Deconstructing the Role of PKC Epsilon in Glucose Homeostasis

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Review

Deconstructing the Role of PKC Epsilon in Glucose Homeostasis Carsten Schmitz-Peiffer1,2,* The failure of insulin to suppress glucose production by the liver is a key aspect of the insulin resistance seen in type 2 diabetes. Lipid-activated protein kinase C epsilon has long been identified as an important mediator of diet-induced glucose intolerance and hepatic insulin resistance and the current view emphasizes a mechanism involving phosphorylation of the insulin receptor by the kinase to inhibit downstream insulin action. However, the significance of this direct effect in the liver has now been challenged by tissue-specific deletion of PKCε, which demonstrated a more prominent role for the kinase in adipose tissue to promote glucose intolerance. New insights regarding the role of PKCε therefore contribute to the understanding of indirect effects on hepatic glucose metabolism.

Highlights Lipid-sensitive protein kinase C (PKC) isoforms are activated upon accumulation of the lipid metabolite diacylglycerol and are strong candidates for mediating insulin resistance. PKCε has been linked to liver insulin resistance and global ablation has demonstrated a key role for this kinase in glucose homeostasis. However, recent genetic and phospho-proteomic studies have generated conflicting data on the tissue-specific function of PKCε and its role in the inhibition of insulin signal transduction.

Protein Kinase C as a Mediator of Insulin Resistance Obesity and a chronic oversupply of fatty acids (FAs) are strongly associated with the development of insulin resistance (see Glossary), a key feature of type 2 diabetes (T2D). The reduced ability of insulin to promote glucose uptake into skeletal muscle and to suppress glucose production by the liver, together with a failure of insulin-secreting pancreatic β-cells to compensate for this insulin resistance, lead to dysregulated glucose homeostasis. This in turn causes complications, such as cardiovascular disease, diabetic nephropathy, and neuropathy, generating a high social and financial burden. In the face of the global obesity epidemic, identification of the mechanisms linking lipid excess to insulin resistance has become critical, to better enable the development of new therapeutic agents for the treatment of T2D. The protein kinase C (PKC) family of lipid-activated signaling enzymes (Box 1) take part in the regulation of a wide range of cellular processes, from cell growth and division to neurotransmission, metabolism, and apoptosis [1,2]. Certain isoforms, especially PKCε, have also been implicated in lipid-induced insulin resistance [3–5]. The conventional and novel subgroups of PKC isoforms are activated, at least in part, by the lipid mediator diacylglycerol (DAG). This binds to C1 domains within the N terminal regulatory region of PKC (Figure 1A), which promotes membrane association and conformational changes that induce activation. DAG molecules are released acutely at the plasma membrane (PM) in response to receptor-mediated activation of phospholipase C (PLC) (Figure 1B), but activation of PKC in this way is generally short lived due to the action of enzymes that degrade DAG to terminate the lipid signal [6]. However, DAG is also generated during FA esterification at the endoplasmic reticulum (ER), being an intermediate in the synthesis of triglyceride, which is stored in lipid droplets (LDs) (Figure 2) [7]. DAG is produced in this way as the sn-1,2 DAG stereoisoform, which can activate PKCs, whereas DAG that is generated by lipolysis at LDs is unable to do so because it is in the sn-2,3 DAG conformation [8]. In addition to the site of DAG accumulation, the subcellular location of PKCs is also determined by their interaction with binding proteins, which include filamentous actin and 14-3-3 proteins [9]. The best-characterized interaction for PKCε, however, is the association with the Receptor for Activated C-Kinase 2 (RACK2), which binds to its C2-like domain (Figure 1A). RACK2 is also known as the coatomer protein COPB2 and is found in a complex Trends in Endocrinology & Metabolism, Month 2020, Vol. xx, No. xx

The role of PKCε impacts on the broader debate concerning the relative importance of direct and indirect actions of insulin on the liver. The measurement of PKCε activation by subcellular translocation assay is open to postextraction artefacts. Determinations in live cells and intact tissue are needed to confirm the response of the kinase to chronic lipid excess and to determine its true cellular location.

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Garvan Institute of Medical Research, Darlinghurst Sydney, NSW 2010, Australia 2 St Vincent’s Clinical School, University of New South Wales, Sydney, NSW 2010, Australia

*Correspondence: [email protected] (C. Schmitz-Peiffer).

https://doi.org/10.1016/j.tem.2020.01.016 © 2020 Elsevier Ltd. All rights reserved.

