Ceramides are necessary and sufficient for diet-induced

impairment of thermogenic adipocytes

Bhagirath Chaurasia et al.

Presented

by
Youssef Shalaby
Department of Genetics & Genomics
College of Medicine & Health Sciences
 CONTENTS
01 02 03
Introduction Objective Study design
Ceramides detrimental effects Aim of the study How the study was conducted

04 05
Methods Results & Conclusion
Laboratory techniques Brief discussion of the results
01 Overview
 Highlights of Lipotoxicity

Ceramides
Ceramides are lipid derivatives,
02 synthesized endogenously in
mammalian cells. They regulate
thermogenic adipocytes to
Adipose tissues influence energy utilization and
Two different types of adipose 01 expenditure.
tissues: White adipose tissue Ceramides
that stores energy in the form effects
of triacylglycerol (TAG) and 03 Ceramides block lipolysis by
Brown adipose tissue that
dissipates energy as heat, inhibiting activation of hormone-
“burning” fatty acids to sensitive lipase (HSL) by
maintain body temperature. isoproterenol.
 Aim of the study

To determine whether ceramides

that accumulate in aging and
overnutrition, are sufficient or
necessary for the metabolic
impairment of thermogenic
adipocytes
 De Novo Pathway

L- serine + palmitoyl-CoA
Serine
palmitoyltransferase

3- ketosphinganine Sphingomyelin hydrolysis Pathway

3- ketosphinganine Sphingomyelin
reductase
Sphinganine SM
aSMase /nSMase
synthase
Acyl-CoA Ceramide
Sphingosine
synthases 1-6
Ceramidase kinase Sphingosine-1
Dihydroceramide Sphingosine
Dihydroceramide Ceramide phosphate
desaturase Ceramide
Sphingosine
Synthases 1-6
Endoplasmic reticulum phosphatase
Glucosyl- Glucosyl-
ceramidase ceramide
synthase Salvage Pathway
Glucosylceramide
Figure by Shalaby YM, Al Aidaros A, Valappil A, Ali BR, Akawi N. Role of
Ceramides in the Molecular Pathogenesis and Potential Therapeutic Strategies of
Cardiometabolic Diseases: What we Know so Far. Front Cell Dev Biol. 2022 Jan
Golgi Apparatus
 Graphical abstract
Ceramides
Ceramides BAT activation Resistance
Synthesis
to diet-induced obesity
SPTLC2

Ceramidase

Ceramides BAT activation Obesity &

Ceramides
degradation low energy expenditure
 Study Design

Animal
Experiments Cell Culture
Six-week-old male C57Bl6/J Primary brown adipocytes were
allowing for the conditional cultured in (DMEM)/F12 plus
ablation of genes required for Glutamax, pen/strep, and 10%
ceramide synthesis (i.e., serine fetal bovine serum (FBS)
palmitoyltransferase subunit 2,
Sptlc2) or degradation (i.e., acid
ceramidase 1, Asah1)
 Methods
RNA Purification and BAT depots obtained from Sptlc2δ /
Gene Expression Analysis Asah1δ Ucp1 mice

qRT-PCR Immunohistochemistry

Insulin levels in serum Protein Extraction and

Blotting Analysis
ELISA kit Western blot

Generation of Sptlc2δ / Asah1δ Lipidomic Analysis

Ucp1 mice
loxP/loxP sites + Cre- UPLC-MS/MS system
recombinase
 Methods cont’d
Metabolic measurements Blood glucose concentrations

CLAMS (Columbus Glucometer

Instruments)

Heat production & Surface

Cellular oxygen consumption
temperature

Seahorse XF24 analyzer Infrared camera

 Results

Regulation of ceramides by Inhibition of ceramide

obesogenic diets and В- biosynthesis in UCP1+ cells
adrenergic stimuli in brown increases energy expenditure
adipocytes in mice

Inducing ceramide Ceramides alter

accumulation in UCP1+ mitochondrial structure and
cells decreases energy bioenergetics
expenditure
Knocking out Sptlc2
 Isoproterenol decreases ceramide synthesis

• Data are generally plotted as mean± SEM. *P < 0.05.

Mice lacking Sptlc2 have lower
ceramide levels
Mice lacking Sptlc2 in UCP1+ cells are resistant to diet-induced obesity

• The Sptlc2 δ Ucp1 mice fed the HFD

were partially protected from obesity.

• Also, exhibited increased VO2,

VCO2, energy expenditure and food
intake compared to the Sptlc2fl/fl
controls.
Mice lacking Sptlc2 in UCP1+ cells are resistant to diet-induced glucose intolerance

• The Sptlc2 δ Ucp1 knockout animals maintained on the obesogenic diet displayed improved glucose tolerance,
enhanced glucose disposal during an insulin-tolerance test and diminished insulin levels.
 Mice lacking Sptlc2 in UCP1+ cells are resistant to diet-induced hepatic
steatosis

• Sptlc2 δ Ucp1 mice exhibited reductions in various liver transcripts associated with
steatosis (e.g., F4/80, Ccl2, Cd36, Cidea, and Pparg)
Knocking out Asah1
(Ceramidase)
Mice lacking Asah1 in UCP1+ cells develop obesity and display reduced energy
expenditure
Mice lacking Asah1 in UCP1+ cells display impaired glucose
metabolism
 Sptlc2 ablation have stimulatory effects on BAT
thermogenesis and mitochondrial structure and function

UCP1+driven Sptlc2 depletion increased expression of several genes implicated in thermogenesis

 Asah1 ablation have inhibitory effects on BAT thermogenesis and mitochondrial structure

UCP1+ driven Asah1 depletion decreased expression of several genes implicated in thermogenesis
 IN VITRO STUDIES
(SPT inhibition by Myriocin)
 Ceramides have cell autonomous effects on fuel metabolism in primary brown
adipocytes

SPT inhibitor myriocin increased expression of genes involved in the thermogenic program
 Statistical analysis

●Differences between the groups were examined for

statistical significance via Student T-test or one way
ANOVA using GraphPad Prism.

●Statistical significance was defined as a P value <0.05

 Limitations
• The study was limited to male mice aged 5-6 weeks and does not
evaluate sex- or age-related differences in ceramide metabolism.
• The researchers did not specify the ratios of ceramides subtype that
possess detrimental/useful effects.
• How could the authors measure total ceramides??!
• The authors should have included the post-hoc test used for their
statistical analysis.
• Larger sample size are needed for clinical implications.
• Typing error in section 2.8
Thanks!!