Muscle and adipose tissue insulin resistance: malady without mechanism?

Under fasting conditions, hepatic glucose output and release of fatty acids from triacylglycerol (TAG) stores in adipose tissue (lipolysis) provide substrates for oxidation and ATP production. In the fed state, increases in circulating amino acids, fatty acids, and glucose stimulate insulin secretion. Insulin suppresses hepatic glucose output and adipose tissue lipolysis, lowering blood glucose and fatty acid levels. It also increases hepatic lipid synthesis for subsequent storage in adipose tissue and stimulates glucose uptake into fat and muscle. The majority of glucose from a meal is deposited in muscle and liver with as little as 5% taken up by adipose tissue (, , ). However, as we describe in more detail below, while adipose tissue does not quantitatively account for much of the acute disposal of glucose at the whole-body level, glucose uptake into adipose tissue may indirectly influence both carbohydrate and lipid metabolism in other tissues, such as liver and muscle.

Insulin elicits these metabolic changes by activating an intracellular signaling cascade largely comprising protein phosphorylation (). This begins with activation of the IR, a tyrosine kinase that phosphorylates IR substrates (IRSs), such as IRS1/IRS2, on multiple tyrosine residues (Fig. 1A). These recruit proteins containing SH2 domains, including phosphoinositide 3-kinase (PI3K) and Grb2. This in turn activates the two major protein kinase signaling pathways found in most eukaryotic cells, those mediated by the Ser/Thr kinases, Akt and MAPK/ERK. In particular, Akt has been intensely studied in the context of metabolism, with its activation being both necessary and sufficient for insulin-stimulated glucose transport (). Akt is recruited to the plasma membrane via binding of its PH domain to PI3K-produced phosphatidylinositol-3,4,5-phosphate (PIP3). It is activated by phosphorylation on Thr308 and Ser473 by PDK1 and mTORC2, respectively [reviewed in ()]. Consequently, these sites are routinely used as markers of Akt activation and subsequently as a measure of the cell's response to insulin. Beyond glucose transport, Akt has more than 100 substrates with a wide array of biological endpoints. These include regulating metabolism, protein synthesis (via mTORC1), transcription (e.g., via FOXO, SREBP1), and cellular proliferation. Here, we focus on the role of Akt in lipid and glucose metabolism.

Insulin rapidly increases glucose transport through the regulated trafficking of the glucose transporter, GLUT4, from intracellular stores to the cell surface in muscle and adipose cells (, ) (Fig. 1A). This is mediated by the phosphorylation of proteins that regulate GLUT4 trafficking, such as TBC1D4/AS160 (). GLUT4 translocation is thought to be the rate-limiting step for insulin-dependent glucose utilization in these tissues (). Once glucose enters muscle and adipose cells, it is rapidly phosphorylated, generating glucose 6-phosphate (G6P). The subsequent metabolism of G6P is coordinated by a series of allosteric and covalent regulatory steps. For example, activation of glycogen synthase () and ATP citrate lyase (, ) promotes glucose storage into glycogen and lipid, respectively. A recent analysis of the insulin-regulated phosphorylation network in adipocytes identified dozens of metabolic enzymes that undergo insulin-regulated phosphorylation (, ), and these likely play a key role in choreographing the ultimate metabolism of glucose in a manner that is more complex than originally anticipated. We recently presented evidence to show that the phosphorylation of these metabolic enzymes precedes the increased delivery of glucose into the cell, thus creating a demand-driven system that primes adipocytes to metabolize glucose in specific ways once glucose transport is fully activated ().

In addition to increasing glucose transport, insulin suppresses adipose tissue lipolysis (Fig. 1A). While this is one of the most important actions of insulin, our understanding of this process is relatively scant. The β-adrenergic receptor agonists activate lipolysis by increasing cAMP, leading to activation of protein kinase A (PKA) and phosphorylation of lipid droplet proteins to promote TAG hydrolysis (). Insulin is thought to inhibit this via Akt-dependent phosphorylation and activation of the phosphodiesterase, PDE3B, lowering cAMP levels and inhibiting PKA (, ). However, recent studies have questioned the role of PDE3B activity and cAMP hydrolysis in this process (). Insulin action in the brain is also reported to contribute to reduced adipose tissue lipolysis by dampening sympathetic innervation of adipose tissue (). Insulin also stimulates adipocyte lipid storage through two concerted processes: 1) lipogenesis via activation of lipogenic enzymes, such as acetyl-CoA carboxylase () and ATP-citrate lyase (, ); and 2) formation of the TAG-glyceride backbone from glucose diverted from glycolysis (). As described below, this latter step represents a key convergence point in metabolic regulation whereby fat cell glucose metabolism may have a profound influence on adipocyte lipid storage independently of de novo lipogenesis.