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Box 1. The PKC Family of Lipid-Activated Enzymes PKC was established as a widely expressed calcium- and DAG-dependent serine/threonine kinase activity 40 years ago [1] and 10 years later the conventional (or classical) PKC isoforms, PKCα, PKCβ, and PKCγ were cloned. These share a similar domain organization, with DAG-binding C1 domains and calcium-binding C2 domains in their N terminal regulatory region and ATP-binding C3 domains and protein substrate-binding C4 domains in their C terminal catalytic regions [2]. The novel PKC isoforms, PKCδ, PKCε, PKCη, and PKCθ, and the atypical isoforms PKCζ and PKCι were cloned thereafter, differing in the organization of their regulatory regions. Novel PKCs are calcium-independent and exhibit a modified C2 domain that performs other functions such as protein and membrane binding. Atypical PKCs are also DAG-independent and are regulated to a large extent by protein interactions. All PKC isoforms contain a pseudosubstrate sequence in their regulatory domain that mediates autoinhibition of the inactive enzyme by interacting with the active site in the catalytic region, masking it from protein substrates. Upon activation by DAG binding, conformational changes uncover the active site. In addition to the hydrophobic nature of DAG, membrane localization of activated PKC isoforms is promoted by their C1 domains that bind phosphatidyl-serine and the C2 domains that bind nonspecifically to anionic phospholipids [10]. PKC isoforms also harbor distinct sequences that bind specific protein partners that serve to locate the activated kinases to particular intracellular locations. Because the catalytic site is highly conserved between isoforms and unable to determine significant substrate selectivity on its own, it is these interactions that promote the phosphorylation of specific proteins [66]. A widely used assay to determine PKC activation in cells and tissues makes use of the redistribution of the kinase, through subjecting homogenates to crude subcellular fractionation by centrifugation in the absence of detergents, generating soluble and particulate fractions normally denoted as the cytosolic and membrane components [67]. The relative amounts of the kinase present in these fractions, determined either by in vitro kinase assay or by immunoblotting with isoform-specific antibodies, is used to compare the extent of activation in different samples.

coating COPI vesicles. These vesicles carry out retrograde transport of proteins between the cis face of the Golgi complex and the ER. Activated PKCε has been localized to the Golgi in many studies, which is partly explained by interaction with RACK2/COPB2 but also by the enrichment of DAG at this site [10] (Figure 2). In addition, COPI vesicle proteins, including COPB2, are essential for the formation of LDs at the ER, regulating ER–LD contact and the transfer of essential LD proteins [11]. Pertinently, PKCε itself has also been localized to LDs [12,13] (Figure 2). Since an oversupply of FAs to insulin target tissues, due to nutrient excess and obesity, would be expected to increase DAG synthesis, it has long been hypothesized that this leads to chronic and aberrant activation of PKC isoforms that subsequently interfere with normal insulin action. This was supported by several correlative studies (reviewed in [3]) showing that the insulin resistance observed in skeletal muscle or liver of obese humans and rodents fed high-fat diets, or in cells treated with FAs, was associated with DAG accumulation and PKC activation, as assessed by the subcellular redistribution of specific isoforms (Box 1). The PKC isoforms most frequently implicated in such studies were PKCθ in skeletal muscle and PKCε in liver [3], although PKCδ also plays a role in hepatic insulin sensitivity [14,15]. While hepatic insulin resistance is often equated with an impairment in proximal insulin signaling (Box 2), physiologically it is defined as a reduced effectiveness of insulin to suppress glucose production. Specifically, in the case of human liver, several studies have associated increased DAG levels with insulin resistance [16–19]. There has, however, been less success in linking specific DAG species, in terms of acyl chain length, degree of saturation, and cellular location, with PKC activation or insulin resistance [5,20].

PKCε and Insulin Resistance in the Liver PKCε translocation and DAG accumulation have been reported in association with hepatic insulin resistance in animal studies using several dietary and genetic approaches. This led to the development of a widely accepted model for the induction of defective insulin action through direct disruption of proximal insulin signaling by the activated kinase [4]. However, alterations in hepatic DAG levels do not always correlate with the expected changes in PKCε translocation or insulin sensitivity [21–24]. Assessment of the role of DAG associated with LDs and the ER has also been equivocal, with increased DAG at this site being linked either to PKCε activation, 2

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Glossary Akt: a serine kinase, also known as protein kinase B, that is central to insulin signaling pathways that regulate glucose and lipid metabolism. It is transiently recruited to the plasma membrane in insulin-stimulated cells, where it is phosphorylated and activated. Coatomer protein: one of several proteins that form a complex around vesicles involved in protein transport, either from the endoplasmic reticulum to the Golgi complex (COPII vesicles) or retrograde transport from the Golgi complex to the endoplasmic reticulum (COPI vesicles). Cre recombinase: an enzyme that catalyzes a site-specific recombination event between two DNA recognition sites (LoxP sites), enabling the excision of intervening DNA, such as a critical exon of a gene of interest. The Cre transgenes used in mice are driven by specific promoters that enable tissuespecific gene deletion, such as the albumin promoter for ablation in the liver. Diacylglycerol (DAG): a neutral lipid composed of two fatty acyl chains linked to a glycerol backbone, which is generated both as an acutely acting second messenger that activates protein kinase C and other signaling molecules and as an intermediate in triglyceride, sphingolipid, and phospholipid synthesis Endoplasmic reticulum (ER): an organelle involved in the synthesis of secretory proteins, at which enzymes of fatty acid esterification and proteins involved in lipid droplet formation are also found. Euglycemic-hyperinsulinemic clamp: a procedure involving the simultaneous infusion of insulin and glucose to determine the amount of glucose necessary to compensate for the effects of insulin on glucose uptake by peripheral tissues and on hepatic glucose production, in order to maintain euglycemia. The glucose infusion rate is a reflection of insulin sensitivity, while the inclusion of glucose tracers enables the determination of glucose uptake into specific tissues and the glucose output of the liver. Glucose effectiveness: the ability of glucose to promote its own disposal in an insulin-independent manner, responsible for approximately 50% of glucose clearance after a meal. Glucose intolerance: a metabolic condition in which blood glucose levels

