo.

Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.

Chromatin remodeling due to degradation of citrate carrier impairs osteogenesis of aged mesenchymal stem cells.

Aging is accompanied by a general decline in the function of many cellular pathways. However, whether these are causally or functionally interconnected remains elusive. Here, we study the effect of mitochondrial-nuclear communication on stem cell aging. We show that aged mesenchymal stem cells exhibit reduced chromatin accessibility and lower histone acetylation, particularly on promoters and enhancers of osteogenic genes. The reduced histone acetylation is due to impaired export of mitochondrial acetyl-CoA, owing to the lower levels of citrate carrier (CiC). We demonstrate that aged cells showed enhanced lysosomal degradation of CiC, which is mediated via mitochondrial-derived vesicles. Strikingly, restoring cytosolic acetyl-CoA levels either by exogenous CiC expression or via acetate supplementation, remodels the chromatin landscape and rescues the osteogenesis defects of aged mesenchymal stem cells. Collectively, our results establish a tight, age-dependent connection between mitochondrial quality control, chromatin and stem cell fate, which are linked together by CiC.

Dnmt3a2/Dnmt3L Overexpression in the Dopaminergic System of Mice Increases Exercise Behavior through Signaling Changes in the Hypothalamus.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

Hippocampal insulin resistance links maternal obesity with impaired neuronal plasticity in adult offspring.

OBJECTIVE: Maternal obesity and a disturbed metabolic environment during pregnancy and lactation have been shown to result in many long-term health consequences for the offspring. Among them, impairments in neurocognitive development and performance belong to the most dreaded ones. So far, very few mechanistic approaches have aimed to determine the responsible molecular events. METHODS: In a mouse model of maternal diet-induced obesity and perinatal hyperinsulinemia, we assessed adult offspring's hippocampal insulin signaling as well as concurrent effects on markers of hippocampal neurogenesis, synaptic plasticity and function using western blotting and immunohistochemistry. In search for a potential link between neuronal insulin resistance and hippocampal plasticity, we additionally quantified protein expression of key molecules of synaptic plasticity in an in vitro model of acute neuronal insulin resistance. RESULTS: Maternal obesity and perinatal hyperinsulinemia result in adult hippocampal insulin resistance with subsequently reduced hippocampal mTor signaling and altered expression of markers of neurogenesis (doublecortin), synaptic plasticity (FoxO1, pSynapsin) and function (vGlut, vGAT) in the offspring. The observed effects are independent of the offspring's adult metabolic phenotype and can be associated with multiple previously reported behavioral abnormalities. Additionally, we demonstrate that induction of insulin resistance in cultured hippocampal neurons reduces mTor signaling, doublecortin and vGAT protein expression. CONCLUSIONS: Hippocampal insulin resistance might play a key role in mediating the long-term effects of maternal obesity and perinatal hyperinsulinemia on hippocampal plasticity and the offspring's neurocognitive outcome.

Matching Dietary Amino Acid Balance to the In Silico-Translated Exome Optimizes Growth and Reproduction without Cost to Lifespan.

Balancing the quantity and quality of dietary protein relative to other nutrients is a key determinant of evolutionary fitness. A theoretical framework for defining a balanced diet would both reduce the enormous workload to optimize diets empirically and represent a breakthrough toward tailoring diets to the needs of consumers. Here, we report a simple and powerful in silico technique that uses the genome information of an organism to define its dietary amino acid requirements. We show for the fruit fly Drosophila melanogaster that such "exome-matched" diets are more satiating, enhance growth, and increase reproduction relative to non-matched diets. Thus, early life fitness traits can be enhanced at low levels of dietary amino acids that do not impose a cost to lifespan. Exome matching also enhanced mouse growth, indicating that it can be applied to other organisms whose genome sequence is known.

Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Experimental analysis of risk factors for ulcerative dermatitis in mice.

Ulcerative dermatitis (UD) is a severe inflammatory skin disorder with an unknown aetiology. Recently, insulin receptor substrate 1 KO mice were shown to be fully resistant to UD. In this study, we showed that high-fat diet (HFD) feeding significantly increased incidence of UD in wild type (WT) C57BL/6 mice, as did lithium-mediated inhibition of GSK3-ß, which is a key negative regulator of IRS1. In contrast to WT mice, resistance to UD was fully preserved in HFD-fed Irs1-KO mice. Our results identify IRS1 as a key determinant of UD pathogenesis and establish a direct link between diet composition, obesity-induced inflammation and chronic ulceration.

Role for insulin signaling in catecholaminergic neurons in control of energy homeostasis.

Dopaminergic midbrain neurons integrate signals on food palatability and food-associated reward into the complex control of energy homeostasis. To define the role of insulin receptor (IR) signaling in this circuitry, we inactivated IR signaling in tyrosine hydroxylase (Th)-expressing cells of mice (IR(ΔTh)). IR inactivation in Th-expressing cells of mice resulted in increased body weight, increased fat mass, and hyperphagia. While insulin acutely stimulated firing frequency in 50% of dopaminergic VTA/SN neurons, this response was abolished in IR(ΔTh) mice. Moreover, these mice exhibited an altered response to cocaine under food-restricted conditions. Taken together, these data provide in vivo evidence for a critical role of insulin signaling in catecholaminergic neurons to control food intake and energy homeostasis.

Enhanced Stat3 activation in POMC neurons provokes negative feedback inhibition of leptin and insulin signaling in obesity.

Leptin-stimulated Stat3 activation in proopiomelanocortin (POMC)-expressing neurons of the hypothalamus plays an important role in maintenance of energy homeostasis. While Stat3 activation in POMC neurons is required for POMC expression, the role of elevated basal Stat3 activation as present in the development of obesity has not been directly addressed. Here, we have generated and characterized mice expressing a constitutively active version of Stat3 (Stat3-C) in POMC neurons (Stat3-C(POMC) mice). On normal chow diet, these animals develop obesity as a result of hyperphagia and decreased POMC expression accompanied by central leptin and insulin resistance. This unexpected finding coincides with POMC-cell-specific, Stat3-mediated upregulation of SOCS3 expression inhibiting both leptin and insulin signaling as insulin-stimulated PIP(3) (phosphatidylinositol-3,4,5 triphosphate) formation and protein kinase B (AKT) activation in POMC neurons as well as with the fact that insulin's ability to hyperpolarize POMC neurons is largely reduced in POMC cells of Stat3-C(POMC) mice. These data indicate that constitutive Stat3 activation is not sufficient to promote POMC expression but requires simultaneous PI3K (phosphoinositide 3-kinase)-dependent release of FOXO1 repression. In contrast, upon exposure to a high-fat diet, food intake and body weight were unaltered in Stat3-C(POMC) mice compared with control mice. Taken together, these experiments directly demonstrate that enhanced basal Stat3 activation in POMC neurons as present in control mice upon high-fat feeding contributes to the development of hypothalamic leptin and insulin resistance.

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.

Signal-dependent control of gluconeogenic key enzyme genes through coactivator-associated arginine methyltransferase 1.

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.