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Download ( 947KB ) Arvind Rajan, Andrea S. Pereyra, Jessica M. Ellis Department of Physiology, Brody School of Medicine, East Carolina Diabetes and Obesity Institute, East Carolina University Determining the Effects of Impaired Muscle Fatty Acid Oxidation on Liver Metabolism Introduction 2. Hepatic Synthesis of Alternative Substrates 5. Effects of Carnitine Supplementation Skeletal muscle plays an extremely critical role in maintaining A. Free Carnitine B. LC-acylcarnitines in Skeletal Muscle C. L-carnitine hepatic synthesis n glucose homeostasis, and is responsible for the majority of 3 +/+ ) Sk LF o ✱ D +/+ i ✱ -/- Sk LF F ✱ s Figure 2. Because whole-body Sk LF n 2.5 ✱ ✱ i +/+ -/- s glucose uptake for energy production. Carnitine +/+ 100000 L Sk HF Sk LF Sk LF e e + t r -/- -/- / +/+ glucose homeostasis is highly ) Sk HF ✱ ✱ + e Sk HF Sk LF p 2.0 o F palmitoyltransferase II (CPT2) is an irreplaceable enzyme for k n ✱ +/+ r 80000 x -/- i 2 L Sk HF S Sk HF t E p regulated by liver metabolism, 14 i + -/- / o 1.5 e n Sk HF t + oxidation of long-chain fatty acids in muscle, converting ✱ g r 60000 v k i u a genes in three main hepatic ✱ d t / S e t a C 1.0 l acylcarnitine into acyl-CoA within the mitochondrial matrix to ✱ z n i o e e 40000 l pathways (GNG: gluconeogenesis, t 1 u e R a r 0.5 o allow for β-oxidation. N m ( F A KG: ketogenesis and β-Oxidation c r 20000 N Defects in CPT2 o n R 0.0 N of fatty acids) were assessed. ( o I impair mitochondrial 0 m Bbox1 Tmlhe Enzyme-coding genes are 0 -Carn +Carn β-oxidation and result D. LiverSkeletal Muscle indicated as red boxes in the Mitochondrial β-oxidation E. Ketogenesis n in the buildup of +/+ n corresponding steps in the o o +/+ i Sk LF +Carn i ) 4 ) 10 Sk LF +Carn s -/- s n acylcarnitines within Sk LF +Carn n -/- pathways. Metabolite s r s r Sk LF +Carn * +/+ e a e Sk HF +Carn a +/+ r the skeletal muscle r Sk HF +Carn * transporters were also evaluated C C 8 p -/- p - * 3 - -/- Sk HF +Carn x Sk HF +Carn x o which are predicted and are indicated as light-blue o E E t t * 6 * e * e * d to impair glucose boxes. Master regulators are d v v e e i 2 * i t z t z i * i depicted as grey 10-point stars. a l a metabolism. l l 4 l a a e e m R m 1 R * r * r 2 * * * o A o A * * N N N Methods * N ( * * ( R 0 R 0 m Cpt1a Cpt2 Acadm Acadl Acadvl m Creb3l3 Bdh1 Hmgcs2 Slc16a1 Fgf21 Real-Time PCR was utilized to determine the abundance of 3. Liver adaptations to muscle-specific CPT2 deficiency codifying RNA (mRNA) involved in mitochondrial β-oxidation, F gluconeogenesis, and ketogenesis in liver samples from 4 Gluconeogenesis Figure 5. Exogenous L-carnitine supplementation C. n A. Mitochondrial -oxidation B. Ketogenesis Gluconeogenesis +/+ obliterated the genotype-specific upregulation of o i Sk LF +Carn experimental groups of 6 mice each: 1) control mice on low fat ) n 5 n n s -/- n ) ) ) o o Sk+/+ Sk+/+ o Sk LF +Carn fatty acid oxidation, gluconeogenesis, and s i r i i ✱ F F diet (Cpt2 LF) 2) control mice on a 60% high-fat diet (Cpt2 5 +/+ F 8 6 +/+ s +/+ e s a s * +/+ L L Sk LF L Sk LF Sk HF +Carn r s s s Sk LF * ketogenic genes in liver. Muscle–specific Cpt2 Sk-/- C -/- + + + -/- 4 ✱ e / p -/- e / e / -/- HF) 3) knockout mice on LF diet (Cpt2 LF) 4) knockout mice Sk LF Sk LF - * r + r r Sk HF +Carn + + Sk LF * 4 x p k k p p k Sk-/- 6 o knockout mice have significantly lower levels of E x t x x S S S on HF diet (Cpt2 HF). To aid the metabolic flux impaired by E 4 E 3 E * e o d o o free, available carnitine in their skeletal muscles t t 3 ✱ t e v * * e e e ✱ acylcarnitine accumulation, free carnitine was supplemented to i v v ✱ v t d ✱ z d d i i i 4 ✱ i t t ✱ t e a * due to high accumulation of LC-acyl-carnitines (A, e e l 2 l ✱ a a ✱ a z z another set of mice with the same grouping as above, and each z 2 l a l l i i i e l l l e e e 2 * B). The muscle carnitine deficit triggers the a a a m R R R group consisted of 3 mice. Gene expression was normalized to R r 2 m m 1 m 1 o r r A A r A A upregulation of genes involved in hepatic carnitine o o the housekeeping enzyme Beta-2-Microglobulin (B2M). o N N N N N * ( N N N R R R ( R ( 0 ( 0 0 0 synthesis like Gamma-Butyrobetaine Hydroxylase 1 m m m Cpt1a Cpt2 Acadm Acadl Acadvl Creb3l3 Bdh1 Hmgcs2Slc16a1 Fgf21 Pck1 G6pc Slc2a2 Pcx Fbp1 m Pck1 G6pc Slc2a2 Pcx Fbp1 (Bbox1) and Trimethyllysine Hydroxylase (Tmlhe) in -/- Cpt2 knockout mice (C). 