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are higher than normal, especially after a meal. This can be tested using a glucose challenge delivered orally, intravenously, or intraperitoneally upon which its clearance from blood is monitored over time. Glucose intolerance can be due to insulin resistance, β-cell dysfunction, or impaired glucose effectiveness. Golgi complex: an organelle composed of a series of flattened membrane-enclosed compartments that receives protein from the endoplasmic reticulum for processing and packaging into vesicles for secretion or use within the cell. Hepatic glucose production: the generation and release of glucose by the liver, which can occur both through the breakdown of stored glycogen (glycogenolysis) and through the synthesis of glucose from substrates such as glycerol (gluconeogenesis). Insulin resistance: the state of the cell, tissue, or whole body when it does not respond adequately to insulin, usually meant in terms of glucose or lipid metabolism. Lipid droplet (LD): a lipid-containing organelle that originates at the endoplasmic reticulum, storing and releasing lipid molecules in a regulated manner. Protein kinase C (PKC): a family of serine/threonine kinases that are mostly activated by diacylglycerol and perform wide ranging roles in cell regulation through the phosphorylation of their protein substrates.

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Figure 1. Domain Structure and Receptor-Mediated Activation of PKCε. The novel PKC isoform PKCε consists of an N terminal regulatory region and a C terminal catalytic region (A). The regulatory region contains C1 domains that bind DAG molecules released from membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-mediated activation of (Figure legend continued at the bottom of the next page.)

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Figure 2. Chronic Activation of Protein Kinase C Epsilon (PKCε) through Accumulation of Intracellular Diacylglycerol (DAG). In cells that are exposed to long-term lipid excess, such as hepatocytes in obese or fat-fed rodents, DAG accumulates due to increased synthesis from internalized fatty acids (FAs). This occurs primarily at the endoplasmic reticulum (ER), where DAG is an intermediate in the sequestration of FAs as triglycerides (TG) that are stored in lipid droplets (LD). This DAG is ostensibly able to activate PKC isoforms, including PKCε, in hepatocytes, promoting the accumulation of activated PKCε at this site, which may be reinforced by association of the kinase with Receptor for Activated C-Kinase 2 (RACK2). RACK2 is also known as the coatomer protein COPB2, which is required for normal LD formation. In addition, RACK2/COPB2 is a component of COPI vesicles that take part in retrograde transport of cargo proteins from the Golgi to the ER. Together with the enrichment of DAG, this promotes localization of PKCε to the Golgi. Abbreviation: FFA, free fatty acid.

redistribution to a total membrane fraction, and insulin resistance [17] or to PKCε sequestration in LDs and maintenance of insulin sensitivity [13]. Such discrepancies have been ascribed to methodological issues, species differences, and to the existence of distinct intracellular pools of DAG, with differing potential to elicit kinase activation or subsequent interference with insulin action [5].

phospholipase C, causing a conformational change in the kinase to activate it (B). Although not calcium-dependent, the regulatory region of PKCε also contains a C2-like domain that takes part in protein–protein interactions, the bestcharacterized being the association with the scaffolding protein RACK2. The pseudosubstrate site located between these domains binds and masks the catalytic site in the C4 domain of the inactive kinase and this autoinhibition is relieved by DAG binding (B). This allows phosphorylation of protein substrates using ATP bound to the C3 domain. Full kinase activity depends on the phosphorylation of three C terminal sites (A), mediated by heterologous kinases and by autophosphorylation, although the precise regulation of these sites is unclear and appears to depend on cell type and context. Abbreviations: DAG, diacylglycerol; PKC, protein kinase C; PLC, phospholipase C; PM, plasma membrane; RACK2, Receptor for Activated C-Kinase 2.

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In addition, PKC activation is frequently measured indirectly by cell or tissue fractionation after tissue homogenization. This assay has not advanced technically for over 30 years and is open to artefacts that will be addressed below. Nevertheless, a causal role for PKCε in liver insulin resistance has been confirmed by independent reports involving either global deletion in mice [25,26] or knockdown using antisense oligonucleotides (ASOs) in rats [27]. In these studies, PKCε ablation protected against diet-induced glucose intolerance or liver insulin resistance, respectively, in the shorter term. An additional β-cell phenotype was revealed in PKCε knockout (KO) mice fed a high-fat diet for longer periods, protecting against the gradual impairment of glucose-stimulated insulin secretion in the pancreas [25,26,28]. Recent work discussed below extends the concept that PKCε regulates glucose homeostasis at extrahepatic sites by identifying a novel role in adipose tissue. In addition, new studies have generated contrasting hypotheses regarding the effects of the kinase on insulin receptor-mediated signaling and glucose metabolism in the liver. These impact on the wider debate concerning the relative importance of the direct regulation of liver glucose and lipid metabolism and indirect effects via other tissues, as well as the role of proximal insulin signaling defects in the establishment of liver insulin resistance (Box 2).

