David James Dyck

Unacylated ghrelin (unAG) is a metabolically active hormone, acutely stimulating fatty acid oxidation in skeletal muscle while promoting lipogenesis and inhibiting lipolysis in adipose tissue. Given these acute effects, we hypothesized that repeated unAG administration would improve skeletal muscle insulin response and that this would be associated with reduced muscle lipid storage. We treated healthy male Sprague–Dawley rats with twice‐daily subcutaneous unAG (200 μg; 400 μg total daily) for 5 days. UnAG treatment did not alter glucose uptake or AKT activation basally, or in response to low or high insulin concentrations when assessed ex vivo in oxidative or glycolytic skeletal muscle, or when muscle was pre‐exposed to a high‐palmitate stress. Protein content of mitochondrial markers (CPT1B, citrate synthase, COXIV) also remained unchanged with treatment in both muscle types. Interestingly, UnAG‐treated animals had a reduced glycemic response to pyruvate administration; however, content of hepatic glucose production‐associated proteins and liver AKT response to insulin remained unchanged. UnAG treatment tended to increase LPL protein content in visceral adipose tissue ( p = 0.07), but did not alter AKT or HSL phosphorylation in response to an insulin bolus in either subcutaneous or visceral adipose tissue. Finally, triacylglycerol content in the red and white gastrocnemius muscle, as well as in the liver, was unchanged by unAG administration. Overall, these findings indicate that short‐term unAG treatment improves pyruvate tolerance, but that it does not exert persistent metabolic changes to skeletal muscle, liver, or adipose tissue substrate metabolism in healthy rats.

Short‐chain fatty acids (SCFAs), derived from peroxisomal metabolism and the gut microbiota, have been proposed as key substrates to support mitochondrial oxidative phosphorylation (OXPHOS) in extrahepatic tissues such as skeletal muscle. However, the extent to which mitochondria can oxidize SCFAs (acetate, propionate and butyrate) and the ability of exercise training and a high‐fat diet (HFD) to modulate this process remains unclear. Here, we show that SCFA‐supported respiration in skeletal muscle is relatively limited (18 ± 6 nmol min ⁻¹ mg ⁻¹ ), accounting for only ∼7% of maximal carbohydrate (pyruvate: 252 ± 41 nmol min ⁻¹ mg ⁻¹ ) and ∼14% of LCFA (palmitoylcarnitine)‐linked respiration. Despite this low capacity, the intrinsic mitochondrial ability to oxidize palmitoylcarnitine, acetate and butyrate increased ( P < 0.05: +50%) following HFD consumption, suggesting HFD rewires mitochondria to optimize lipid oxidation. By contrast, exercise training prevented these HFD‐induced intrinsic mitochondrial responses. Although intrinsic changes are biologically relevant, skeletal muscle adaptation to metabolic stress also involves mitochondrial biogenesis and an expansion of the mitochondrial proteome. Proteomic analysis and citrate synthase activity revealed that, although HFD independently did not alter mitochondrial protein abundance, exercise training increased mitochondrial proteins, a response amplified in the presence of a HFD. Consequently, although exercise did not directly enhance mitochondrial SCFA‐supported respiration, the combined effect of HFD and exercise predicted a greater overall capacity for SCFA oxidation because of increased mitochondrial abundance. Collectively, although SCFAs contribute minimally to mitochondrial respiration in skeletal muscle, combined HFD and exercise synergistically enhance overall OXPHOS capacity across diverse substrates, including SCFAs, primarily through increased mitochondrial protein abundance rather than intrinsic mitochondrial remodelling. image Key points: Peroxisome and gut derived short‐chain fatty acids (SCFA) have been proposed as an alternative metabolic fuel source to support skeletal muscle oxidative phosphorylation. The capacity and adaptability of mitochondrial SCFA oxidation remains unknown. SCFA‐supported mitochondrial respiration is limited (<15%) compared to carbohydrate (pyruvate) and long‐chain fatty acid linked substrates. High‐fat feeding increased the intrinsic capacity of mitochondria to utilize palmitoylcarnitine, acetate and butyrate− effects prevented by 4 weeks of exercise training. Combined high‐fat diet and exercise training increased skeletal muscle mitochondrial protein content in an additive manner, increasing oxidative capacity and ability to utilize both long‐ and SCFAs as a fuel source.