Decreased IR expression, tyrosine phosphorylation, and kinase activity have been observed in a variety of tissues from insulin-resistant animals (, ) and humans (, , , , , , , ). Similarly, alterations in IRS1 in insulin-resistant humans and animals have been reported, including altered phosphorylation (, , , , , ), expression (), degradation (), and protein-protein interactions (). Studies in humans undergoing hyperinsulinemic-euglycemic clamps have also reported decreases in Akt phosphorylation by up to 50% in insulin-resistant subjects relative to controls (, , , , ). However, there are a number of cases in insulin-resistant humans and animals where no change in phosphorylation of the IR, IRS1, or Akt has been detected (, , , , ) (Fig. 1B). Importantly, studies that have examined signaling downstream of Akt in insulin-resistant humans undergoing hyperinsulinemic-euglycemic clamps found no changes in TBC1D4/AS160 Thr642 phosphorylation (), FoxO1 Ser256 phosphorylation (), or glycogen synthase activity () (Fig. 1B). Therefore, although impaired signaling has been reported at proximal sites in insulin signaling in insulin resistance, there is ample evidence that insulin resistance occurs in the absence of such changes in signaling.

Why is impaired signaling at proximal components of the insulin signaling pathway, like IR/IRS/Akt, not transmitted to distal elements, like FOXO or TBC1D4/AS160? To rationalize this, it is important to consider how the insulin dose response curve varies between these components. Decades ago, studies described a “Spare Receptor” hypothesis, where it was shown that maximal insulin responses require engagement of a relatively small subset of the available total IR pool at the cell surface (, ). More recent studies have shown that considerable spareness exists in many proximal components of the insulin signaling pathway, including IR, IRS, PI3K, and Akt. For example, in muscle cells and adipocytes, the IR and IRS1 are expressed at much higher levels than required for a maximal downstream response to insulin (, , ). This is exemplified by the fact that heterozygous deletion and resultant lower expression of each of these components has no effect on insulin-regulated metabolism and, in the case of PI3K, it was necessary to delete all major isoforms of PI3K simultaneously before reduced downstream signaling events or effects on metabolism were evident (). Therefore, it is likely that only drastic changes in expression of proximal signaling elements could impair insulin signaling and drive insulin resistance.

In addition to spareness in protein expression, only a small proportion of Akt needs to be active in order for its substrates to be maximally phosphorylated. For instance, in L6 myotubes, insulin had a maximal effect on GLUT4 translocation at concentrations where only 5% of the total available Akt pool was phosphorylated () (Fig. 1A). In 3T3-L1 adipocytes, reducing insulin-simulated Akt phosphorylation by 85–90% using small molecule inhibitors only had modest effects on Akt substrate phosphorylation (). Together, this implies a system whereby an excess of each signaling component enables a small change in the proximal signal to be amplified to the distal components, providing a rapid response to insulin and buffering minor decreases in activity in proximal components of the pathway. This has several implications for insulin resistance: 1) a severe defect in proximal signaling (e.g., a genetic mutation or saturating doses of signaling pharmaceuticals that are currently in use for cancer) may be required to translate to distal signaling components, where a more modest impairment, as observed in common insulin resistance, would not; 2) the inconsistent relationship between Akt and substrate phosphorylation in human studies (Fig. 1B) may be due to high doses of insulin obscuring subtle changes in substrate phosphorylation; and 3) the dose response characteristics of insulin action should be considered when interrogating insulin signaling in the context of insulin resistance.