PKCε-Mediated Interference with Insulin Receptor Signaling Several protein kinases reduce insulin signal transduction through Ser/Thr phosphorylation of IRS-1, which impairs subsequent insulin receptor-mediated Tyr phosphorylation [29]. Thus PKCε translocation in the liver was initially associated with diminished tyrosine phosphorylation of IRS-1 in fat-fed rats [30]. More recently, however, attention has been focused on direct phosphorylation of the insulin receptor by the kinase. Analysis by mass spectrometry of phosphopeptides, generated by in vitro incubation of recombinant human insulin receptor and PKCε, identified Thr1160 as a target site for the kinase and phosphorylation of this site was also detected in receptors isolated from cell extracts [31]. Residing in the tyrosine kinase activation loop, phosphorylation of this site impaired in vitro tyrosine kinase activity of the receptor, as did a phosphomimicking mutation (Thr1160Glu). Furthermore, fat-fed transgenic mice expressing a mutant insulin receptor, nonphosphorylatable at the equivalent site in the murine receptor Box 2. Direct and Indirect Regulation of Liver Metabolism Insulin promotes the net synthesis of glycogen and diminishes the rate of gluconeogenesis, to reduce overall hepatic glucose production [68]. In addition, insulin stimulates de novo lipogenesis (DNL) and the esterification of FAs, so that lipid accumulation is enhanced [68]. These effects are differentially affected in the insulin resistant state, so that the suppression of glucose production is defective, whereas the accumulation of lipid is further increased, termed selective hepatic insulin resistance [69]. This phenomenon may, however, be explained by the fact that liver metabolism can be regulated both directly and also indirectly, involving other tissues. Direct effects of insulin are mediated by the binding of insulin to its receptor at the plasma membrane (PM) of hepatocytes (Figure I). This activates the receptor tyrosine kinase to phosphorylate insulin receptor substrate proteins, especially IRS1, which recruit phosphatidylinositide 3-kinase (PI3K), leading to the acute generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the PM. The kinase Akt is transiently recruited to the PM by PIP3, where it is activated in part through phosphorylation by phosphoinositide-dependent kinase 1 (PDK1) [70]. Akt activation is essential to stimulate DNL, to promote net glycogen synthesis and to suppress gluconeogenic enzyme expression [68]. It is possible that an insulin signaling defect in the liver lies downstream of the branch point separating the control of lipid and glucose metabolism, so that only glucose production is rendered insulin resistant [43]. However, evidence has accumulated to suggest that the acute suppression of gluconeogenesis is significantly affected by insulin action in adipose tissue [71]. This involves the acute inhibition of lipolysis by the hormone and hence the release of the gluconeogenic substrate glycerol as well as of FAs, which also promote gluconeogenesis through allosteric effects [72] and through increased gluconeogenic enzyme expression [73,74]. Defective insulin action in adipose tissue, and a resultant failure to restrict the release of glycerol and FAs, will therefore promote gluconeogenesis in the liver [43]. In this case, DNL in the liver could proceed in the absence of any hepatic insulin signaling defect, accounting for the apparent selective insulin resistance. Alternatively, defective insulin receptor activation in the liver, mediated by lipid-induced activation of PKCε, leads to net glycogenolysis that contributes significantly to the failure of insulin to suppress hepatic glucose production [5]. In this case, although all hepatic insulin responses are defective, increased hepatic lipid accumulation may arise due to increased FA supply and carbohydrate-driven DNL [5]. The importance of a PKCε-mediated insulin signaling defect in liver, and the relative contributions of the direct and indirect effects of insulin on liver glucose and lipid metabolism, are currently under debate [5,43,68,75,76].

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Figure I. Proximal Signaling, Elicited by Binding of Insulin to Surface Receptors on Hepatocytes, Leading to Direct Effects of the Hormone in Liver. Abbreviations: PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositide 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5trisphosphate; PM, plasma membrane.

(Thr1150Ala), were protected against hepatic insulin resistance as assessed in euglycemichyperinsulinemic clamps [31]. While consistent with the in vitro data, the cell-based and in vivo studies do not confirm PKCε as the kinase responsible for physiological Thr1160/1150 phosphorylation. This requires demonstration of changes in phosphorylation state under conditions when the kinase is activated or depleted, because many protein kinases can phosphorylate proteins that are not their physiological substrates in vitro [32]. PKCε substrate specificity is determined in large part by its localization, controlled in turn by its binding partners (Box 1). Crucially, whether an expected increase in Thr1150 phosphorylation occurred in fat-fed mice was not reported. Subsequently, independent phosphoproteomic studies using recombinant PKCε to phosphorylate insulin receptors immunoprecipitated from mouse liver did not identify Thr1150 as a phosphorylation site of the kinase, although in vitro activity towards other sites was detected [33]. Importantly, insulin receptors isolated from livers of fat-fed global PKCε KO mice did not exhibit differential phosphorylation when compared with receptors from control mice and Thr1150 phosphorylation was not observed [33]. Similarly, Thr1160/1150 phosphorylation was not detected in a recent phosphoproteomic investigation of liver from fat-fed rats [34] or in an independent study of lipid-treated hepatocytes [35]. However, these discrepancies may be explained by the low abundance of the Thr1160/1150-containing peptide and technical difficulties arising from the multiple phospho-sites within it [36]. 6

