Long–echo time MR spectroscopy for skeletal muscle acetylcarnitine detection

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Abstract

Animal models suggest that acetylcarnitine production is essential for maintaining metabolic flexibility and insulin sensitivity. Because current methods to detect acetylcarnitine involve biopsy of the tissue of interest, noninvasive alternatives to measure acetylcarnitine concentrations could facilitate our understanding of its physiological relevance in humans. Here, we investigated the use of long–echo time (TE) proton magnetic resonance spectroscopy (¹H-MRS) to measure skeletal muscle acetylcarnitine concentrations on a clinical 3T scanner. We applied long-TE ¹H-MRS to measure acetylcarnitine in endurance-trained athletes, lean and obese sedentary subjects, and type 2 diabetes mellitus (T2DM) patients to cover a wide spectrum in insulin sensitivity. A long-TE ¹H-MRS protocol was implemented for successful detection of skeletal muscle acetylcarnitine in these individuals. There were pronounced differences in insulin sensitivity, as measured by hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial function, as measured by phosphorus-MRS (³¹P-MRS), across groups. Insulin sensitivity and mitochondrial function were highest in trained athletes and lowest in T2DM patients. Skeletal muscle acetylcarnitine concentration showed a reciprocal distribution, with mean acetylcarnitine concentration correlating with mean insulin sensitivity in each group. These results demonstrate that measuring acetylcarnitine concentrations with ¹H-MRS is feasible on clinical MR scanners and support the hypothesis that T2DM patients are characterized by a decreased formation of acetylcarnitine, possibly underlying decreased insulin sensitivity.

Authors

Lucas Lindeboom, Christine I. Nabuurs, Joris Hoeks, Bram Brouwers, Esther Phielix, M. Eline Kooi, Matthijs K.C. Hesselink, Joachim E. Wildberger, Robert D. Stevens, Timothy Koves, Deborah M. Muoio, Patrick Schrauwen, Vera B. Schrauwen-Hinderling

PPARγ ablation sensitizes proopiomelanocortin neurons to leptin during high-fat feeding

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Abstract

Activation of central PPARγ promotes food intake and body weight gain; however, the identity of the neurons that express PPARγ and mediate the effect of this nuclear receptor on energy homeostasis is unknown. Here, we determined that selective ablation of PPARγ in murine proopiomelanocortin (POMC) neurons decreases peroxisome density, elevates reactive oxygen species, and induces leptin sensitivity in these neurons. Furthermore, ablation of PPARγ in POMC neurons preserved the interaction between mitochondria and the endoplasmic reticulum, which is dysregulated by HFD. Compared with control animals, mice lacking PPARγ in POMC neurons had increased energy expenditure and locomotor activity; reduced body weight, fat mass, and food intake; and improved glucose metabolism when exposed to high-fat diet (HFD). Finally, peripheral administration of either a PPARγ activator or inhibitor failed to affect food intake of mice with POMC-specific PPARγ ablation. Taken together, our data indicate that PPARγ mediates cellular, biological, and functional adaptations of POMC neurons to HFD, thereby regulating whole-body energy balance.

Authors

Lihong Long, Chitoku Toda, Jing Kwon Jeong, Tamas L. Horvath, Sabrina Diano

Ventromedial hypothalamus–specific Ptpn1 deletion exacerbates diet-induced obesity in female mice

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Abstract

Protein-tyrosine phosphatase 1B (PTP1B) regulates food intake (FI) and energy expenditure (EE) by inhibiting leptin signaling in the hypothalamus. In peripheral tissues, PTP1B regulates insulin signaling, but its effects on CNS insulin action are largely unknown. Mice harboring a whole-brain deletion of the gene encoding PTP1B (Ptpn1) are lean, leptin-hypersensitive, and resistant to high fat diet–induced (HFD-induced) obesity. Arcuate proopiomelanocortin (POMC) neuron–specific deletion of Ptpn1 causes a similar, but much milder, phenotype, suggesting that PTP1B also acts in other neurons to regulate metabolism. Steroidogenic factor-1–expressing (SF-1–expressing) neurons in the ventromedial hypothalamus (VMH) play an important role in regulating body weight, FI, and EE. Surprisingly, Ptpn1 deletion in SF-1 neurons caused an age-dependent increase in adiposity in HFD-fed female mice. Although leptin sensitivity was increased and FI was reduced in these mice, they had impaired sympathetic output and decreased EE. Immunohistochemical analysis showed enhanced leptin and insulin signaling in VMH neurons from mice lacking PTP1B in SF-1 neurons. Thus, in the VMH, leptin negatively regulates FI, promoting weight loss, whereas insulin suppresses EE, leading to weight gain. Our results establish a novel role for PTP1B in regulating insulin action in the VMH and suggest that increased insulin responsiveness in SF-1 neurons can overcome leptin hypersensitivity and enhance adiposity.