One of the most prominent hypotheses for how insulin signaling may be impaired and so contribute to insulin resistance is via serine/threonine phosphorylation of IR or IRS1. Here, feedback signaling from distal kinases of the insulin signaling pathway (e.g., mTORC1/S6K) or lipid- or stress-activated kinases (e.g., PKC isoforms, JNK, p38 MAPK) leads to phosphorylation of the IR to reduce its kinase activity, or of IRS1 to elicit 14-3-3 protein binding and degradation. In support of this, ablation of a proposed PKCε-mediated feedback phosphorylation site on the IR protected mice from high-fat diet-induced insulin resistance in liver (), implicating a role for feedback involving the IR in altering insulin responses in this tissue. However, recent studies have shown that liver-specific deletion of PKCε has no detectable effect on insulin resistance, questioning the relevance of this pathway (Schmitz-Peiffer, personal communication). The necessity for impaired signaling via IR/IRS1 in insulin resistance in adipose and muscle tissue was directly tested using mice ectopically expressing the PDGF receptor, which can facilitate GLUT4 translocation to the plasma membrane independently of the IR and IRS1. Although we cannot rule out the presence of feedback to ectopically expressed PDGF receptors in these experiments, a high-fat diet diminished the ability of both insulin and PDGF to activate glucose transport in skeletal muscle, suggesting that insulin resistance can occur independently of the IR or IRS1 (). Similarly, disarming of the major negative feedback phosphorylation site, Ser307, in IRS1 by mutating it to Ala failed to protect mice against developing insulin resistance (). As to whether other sites in IRS1 replace this negative feedback function remains to be investigated. Collectively, these data suggest that it is unlikely that common insulin resistance in different metabolic tissues is fully accounted for by feedback to IR or IRS1.

This term was originally used to describe the observation that under insulin-resistant conditions, MAPK signaling remains intact (), implying that insulin resistance was not due to global inhibition of insulin-dependent signaling. The term has subsequently been repurposed to describe hepatic insulin resistance, where insulin-regulated suppression of hepatic glucose output, but not triglyceride synthesis, is impaired (). In light of the emerging picture concerning non-cell autonomous effects of insulin in liver (, ), it is tempting to postulate that hepatic selective insulin resistance is due to selective impairments in the adipose tissue-lipolysis-hepatic glucose production axis, while cell-autonomous effects on hepatic lipogenesis remain intact ().

Recently, an alternate form of selective insulin resistance has been reported. Although insulin/Akt signaling regulates several cellular processes (e.g., glucose transport, protein synthesis, antilipolysis), insulin resistance is characterized by selective downregulation of insulin/Akt-dependent processes within the same cell (Fig. 1A). We refer to this as “cis” selective insulin resistance. This contrasts with “trans” selective insulin resistance that may occur between cells or tissues, such as the adipose-lipolysis-liver-glucose output pathway described above. The cis selective insulin resistance challenges the notion that insulin resistance arises from a generalized impairment in insulin signaling, which would be expected to affect all insulin/Akt-regulated processes to a similar extent. Hence, we will summarize recent examples of cis selective resistance within adipose and muscle tissue.

Alternatively, different stresses may conspire to disrupt insulin action by convergence upon a common stress or mechanism. Several laboratories, including our own, have reported an important role for increased production or lower scavenging of ROS in mitochondria in insulin resistance. This is based on three major lines of evidence. First, mitochondrial ROS are a characteristic feature of cells and tissues exposed to excess nutrients and a number of other models of insulin resistance (, , , , ), and oxidative stress is associated with the onset of insulin resistance in humans (, ). Second, interventions, either pharmacologic or genetic, that prevent increases in ROS specifically in mitochondria improve insulin sensitivity (, , , , , ). Third, acute induction of mitochondrial ROS causes insulin resistance in muscle and adipose tissue (, ). Further, the insulin-sensitizing drugs, thiazolidinediones, metformin, and berberine, may mediate part of their beneficial effects by modulating mitochondrial function (, ) and lowering ROS (, , ).