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The hypothesis that PKCε promotes significant defects in the suppression of hepatic glucose production through phosphorylation of the insulin receptor kinase [31], or other proximal components of the insulin signaling cascade [34], is consistent with a major contribution of direct insulin action in the liver to regulate glucose metabolism (Figure 3, Key Figure). It has been proposed that this direct action primarily involves the inhibition of glycogenolysis and stimulation of net glycogen synthesis via the activation of Akt (Box 2) and that this is negatively impacted by lipid-mediated activation of PKCε and inhibition of insulin signal transduction [5]. However, the significance of this effect of PKCε is questioned by the observations that hepatic insulin resistance and glucose intolerance induced by dietary lipid excess arise initially in the absence of proximal defects in hepatic insulin signaling [26,37] and that global PKCε deletion is already protective of glucose tolerance at this point [26]. In addition, PKCε deletion does not alter the dosedependent response of insulin receptor tyrosine phosphorylation or downstream Akt and ERK phosphorylation to insulin in primary hepatocytes [25]. Furthermore, an impaired insulin response that is generated by PKCε-mediated phosphorylation of the insulin receptor [31], or other upstream elements [34], is difficult to reconcile with the excess capacity observed in many components of the insulin signaling cascade using a variety of systems [38]. Indeed, robust Akt activation is still observed with a 40% reduction in insulin receptor protein levels [39], which would reduce insulin receptor kinase activity to a similar extent to that seen upon 1:1 incubation of recombinant receptor with PKCε in vitro [31]. Similarly, Akt can be fully activated in the liver of mice mostly deficient in PI3K, despite insulin-stimulated PIP3 levels being reduced by 50% [40]. A high level of spare capacity of Akt itself has been demonstrated in liver by the observation that an 85–90% reduction in Akt still permits normal downstream insulin signaling in response to feeding and normal glucose tolerance [41]. Taken together, these observations suggest that PKCε-mediated events that have physiologically relevant effects on glucose homeostasis are unlikely to be exerted purely at the level of proximal insulin signaling in the liver. Similar findings, arguing against the hypothesis that insulin resistance is generated by insulin signaling defects at or prior to Akt activation, have also been reported for adipose tissue and skeletal muscle [38,42], indicating that insulin action in general is little affected by diminished signaling. In the case of the liver, this is consistent with the view that hepatic insulin resistance in obesity is predominantly elicited further downstream or at other sites entirely [43], while reduced signaling may in fact be a consequence of insulin resistance [38].

Tissue-Specific PKCε Deletion Highlights Extrahepatic Roles Although the conventional whole body PKCε KO mouse provided evidence that PKCε played a causal role in the impaired glucose tolerance observed in fat-fed mice [25], because the kinase was deleted in every cell type, a direct effect in liver was not definitively established. Similarly, knockdown of PKCε with ASOs led to the abolition of PKCε expression not only in liver, but also in adipose tissue and possibly in several other additional tissues [27]. In order to determine the contribution that PKCε action in hepatocytes makes to a diet-induced impairment of glucose homeostasis, tissue-specific deletion of the kinase has recently been investigated. Mice harboring a PKCε gene in which exon 1 was flanked by LoxP sites were crossed with transgenic mice expressing Cre recombinase, under the control of tissue-specific promoters or a ubiquitously expressed CMV promoter [33]. The validity of this approach was verified by the demonstration that CMV-Cre-mediated global PKCε deletion phenocopied conventional whole-body PKCε KO mice. This applied to both shorter and longer term high-fat diet feeding, when benefits related respectively to insulin sensitivity and insulin secretion [26] were reconfirmed in mice subjected to glucose tolerance tests [33]. Unexpectedly, however, given the previous emphasis on direct PKCε action in the liver, there was no beneficial effect on glucose tolerance in fat-fed mice when PKCε was deleted specifically in the liver using an albumin promoter-driven Cre transgene, Trends in Endocrinology & Metabolism, Month 2020, Vol. xx, No. xx

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Key Figure

Tissue-Specific Roles of Protein Kinase C Epsilon (PKCε) Promoting Insulin Resistance and Glucose Intolerance

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Figure 3. (A) Studies showing increased diacylglycerol (DAG) levels and PKCε translocation in insulin resistant liver, as well as proteomic studies of PKCε-mediated insulin receptor phosphorylation, support a direct action of the kinase in this tissue. In this model, the accumulation of DAG in hepatocytes leads to the chronic activation of PKCε and inhibition of insulin signaling through phosphorylation of the insulin receptor. This impairs the acute response to insulin by reducing the ability of the hormone to stimulate net glycogen synthesis via Akt activation. The indirect effect of insulin to curb gluconeogenesis (GNG) acutely, mediated by a reduction in the supply of glycerol and FAs from adipose tissue, may also be impaired in the insulin resistant state, but this is not affected by PKCε action in the liver. In contrast, longer term effects of insulin on GNG through the suppression of gluconeogenic enzyme expression are affected by the kinase. (B) Observations of tissue(Figure legend continued at the bottom of the next page.)