1. Cpt2Sk Mouse Model Figure 3. Hepatic adaptations to skeletal muscle-specific CPT2 deficiency involves upregulation of metabolic pathways aiming to produce alternative fuel sources. In order to attempt restoring metabolic flux, free carnitine was supplemented to the drinking A ) 1.5 CPT2 n A. N Tub B. -/- water of mice for 16 weeks and liver gene expression for FAO (D), KG (E) and GNG (F) was o R i Upon abolishment of fatty acid oxidation in the skeletal muscle, the liver of Cpt2 knockout mice (Sk LF) s +/+ m Sk assessed again. Data was normalized to the respective Non-Carnitine groups (dashed orange s 2 e significantly upregulated the expression of enzymes involved in the production of ketone bodies (B) and glucose t r 1.0 -/- p p Sk line). Data is shown as average ± SEM. *p<0.05. x C (C) that can be utilized by peripheral tissues like muscles as alternative fuel sources. Ketogenesis is dependent on e e l e c v 0.5 high rates of FAO, thus genes of FAO are upregulated in knockout (A). Data is shown as average ± SEM. *p<0.05. s i t u a l M e $ r k ( Conclusions S 0.0 Sk+/+ Sk-/- Relative Abundance of Long-Chain Acylcarnitines Sk+/+ LFD Sk-/- 4 * * 4. Liver adaptations under high fat feeding C. 6×10 -/- ➢Our novel Cpt2 mouse model can modify expression of genes involved in Sk LFD Sk+/+ HFD 4 * 4×10 -/- n Sk HFD i hepatic biosynthetic pathways in order to provide alternative substrates to the e t * * * * o r A. 2×10 4 B. C. p g mtFAO–deficient skeletal muscle, demonstrating the role of multi-organ u * * / * * Ketogenesis t Gluconeogenesis 3 Mitochondrial -oxidation n 7×10 u n n o physiological cross talk as a regulator of metabolic homeostasis. 3 4.5×10 * n ✱ ) ) C o ✱ o ) o i i n ✱ F 3 8 F * * i 2×10 * F 6 +/+ o s 5 s I * * +/+ +/+ L s * L Sk HF 2 L Sk HF s 7.5×10 Sk HF s * * * s -/- + 2 + Sk-/- e -/- / + -/- e 5×10 ✱ / Sk HF e / r + ➢ Sk HF Sk HF r + Free carnitine levels in Cpt2 mice are significantly reduced in the skeletal 2 r 2.5×10 + ✱ p k p 4 6 k p 0 k x x 0 0 1 2 0 1 2 S ✱ C H H C S x : : : : : : : S D 4 6 8 8 0 0 D 6 O O E ✱ E 4 - - muscle and hepatic synthesis of L-carnitine is consequently upregulated. - - E 1 1 1 1 2 2 1 o o 0 0 1 2 o t : : C C C C C C : : C t ✱ e / e t 3 4 6 6 6 / ✱ e 1 1 v 1 1 1 v d : i v 4 d C C C C i d 6 / i t / e t 1 e t 0 e a z : C a l ✱ z a i z 8 l ➢ 2 i l Further supplementation of L-Carnitine in the drinking water aids the flux of l i 1 e l l e 2 ✱ e a C a R a R R 2 m +/+ m m r metabolic intermediaries thus eliminating the genotypic upregulation of these 1 Sk A r A r A Figure 1. To assess the specific effects of Cpt2-deficiency on o N o o LF N N N R N ( N R R pathways in knockout. ( muscle and whole-body physiology, we generated a skeletal ( 0 0 0 m m m Creb3l3 Bdh1 Hmgcs2Slc16a1 Fgf21 muscle specific CPT2-knockout mouse model (Cpt2Sk-/-). Deletion Cpt1a Cpt2 Acadm Acadl Acadvl Pck1 G6pc Slc2a2 Pcx Fbp1 of skeletal muscle Cpt2 was confirmed in the soleus muscle both Figure 4. Under a high fat diet, hepatic adaptations to skeletal muscle-specific CPT2 deficiency involves increased in the proteome (Western Blot) and transcriptome (RT-PCR) (A). upregulation of metabolic pathways aiming to produce alternative fuel sources. References Compared to controls, the skeletal muscles of Cpt2Sk-/- mice have Due to very low glucose availability from the diet coupled with the inhibition of fatty acid oxidation in the skeletal 1. Lee, Jieun, et al. “Hepatic Fatty Acid Oxidation Restrains Systemic Catabolism during Starvation.” Cell Reports, vol.
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