There is also evidence that many different intracellular stresses, including ceramides () and endoplasmic reticulum stress (), converge upon mitochondria to cause increased ROS production. Hence, mitochondrial ROS production is intimately linked to many forms of cellular stress and thus poised to act as a sentinel of general stress. Based on the role of the mitochondrion in balancing cellular energy supply with demand, which is unbalanced in obesity where supply outweighs demand, and in mediating redox signaling, it represents an ideal candidate to coordinate insulin resistance in response to nutrient oversupply as occurs in diet/obesity-induced insulin resistance (). Whether different stresses are linked linearly or cyclically is a point for debate and future research, but certainly if the latter, this could explain how disorders like insulin resistance could occur almost via an autocatalytic mechanism and why breaking the cycle at just one point (e.g., ROS, ceramides) may correct the entire cycle. There are currently no studies that have systematically assessed the temporal relationship between ROS, DAGs, ceramides, and ER stress, and such analyses have been invaluable in placing adipose tissue inflammation as a relatively late contributor to insulin resistance (, ). Understanding the interrelationship between stresses that cause insulin resistance is an important step in implementing rational therapies to overcome insulin resistance.

Increased availability of nutrients, such as fatty acids, which have a propensity to generate more ROS than other substrates, leads to increased mitochondrial ROS production (, , , ). Thus, at one level it seems logical that increased ROS will be a consequence of the increased fatty acid supply in obesity. However, mitochondria isolated from insulin-resistant adipose or muscle tissue generate higher rates of ROS production per unit of substrate (), suggesting that there may be changes within mitochondria that promote ROS production or lower ROS scavenging. Defects in oxidative phosphorylation can increase ROS production, yet a number of studies have reported no change in or improved mitochondrial oxidative capacity in insulin resistance (, , ) [reviewed in (, , )], suggesting that changes in the oxidative phosphorylation pathway are unlikely to be a primary driver of ROS under these conditions. We recently identified a deficiency in coenzyme Q, a cofactor that transfers electrons from complexes I and II to complex III of the electron transport chain, in insulin-resistant adipose and muscle tissue (). This loss of coenzyme Q, specifically in mitochondria, drove increased mitochondrial ROS production and insulin resistance (). The mechanism behind loss of coenzyme Q from mitochondria remains unclear, but these data highlight that there are chronic changes in mitochondria under insulin-resistant conditions, beyond substrate selection, that contribute to ROS production. Insulin resistance may also be accompanied by changes in substrate preference for oxidation, which remains a subject to future investigation.

In mitochondria, ROS, in the form of superoxide, are generated at several defined sites within the respiratory chain (), and the overall ROS burden is dependent on the rates of production and scavenging. The proximal ROS from the electron transport chain is superoxide, but this is rapidly dismutated by SOD2 to hydrogen peroxide, and both superoxide and hydrogen peroxide are implicated in causing insulin resistance (, ). It is not clear whether a specific form of ROS or a specific site of ROS production in mitochondria is responsible for conferring insulin resistance. Because multiple interventions that scavenge either superoxide or hydrogen peroxide are insulin sensitizing and multiple sites of ROS production have been implicated in causing insulin resistance (, , , ), it may be that the overall redox state of mitochondria may be more important than the exact species of ROS or site of ROS production, as hypothesized by Fisher-Wellman and Neufer ().

In addressing this question, it is crucial to emphasize the importance of the site of ROS production, namely mitochondria, in the progression to insulin resistance. Indeed, cytosolic ROS production plays a central role in propagating critical signaling pathways like the insulin signaling pathway and, as such, these pathways positively influence insulin action [reviewed in ()]. Moreover, the temporal nature of elevated ROS may also be a crucial determinant of the long-term outcome. It probably does not require long-term adaptive changes such as altered transcription () but rather either allosteric modifications possibly due to concomitant metabolic changes or posttranslational changes. In the case of the latter, ROS can react with a range of macromolecules, for instance, oxidizing exposed cysteine residues within proteins (, ). While the precise mechanism is unclear, ROS have been shown to activate a range of signaling molecules, particularly members of the MAPK family [reviewed in (, )], such as JNK () and p38 MAPK (). However, recent evidence suggests that increased JNK/p38 activity is associated with increased, rather than decreased, insulin sensitivity (); so it seems unlikely that these molecules are major purveyors of insulin resistance. Similarly, ROS have been shown to activate other kinases, such as LYN and SYK, in mitochondria, but their role in insulin resistance has not been established (). Hence, currently, the mechanism by which mitochondrial ROS trigger insulin resistance is not known.

In going forward, we propose that the following three questions might provide a useful framework to establish the link between mitochondrial ROS and insulin resistance: