Insulin rapidly increases skeletal muscle mitochondrial ADP sensitivity in the 1 absence of a high lipid environment 2

: Reductions in mitochondrial function have been proposed to cause insulin 18 resistance, however the possibility that impairments in insulin signaling negatively affects 19 mitochondrial bioenergetics has received little attention. Therefore, we tested the 20 hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key 21 process linked to oxidative phosphorylation and redox balance, and if this phenomenon 22 would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely 23 (60 minutes post I.P.) increased submaximal (100-1000 μM ADP) mitochondrial 24 respiration ~2-fold without altering maximal (>1000 μM ADP) respiration, suggesting 25 insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired 26 submaximal ADP-supported respiration ~50%, however, despite the induction of insulin 27 resistance, the ability of acute insulin to stimulate ADP sensitivity and increase 28 submaximal respiration persisted. While these data suggest that insulin mitigates HFD- 29 induced impairments in mitochondrial bioenergetics, the presence of a high intracellular 30 lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely 31 prevented the beneficial effects of insulin. Altogether, these data show that while insulin 32 rapidly stimulates mitochondrial bioenergetics through an improvement in ADP 33 sensitivity, this phenomenon is possibly lost following HFD due to the presence of 34 intracellular lipids.


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Insulin is a potent anabolic hormone that regulates several metabolic ATP-55 dependent processes (7). As a result, the absence of insulin action (i. e. insulin deficiency, 56 Akt2 ablation) causes reductions in mitochondrial content, oxidative capacity and ATP 57 concentration while also increasing ROS production (8-10). Moreover, insulin stimulates 58 mitochondrial NADH activity, improves mitochondrial coupling, and upregulates ATP 59 synthesis in cell culture, mice, and humans (11-15), providing compelling evidence that 60 insulin influences mitochondrial bioenergetics. Notably, the effects of insulin on 61 mitochondrial function are partially lost after high fat-or palmitate-induced insulin 62 resistance (11,16), which suggests a bidirectional interaction between reduced insulin 63 sensitivity and abnormalities in mitochondrial function. Therefore, while it has been 64 proposed that chronic insulin stimulates ATP synthesis and increases oxidative 3 phosphorylation, there is a lack of experimental evidence focusing on the rapid effects of 66 insulin on mitochondrial function, or the influence of insulin resistance in these processes. 67 Therefore, the present study aimed to investigate the acute effects of insulin on 68 mitochondrial bioenergetics, ADP sensitivity, and ROS emission in skeletal muscle from 69 control and high-fat diet (HFD)-induced insulin resistant mice. We tested the hypothesis 70 that insulin could rapidly improve mitochondrial ADP sensitivity, a dynamic 71 phenomenon that is acutely modulated in several physiological contexts (1,16,17 113 In a separate cohort of mice, we determined the direct ability of insulin to stimulate 114 mitochondrial ADP sensitivity in an incubated muscle preparation. Specifically, mice 115 were fed a control or an HFD for 8 weeks. Then, mice were anesthetized with an 116 intraperitoneal injection of sodium pentobarbital and the hindlimb was skinned, quickly   137 Following the addition of 5 mM pyruvate (cat. number P5280) and 2 mM malate (cat. curve-fit with an R 2 greater than 0.95, and the adjusted R 2 (from 0 to 1) and Sy.x (in pmol 150 · sec -1 · mg -1 dry wt) are reported in each figure legend to specify the goodness-of-fit. To 151 evaluate the effect of ADP recycling/ATPase activity on mitochondrial respiration, 152 mitochondrial respiration was also measured in the presence/absence of a hexokinase-153 ADP clamp (0.5 U/mL hexokinase; 3 mM 2-deoxyglucose) as previously described (19).

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The content of insulin signaling proteins were examined by Western blotting in red 156 gastrocnemius samples snap-frozen in liquid nitrogen as previously described (1).

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Samples were processed and loaded equally using standard SDS-PAGE methodology,   175 Data are expressed as mean ± SD for the kinetic experiments or individual observations 176 with mean ± SD highlighted. Unpaired two-tailed student's t-test was used to analyze data 177 between control and HFD-fed groups. To test the interaction between HFD and insulin, 178 we used two-way ANOVA followed by LSD post-hoc analysis. Since different cohorts 179 were used to test specific questions within the study, the specific sample size is provided 180 in each figure/experimental design. Statistical significance was considered when P<0.05. 183 We first aimed to determine if acute insulin administration would affect Thr308), CaMK-II phosphorylation was increased in the HFD-fed group ( Figure 2F).