Ketogenic diets (KDs) improve glucose metabolism in humans and rodents that are insulin resistant, but their impact is unclear in a healthy context. Furthermore, standard KDs typically lack beneficial omega-3 long-chain polyunsaturated fatty acids (n3-LCPUFA). This study assessed whether supplementing a KD with n3-LCPUFA could alter glucose homeostasis or skeletal muscle insulin response. No differences were observed between a standard KD and a KD with n3-LCPUFA when energy intake was controlled.

While a definitive mechanism‐of‐action remains to be identified, recent findings indicate that ghrelin, particularly the unacylated form (UnAG), stimulates skeletal muscle fatty acid oxidation. The biological importance of UnAG‐mediated increases in fat oxidation remains unclear, as UnAG peaks in the circulation before mealtimes, and decreases rapidly during the postprandial situation before increases in postabsorptive circulating lipids. Therefore, we aimed to determine if the UnAG‐mediated stimulation of fat oxidation would persist long enough to affect the oxidation of meal‐derived fatty acids, and if UnAG stimulated the translocation of fatty acid transporters to the sarcolemma as a mechanism‐of‐action. In isolated soleus muscle strips from male rats, short‐term pre‐treatment with UnAG elicited a persisting stimulus on fatty acid oxidation 2 h after the removal of UnAG. UnAG also caused an immediate phosphorylation of AMPK, but not an increase in plasma membrane FAT/CD36 or FABPpm. There was also no increase in AMPK signaling or increased FAT/CD36 or FABPpm content at the plasma membrane at 2 h which might explain the sustained increase in fatty acid oxidation. These findings confirm UnAG as a stimulator of fatty acid oxidation and provide evidence that UnAG may influence the handling of postprandial lipids. The underlying mechanisms are not known.

Skeletal muscle is the primary location of insulin-stimulated glucose uptake. EPA and DHA supplementation has been observed to improve skeletal muscle insulin-stimulated glucose uptake in models of metabolic dysfunction. Fads2^−/− knockout mice cannot endogenously produce long-chain n-3 polyunsaturated fatty acids. Our results show that the absence of DHA in skeletal muscle is not detrimental to whole body glucose homeostasis in healthy mice.

Ghrelin is released from the stomach as an anticipatory signal prior to a meal and decreases immediately after. Previous research has shown that both acylated (AG) and unacylated (UnAG) ghrelin blunt adrenoreceptor-stimulated lipolysis in rat white adipose tissue (WAT) ex vivo. We investigated whether acute or chronic consumption of a high fat diet (HFD) impaired the ability of ghrelin to regulate adipose tissue lipolysis, and if this impairment could be restored with exercise. After 5 days (5d) of a HFD, or 6 weeks (6 w) of a HFD (60% kcal from fat) with or without exercise training, inguinal and retroperitoneal WAT was collected from anesthetized rats for adipose tissue organ culture. Samples were treated with 1 μM CL 316,243 (CL; lipolytic control), 1 μM CL+150 ng/ml AG or 1 μM CL+150 ng/ml UnAG. Incubation media and tissue were collected after 2 hours. Colorometric assays were used to determine glycerol and free fatty acid (FFA) concentrations in media. Western blots were used to quantify the protein content of lipolytic enzymes and ghrelin receptors in both depots. CL stimulated lipolysis was evidenced by increases in glycerol (p < 0.0001) and FFA (p < 0.0001) concentrations in media compared to control. AG decreased CL-stimulated glycerol release in inguinal WAT from 5d LFD rats (p = 0.0097). Neither AG nor UnAG blunted lipolysis in adipose tissue from 5d or 6 w HFD-fed rats, and exercise did not restore ghrelin’s anti-lipolytic ability in 6 w HFD-fed rats. Overall, this study demonstrates that HFD consumption impairs ghrelin’s ability to regulate adipose tissue lipolysis.