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even though a clear protection was observed under the same conditions using the CMV-Cre global PKCε KO mice [33]. Furthermore, euglycemic-hyperinsulinemic clamps did not demonstrate any increase in insulin sensitivity in fat-fed mice when PKCε was ablated in the liver [33]. While the specific deletion of PKCε in the liver in this way was congenital and, unlike the use of ASOs, open to the induction of compensatory mechanisms during development, this is unlikely to explain the lack of phenotype because such compensation was not observed in the CMVCre global PKCε KO mice nor in other tissue-specific mice discussed below. PKCε has been proposed to inhibit the insulin-mediated suppression of glycogenolysis [5] (Box 2) and it has been suggested that the effects of the kinase may not be observed under conditions of fastinginduced glycogen depletion [36]. In these studies, however, both glucose tolerance tests and clamps were undertaken after very limited fasting [33], when glycogen levels in mice are not significantly diminished [44], excluding this possibility. These findings therefore suggest that the relative contribution of any direct action of PKCε in the liver is minimal compared with that observed upon whole-body deletion, when clear phenotypes can be demonstrated [33]. The absence of an expected phenotype in liver-specific PKCε KO mice indicated that the major effects of the kinase on glucose metabolism are in fact exerted elsewhere. This is consistent with the importance of extrahepatic effects on glucose production by the liver, mediated to a large extent via adipose tissue (Box 2). To investigate a role for PKCε action in this compartment, a Cre recombinase transgene driven by the adiponectin promoter, which is highly expressed in adipocytes [45], was used. Adipose-specific PKCε KO mice were subjected to high-fat feeding over 16 weeks and glucose tolerance tests revealed a significant contribution of the kinase in this tissue to promote whole-body glucose intolerance. This was independent of any effects on insulin release under conditions of both short- and long-term diet duration [33]. Adipose tissue itself makes a quantitatively limited contribution to whole-body glucose disposal, and liver rather than skeletal muscle accounts for most of the defect in glucose homeostasis at the early stages of dietary lipid oversupply [37,46]. The improved glucose tolerance therefore most likely involved an indirect effect on hepatic glucose production. In agreement, transcriptomics revealed that the expression of a large cohort of genes in the liver, including genes associated with lipid metabolism, were significantly affected by PKCε deletion in adipose tissue, whereas only limited changes were observed in adipose tissue itself [33] (Figure 3). Despite the improved glucose tolerance, no protection against insulin resistance, as determined under euglycemic-hyperinsulinemic clamp conditions, was observed upon adipose-specific deletion of PKCε [33]. Fortuitously, the sensitivity of the clamp measurements was verified by the observation of a strong phenotype, as expected in littermate mice in which PKCε was deleted globally by random germline expression of the Cre transgene [33]. The discrepancy between findings in adipose-specific PKCε KO mice made during glucose tolerance tests, when glucose levels rise and fall similarly to the absorptive state after a meal, and during the clamps where low glucose levels are maintained, is most likely explained by changes in ‘glucose effectiveness’ secondary to PKCε deletion in adipose tissue. Glucose enhances its own disposal through several insulinindependent mechanisms, which account for approximately 50% of glucose clearance during

specific deletion of PKCε in liver and adipose tissue support a more significant role of the kinase in fat cells to promote glucose intolerance upon diet-induced obesity. In this model, PKCε promotes the phosphorylation of nuclear proteins and proteins involved in endosome function and cell adhesion, leading to a remodeling of adipose tissue and accumulation of larger adipocytes. This in turn leads to altered gene expression in the liver, most likely mediated by a change in the profile of secreted adipokines, promoting glucose production. PKCε may also increase the supply of fatty acid (FA) and glycerol from adipose tissue to the liver upon nutrient oversupply to promote GNG, as it does in the fasting state to enable ketone synthesis, but this remains to be demonstrated.