Authors

Franck Chiappini, Karyn J. Catalano, Jennifer Lee, Odile D. Peroni, Jacqueline Lynch, Abha S. Dhaneshwar, Kerry Wellenstein, Alexandra Sontheimer, Benjamin G. Neel, Barbara B. Kahn

Altered miRNA processing disrupts brown/white adipocyte determination and associates with lipodystrophy

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Abstract

miRNAs are important regulators of biological processes in many tissues, including the differentiation and function of brown and white adipocytes. The endoribonuclease dicer is a major component of the miRNA-processing pathway, and in adipose tissue, levels of dicer have been shown to decrease with age, increase with caloric restriction, and influence stress resistance. Here, we demonstrated that mice with a fat-specific KO of dicer develop a form of lipodystrophy that is characterized by loss of intra-abdominal and subcutaneous white fat, severe insulin resistance, and enlargement and “whitening” of interscapular brown fat. Additionally, KO of dicer in cultured brown preadipocytes promoted a white adipocyte–like phenotype and reduced expression of several miRNAs. Brown preadipocyte whitening was partially reversed by expression of miR-365, a miRNA known to promote brown fat differentiation; however, introduction of other miRNAs, including miR-346 and miR-362, also contributed to reversal of the loss of the dicer phenotype. Interestingly, fat samples from patients with HIV-related lipodystrophy exhibited a substantial downregulation of dicer mRNA expression. Together, these findings indicate the importance of miRNA processing in white and brown adipose tissue determination and provide a potential link between this process and HIV-related lipodystrophy.

Authors

Marcelo A. Mori, Thomas Thomou, Jeremie Boucher, Kevin Y. Lee, Susanna Lallukka, Jason K. Kim, Martin Torriani, Hannele Yki-Järvinen, Steven K. Grinspoon, Aaron M. Cypess, C. Ronald Kahn

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity

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Abstract

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a person’s microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

Authors

Zhongyi Chen, Lilu Guo, Yongqin Zhang, Rosemary L. Walzem, Julie S. Pendergast, Richard L. Printz, Lindsey C. Morris, Elena Matafonova, Xavier Stien, Li Kang, Denis Coulon, Owen P. McGuinness, Kevin D. Niswender, Sean S. Davies

Leptin-promoted cilia assembly is critical for normal energy balance

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Abstract

The majority of mammalian cells have nonmotile primary cilia on their surface that act as antenna-like sensory organelles. Genetic defects that result in ciliary dysfunction are associated with obesity in humans and rodents, which suggests that functional cilia are important for controlling energy balance. Here we demonstrated that neuronal cilia lengths were selectively reduced in hypothalami of obese mice with leptin deficiency and leptin resistance. Treatment of N1 hypothalamic neuron cells with leptin stimulated cilia assembly via inhibition of the tumor suppressors PTEN and glycogen synthase kinase 3β (GSK3β). Induction of short cilia in the hypothalamus of adult mice increased food intake and decreased energy expenditure, leading to a positive energy balance. Moreover, mice with short hypothalamic cilia exhibited attenuated anorectic responses to leptin, insulin, and glucose, which indicates that leptin-induced cilia assembly is essential for sensing these satiety signals by hypothalamic neurons. These data suggest that leptin governs the sensitivity of hypothalamic neurons to metabolic signals by controlling the length of the cell’s antenna.