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Combined, these data suggest that 8 weeks of HFD-feeding caused obesity, whole-body  In vitro effects of insulin on mitochondrial ADP sensitivity 233 Since insulin influences the metabolism of glucose, lipids, and amino acids in vivo, we 234 decided to test the effects of insulin on mitochondrial bioenergetics in a more controlled system. To determine this, we excised mouse hindlimbs from control-and HFD-fed mice 236 and incubated muscles in the presence and absence of insulin for 60 minutes to determine     293 In the present study, our data highlight that insulin acutely increases mitochondrial ADP   (25)). It therefore 326 remains possible that insulin increased cytosolic Ca 2+ following HFD to stimulate 327 respiration, but it is unlikely to occur directly from CaMK-II activation. An additional 328 hypothesis to consider is that insulin may trigger SR-mitochondrial physical interactions, 329 which would impact energy metabolism and mitochondrial function (27). 330 Although Akt has been implicated as a central regulator of insulin action, recently, 331 its role has been challenged since chronic skeletal muscle-specific Akt2 ablation did not 332 result in insulin resistance or reductions in glucose uptake (9). Importantly, Akt2 accounts 333 for ~90% of Akt within skeletal muscle, which suggest that either a lower phosphorylation fraction could be sufficient to drive insulin-stimulated mitochondrial 335 responses or Akt1 could be the mediator of this response. Moreover, based on the findings 336 of triple Akt knock-out, a non-canonical insulin pathway through atypical protein kinase 337 Cλ (aPKCλ) has been proposed (28). In liver, aPKCλ can be activated by IRS-PI3K-338 PDPK1 cascade, in an Akt-independent manner. Moreover, as discussed prior, insulin-339 stimulated SR Ca 2+ release has been proposed to be independent of Akt activation (24),  Figure 7C, left panel), the phosphorylation status of insulin targets is low ( Figure 2F, 366 3A), and baseline mitochondria activity is low (Figure 3B, 4D-G). In response to a meal  375 Excessive lipid availability is suggested as an underlying mechanism driving 376 insulin resistance. Currently, it is extensively debated whether intramyocellular lipid 377 accumulation is a result of mitochondrial dysfunction or rather if these lipids can impair 378 mitochondrial function (4, 5, 26, 27). In the present study, our data highlight the robust 379 negative effects of HFD on mitochondrial bioenergetics, as mitochondrial ADP 380 sensitivity was decreased in HFD-fed mice. These observed effects are strongly related 381 to mitochondrial bioenergetics as opposed to other endogenous ATP-consuming 382 processes, as the addition of blebbistatin prevents myosin ATPase activity, and we have 383 shown using a hexokinase-clamp that changes in ADP sensitivity are not influenced by ATPase-recycling (present study and (35)). Furthermore, the presence of P-CoA 385 completely mitigated the ability of insulin to stimulate mitochondrial bioenergetics, a 386 process that is directly related to reductions in mitochondrial ADP transport (21). This 387 would suggest an impaired ability for ADP to rapidly modify flux through the electron 388 transport, which has implications to oxidative phosphorylation and redox balance. In 389 further support of our findings that lipids impair mitochondrial bioenergetics, in rat and  Alternatively, lipids display a high propensity for mitochondrial ROS production when transported into the matrix (36) and therefore may also indirectly inhibit ANT, 410 contributing to the reduction in submaximal respiration following HFD in the absence of 411 P-CoA in the respiratory chamber. In support of this, ANT has three known cysteine sites 412 and evidence has accumulated to suggest that ANT is impaired in situations of redox 413 stress (e.g. high fat diet and aging) (1,37). Interestingly, insulin can acutely increase  Although the present data suggests a high lipid environment prevents insulin-mediated 432 improvements in mitochondrial function, the interplay between lipids, insulin, and 433 mitochondrial function warrants further investigation.

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The notion that impaired mitochondrial bioenergetics contributes to the induction 436 of insulin resistance has been extensively studied, however in the present study we 437 provide evidence that insulin rapidly regulates mitochondrial bioenergetics by increasing 438 ADP respiratory sensitivity. Specifically, we show that i) insulin rapidly increases 439 submaximal oxidative phosphorylation, and while ii) HFD impairs submaximal ADP-