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a glucose tolerance test or after a meal [47,48] but are less evident under the conditions of a euglycemic clamp. Several pathways may be involved, including the stimulation of glucokinase and glycogen synthase activity in the liver to promote glycogen synthesis and the suppression of lipolysis and FA release in adipose tissue to reduce gluconeogenesis [49] (Box 2). Pertinently, PKCε deletion has been shown to inhibit the supply of FAs to the liver under fasting conditions [50] and a reduction of FA release from adipose tissue in the fed state would be consistent with the improved glucose tolerance observed in fat-fed adipose-specific PKCε KO mice. While alterations in adipose tissue lipolysis were not observed in these animals, as judged by plasma FA levels or ex vivo incubations [33], more sensitive in vivo measurements of FA flux using lipid tracers [5] may reveal PKCε-dependent alterations (Figure 3). Such a mechanism would be in keeping with the known functions of PKC isoforms as regulators of lipid metabolism (as well as effectors of lipid second messengers) [51] and with a RACK2-mediated localization of PKCε at LDs (Figure 2). In agreement with the similar insulin sensitivity observed in adipose-specific PKCε KO and control mice under euglycemic-hyperinsulinemic clamp conditions, no alterations in the response of glucose or lipid metabolism to insulin were seen in PKCε-deficient adipose tissue explants [33]. This is also consistent with the comparable dose-dependency of proximal insulin signaling in wild type and PKCε-deficient primary hepatocytes previously reported [25]. In addition, phosphoproteomic analysis of primary adipocytes revealed no alterations in insulin-stimulated phosphorylation of the insulin receptor, or a wide range of well-characterized components of proximal insulin signaling [33]. However, alterations in the phosphorylation of small monomeric G-proteins, proteins involved in endosomal function and cell adhesion, were observed and such changes may underlie a remodeling of adipose tissue and beneficial modifications in adipokine release that affect hepatic glucose production (Figure 3). In agreement, the adipocyte cell size profile was altered in favor of smaller cells by deletion of PKCε [33]. These differences may be mediated in part by PKCε action at the Golgi (Figure 2) and are again consistent both with an indirect role for PKCε on hepatic glucose metabolism (Figure 3) and with an effect of adipose tissue on the liver through the release of adipokines or metabolites that remain to be identified. The beneficial effects on glucose tolerance observed upon PKCε deletion in adipose tissue further validate the negative findings made using liver-specific PKCε KO mice [33]. Similarly, the striking improvement in liver insulin sensitivity measured in clamp studies of global PKCε KO mice indicate that indirect effects of PKCε deletion on hepatic glucose production are readily evident [33] and any significant contribution by direct PKCε action in the liver would have been detected in liverspecific PKCε KO mice. However, the ability of global, but not liver- or adipose-specific, deletion of PKCε to enhance insulin sensitivity measured during euglycemic-hyperinsulinemic clamps suggests that further sites of action of the kinase, which impact on whole body glucose homeostasis, remain to be identified.

Measuring PKCε Activation PKC translocation from soluble to particulate fractions of cells and tissues has long been used as a convenient surrogate measure for enzyme activation (Box 1). Activation of PKC is mediated by an accumulation of intracellular DAG molecules that have access to the kinase and recruit it to membranes. It should be noted, however, that this procedure may be open to postextraction artefacts, because pools of DAG that are not in contact with PKC molecules in intact cells could promote membrane association during homogenization. Changes in pools of DAG may result from chronic FA oversupply or from alterations in glycerolipid, sphingolipid, or phospholipid metabolism, because all of these involve DAG as an intermediate [7,52]. These pools may then contribute to a membrane partitioning of PKCε whether associated with the kinase in intact 10

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cells or not. A more recent measure that has been used to investigate PKC activation, which does not rely on subcellular fractionation, relates to the level of phosphorylation of the kinase itself. Thus the extent of Ser729 phosphorylation detected by immunoblotting with phospho-specific antibodies has also been used to infer PKCε activation in models of insulin resistance [53,54]. In conventional PKC isoforms, coordinated phosphorylation events serve to prime and stabilize the mature enzymes, prior to activation [2]. In the case of novel PKCs, phosphorylation of the kinases can accompany activation itself, but the role of this is less well understood [2,55]. The PKCε C terminal Ser729 site, known as the hydrophobic motif (Figure 1A), is targeted after PDK1-dependent phosphorylation of the kinase activation loop (Thr536). However, the levels of Ser729 phosphorylation can reflect autophosphorylation or the activity of heterologous kinases, integrating signaling from several inputs rather merely the activation of PKCε itself by DAG [56–58]. Phosphorylation at Ser729 has been linked to Golgi localization, whereas the DAG analog 12-O-tetradecanoylphorbol-13-acetate, a pharmacological activator of conventional and novel PKC isoforms, can in fact promote Ser729 dephosphorylation and nuclear accumulation of PKCε in fibroblasts [59]. Overall, therefore, Ser729 phosphorylation of PKCε is not an easily interpreted marker for activation and does not necessarily correspond to PM localization of the active kinase, which would be required for interaction with proximal insulin signaling components such as the insulin receptor. To circumvent these difficulties in determining the true cellular distribution of PKCε and the extent of kinase activation, the enzyme would ideally be investigated in live cells and intact tissues exposed to lipid excess. This has been pursued in the case of PKCδ using tagged constructs that undergo conformationally dependent changes in fluorescence [60,61] and, conceivably, this could be extended to the determination of PKCε activation. Such reporters would not only indicate PKCε activation, but also at which intracellular sites this occurs [60], so that appropriate localization of the active kinase for the phosphorylation of putative targets, such as the insulin receptor at the PM, can be investigated. This is important in the case of PKCε since the active enzyme is in fact most commonly reported to be localized at the Golgi, associated with RACK2 (Figure 2), although chronic FA oversupply may induce an alternative distribution. Finally, biosensors of this kind also lend themselves to intravital imaging [62], so that expression of PKCε reporters could be used to determine PKCε activation in vivo in specific tissues, using animal models of obesity and insulin resistance.

