Ceramides are necessary and sufficient for diet-induced impairment of thermogenic adipocytes
Authors: Scott A Summers
Li Ying
Yoshio Hirabayashi
Bhagirath Chaurasia
Chad Lamar Talbot
John Alan Maschek
William L Holland
James Cox
Edward H Schuchman
Journal: Molecular Metabolism (2021)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Ceramides; Diabetes; Insulin resistance; Obesity; Sphingolipids
Affiliations: Division of Endocrinology, Department of Internal Medicine, Carver College of Medicine, Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, 52242, USA; Department of Nutrition and Integrative Physiology and the Diabetes and Metabolism Research Center, University of Utah, Salt Lake City, UT, USA. Electronic address: ., Department of Nutrition and Integrative Physiology and the Diabetes and Metabolism Research Center, University of Utah, Salt Lake City, UT, USA., Department of Biochemistry, University of Utah, Salt Lake City, UT, USA., Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, USA., RIKEN Brain Science Institute, Wako-Shi, Saitama Japan.

Abstract:  Objective: Aging and weight gain lead to a decline in brown and beige adipocyte functionality that exacerbates obesity and insulin resistance. We sought to determine whether sphingolipids, such as ceramides, a class of lipid metabolites that accumulate in aging and overnutrition, are sufficient or necessary for the metabolic impairment of these thermogenic adipocytes. Methods: We generated new mouse models allowing for the conditional ablation of genes required for ceramide synthesis (i.e., serine palmitoyltransferase subunit 2, Sptlc2) or degradation (i.e., acid ceramidase 1, Asah1) from mature, thermogenic adipocytes (i.e., from cells expressing uncoupling protein-1). Mice underwent a comprehensive suite of phenotyping protocols to assess energy expenditure and glucose and lipid homeostasis. Complementary studies were conducted in primary brown adipocytes to dissect the mechanisms controlling ceramide synthesis or action. Results: Depletion of Sptlc2 increased energy expenditure, improved glucose homeostasis, and prevented diet-induced obesity. Conversely, depletion of Asah1 led to ceramide accumulation, diminution of energy expenditure, and exacerbation of insulin resistance and obesity. Mechanistically, ceramides slowed lipolysis, inhibited glucose uptake, and decreased mitochondrial respiration. Moreover, β-adrenergic receptor agonists, which activate thermogenesis in brown adipocytes, decreased transcription of enzymes required for ceramide synthesis. Conclusions: These studies support our hypothesis that ceramides are necessary and sufficient for the impairment in thermogenic adipocyte function that accompanies obesity. Moreover, they suggest that implementation of therapeutic strategies to block ceramide synthesis in thermogenic adipocytes may serve as a means of improving adipose health and combating obesity and cardiometabolic disease. 

Retracted: No

PubPeer comments:
 Fig. 1 B-D: Here authors treated the brown adipocytes with beta-adrenergic receptors agonists, isoproterenol (1 uM and 5 uM) and 13C-15N-Serine labelled serine, and measured the cellular abundance of 13C-15N labeled ceramide. These are very potent drugs that work effectively even at low nanomolar range (lipolysis in fat cells 1-10 nM). At the concentrations used here, the response they see could be some nonspecific or off-target effects. At very high concentrations as the ones used here, Isoproterenol treatment would induce lipolysis and block lipid synthesis (palmitate synthesis) and possibly blocks nutrient uptake. If it blocked de novo palmitate synthesis or serine uptake, which also would also cause severe reduction in cellular abundance of 13C-15N-Serine labelled Ceramide and low labled-Ceramide unlabeled-Ceramide as seen here. They should show absolute ceramide concentrations for labeled and unlabeled forms, total cellular label concentrations for all the samples so we can be sure that the serine uptake was not reduced in Iso-treated cells. In addition, readers would also like to see palmitate concentrations in vehicle and Iso-treated cells. A direct proof for isoproterenol activating SPT enzyme activity would be measuring the activity SPT immunoprecipitate prepared from isoproterenol and vehicle u2013treated cells and testing the specificity by conducting this assay in the presence and absence of myriocin, a SPT enzyme inhibitor. Figure 1F. The serum ceramide concentrations in NCD fed control C57BL 6 mice shown in this article is about 10 times more than concentrations previously reported by the authors (Summers lab) (Figure 4A, https: science.sciencemag.org content sci suppl 2019 07 02 science.aav3722.DC1 aav3722_Chaurasia_SM.pdf). Similar discrepancy was also seen in tissue ceramide concentrations. This problem is mainly due to inefficient metabolite extraction and quantification, which is always a major problem with quantitative lipidomics. Such issues questions the credibility entire lipidomics data presented in these papers and the conclusions drawn based on these data. Fig. 2B. NCD controls did not respond to insulin during ITT. Were they also insulin resistant? Fig 4 A and K: FLIR infrared camera used for measuring the surface temperature of BAT in mice. In case of infrared camera imaging, object distance influences the recorded temperature of an object. To perform accurate recording of temperatures, same object distance should be maintained for all experimental groups. Since this article lacks that information, it is difficult to know whether authors are aware of this fact. Fig. 4 D N- the UPC1 signal intensity in HFD fed control BAT micrographs appear to change according to authorsu2019 expectations and needs. When they want to prove that HFD fed sptlc KO BAT show better UCP1 staining when compared with HFD fed controls, they show a lighter micrograph for HFD controls (Fig. 4D) and it was reversed in case of HFD Asah KO and HFD controls (Fig. 4N). However, it is known that HFD feeding does not reduce UCP1 expression in BAT (Am J Physiol Regul Integr Comp Physiol 300: R1u2013R8, 2011). Fig 4 E and O: Mitochondrial complex activities in BAT homogenates using O2K respirometer. BAT is a heterogeneous tissue with multiple cell types that differ in mitochondrial content. In addition to that, each BAT explant would differ in number of brown adipocytes and amount of mitochondria from that cell type. So it would be incorrect to assume as the authors do that changes seen mitochondrial complexes in knockouts are solely due to changes in ceramide expression in Brown adipocytes. ,