Authors

Yu Mi Han, Gil Myoung Kang, Kyunghee Byun, Hyuk Wan Ko, Joon Kim, Mi-Seon Shin, Hyun-Kyong Kim, So Young Gil, Ji Hee Yu, Bonghee Lee, Min-Seon Kim

Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk

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Abstract

The nuclear bile acid receptor farnesoid X receptor (FXR) is an important transcriptional regulator of bile acid, lipid, and glucose metabolism. FXR is highly expressed in the liver and intestine and controls the synthesis and enterohepatic circulation of bile acids. However, little is known about FXR-associated proteins that contribute to metabolic regulation. Here, we performed a mass spectrometry–based search for FXR-interacting proteins in human hepatoma cells and identified AMPK as a coregulator of FXR. FXR interacted with the nutrient-sensitive kinase AMPK in the cytoplasm of target cells and was phosphorylated in its hinge domain. In cultured human and murine hepatocytes and enterocytes, pharmacological activation of AMPK inhibited FXR transcriptional activity and prevented FXR coactivator recruitment to promoters of FXR-regulated genes. Furthermore, treatment with AMPK activators, including the antidiabetic biguanide metformin, inhibited FXR agonist induction of FXR target genes in mouse liver and intestine. In a mouse model of intrahepatic cholestasis, metformin treatment induced FXR phosphorylation, perturbed bile acid homeostasis, and worsened liver injury. Together, our data indicate that AMPK directly phosphorylates and regulates FXR transcriptional activity to precipitate liver injury under conditions favoring cholestasis.

Authors

Fleur Lien, Alexandre Berthier, Emmanuel Bouchaert, Céline Gheeraert, Jeremy Alexandre, Geoffrey Porez, Janne Prawitt, Hélène Dehondt, Maheul Ploton, Sophie Colin, Anthony Lucas, Alexandre Patrice, François Pattou, Hélène Diemer, Alain Van Dorsselaer, Christophe Rachez, Jelena Kamilic, Albert K. Groen, Bart Staels, Philippe Lefebvre

Relative acidic compartment volume as a lysosomal storage disorder–associated biomarker

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Abstract

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency–approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.

Authors

Danielle te Vruchte, Anneliese O. Speak, Kerri L. Wallom, Nada Al Eisa, David A. Smith, Christian J. Hendriksz, Louise Simmons, Robin H. Lachmann, Alison Cousins, Ralf Hartung, Eugen Mengel, Heiko Runz, Michael Beck, Yasmina Amraoui, Jackie Imrie, Elizabeth Jacklin, Kate Riddick, Nicole M. Yanjanin, Christopher A. Wassif, Arndt Rolfs, Florian Rimmele, Naomi Wright, Clare Taylor, Uma Ramaswami, Timothy M. Cox, Caroline Hastings, Xuntian Jiang, Rohini Sidhu, Daniel S. Ory, Begona Arias, Mylvaganam Jeyakumar, Daniel J. Sillence, James E. Wraith, Forbes D. Porter, Mario Cortina-Borja, Frances M. Platt

Targeting the cell cycle inhibitor p57^Kip2 promotes adult human β cell replication

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Abstract

Children with focal hyperinsulinism of infancy display a dramatic, non-neoplastic clonal expansion of β cells that have undergone mitotic recombination, resulting in paternal disomy of part of chromosome 11. This disomic region contains imprinted genes, including the gene encoding the cell cycle inhibitor p57^Kip2 (CDKN1C), which is silenced as a consequence of the recombination event. We hypothesized that targeting p57^Kip2 could stimulate adult human β cell replication. Indeed, when we suppressed CDKN1C expression in human islets obtained from deceased adult organ donors and transplanted them into hyperglycemic, immunodeficient mice, β cell replication increased more than 3-fold. The newly replicated cells retained properties of mature β cells, including the expression of β cell markers such as insulin, PDX1, and NKX6.1. Importantly, these newly replicated cells demonstrated normal glucose-induced calcium influx, further indicating β cell functionality. These findings provide a molecular explanation for the massive β cell replication that occurs in children with focal hyperinsulinism. These data also provided evidence that β cells from older humans, in which baseline replication is negligible, can be coaxed to re-enter and complete the cell cycle while maintaining mature β cell properties. Thus, controlled manipulation of this pathway holds promise for the expansion of β cells in patients with type 2 diabetes.

Authors

Dana Avrahami, Changhong Li, Ming Yu, Yang Jiao, Jia Zhang, Ali Naji, Seyed Ziaie, Benjamin Glaser, Klaus H. Kaestner

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

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Abstract

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.

Authors

Lijun Liu, Minwoo Nam, Wei Fan, Thomas E. Akie, David C. Hoaglin, Guangping Gao, John F. Keaney Jr., Marcus P. Cooper