Outstanding Questions What are the direct protein substrates for PKCε in adipose tissue that mediate its effects on whole-body glucose tolerance? What are the intracellular sites of action of PKCε in adipocytes and what part is played by binding partners such as RACK2? How does PKCε action in adipose tissue affect crosstalk with the liver to modulate hepatic glucose and lipid metabolism? In what other tissues does PKCε respond to lipid oversupply to have significant effects on glucose tolerance? Can PKCε activation by chronic lipid oversupply be demonstrated in live cells and tissues, as suggested from subcellular fractionation assays?

Concluding Remarks There is broad agreement that PKCε plays a significant role in the generation of lipid-induced insulin resistance and glucose intolerance. Until recently, the major focus has been on a direct effect of the kinase in the liver and a mechanism involving inhibitory phosphorylation of the insulin receptor and impaired downstream signaling. However, PKCε is ubiquitously expressed and earlier work had already elucidated a role for this isoform in the β-cell dysfunction that develops after insulin resistance in fat-fed rodents [26,28]. Now, a liver-autonomous role for PKCε has been challenged using tissue-specific deletion of PKCε. This has further revealed an effect of the kinase in adipose tissue to promote glucose intolerance, although the precise mechanism involved requires further clarification (see Outstanding Questions). Because the active kinase is frequently found targeted to the Golgi complex and potentially to LDs, a role in the regulation of endocrine function and lipid metabolism itself is likely. Further action on glucose metabolism mediated in additional tissues is also implicated by the discrepancies between the effects caused by wholebody PKCε deletion and by liver- and adipose-specific deletion. One potential site is the brain, which modulates both adipose tissue lipolysis and hepatic glucose production [63]. Lipid levels in the hypothalamus are also sensitive to diet [64] and promote novel PKC activation [65]. Finally, the PKC translocation assay, which may not accurately reflect PKCε activation in intact cells, Trends in Endocrinology & Metabolism, Month 2020, Vol. xx, No. xx

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requires validation by the determination of PKCε activation in live cells and also tissues in vivo. This may be achieved using PKCε fluorescent biosensors to examine the effects of FA oversupply on the kinase. In summary, although consideration of the role of PKCε in insulin resistance and glucose homeostasis is frequently confined to kinase action in the liver, new data has broadened this paradigm and opened new avenues for investigation.

Acknowledgments Studies in the author’s laboratory have been supported by grants from the National Health and Medical Research Council of Australia (APP535917, APP1081869) and Diabetes Australia Research Program (Y15G-SCHC, Y18G-SCHC, Y19GSCHC). The author is grateful to Prof. Trevor J. Biden for critical reading of the manuscript.

References 1.

2. 3.

4.

5. 6.

7.

8.

9. 10.

11. 12. 13.

14.

15.

16.

17.

18.

19.

20.

12

Battaini, F. and Mochly-Rosen, D. (2007) Happy birthday protein kinase C: past, present and future of a superfamily. Pharmacol. Res. 5, 461–466 Newton, A.C. (2018) Protein kinase C: perfectly balanced. Crit. Rev. Biochem. Mol. Biol. 53, 208–230 Schmitz-Peiffer, C. and Biden, T.J. (2008) Protein kinase C function in muscle, liver, and beta-cells and its therapeutic implications for type 2 diabetes. Diabetes 57, 1774–1783 Jornayvaz, F.R. and Shulman, G.I. (2012) Diacylglycerol activation of protein kinase Cε and hepatic insulin resistance. Cell Metab. 15, 574–584 Petersen, M.C. and Shulman, G.I. (2018) Mechanisms of insulin action and insulin resistance. Physiol. Rev. 98, 2133–2223 Massart, J. and Zierath, J.R. (2019) Role of diacylglycerol kinases in glucose and energy homeostasis. Trends Endocrinol. Metab. 30, 603–617 Coleman, R.A. and Mashek, D.G. (2011) Mammalian triacylglycerol metabolism: synthesis, lipolysis, and signaling. Chem. Rev. 111, 6359–6386 Eichmann, T.O. and Lass, A. (2015) DAG tales: the multiple faces of diacylglycerol–stereochemistry, metabolism, and signaling. Cell. Mol. Life Sci. 72, 3931–3952 Newton, P.M. and Messing, R.O. (2010) The substrates and binding partners of protein kinase Cε. Biochem. J. 427, 189–196 Gallegos, L.L. and Newton, A.C. (2008) Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm. IUBMB Life 60, 782–789 Kory, N. et al. (2016) Targeting fat: mechanisms of protein localization to lipid droplets. Trends Cell Biol. 26, 535–546 Suzuki, M. et al. (2013) Protein kinase Cη is targeted to lipid droplets. Histochem. Cell Biol. 139, 505–511 Cantley, J.L. et al. (2013) CGI-58 knockdown sequesters diacylglycerols in lipid droplets/ER-preventing diacylglycerol-mediated hepatic insulin resistance. Proc. Natl. Acad. Sci. U. S. A. 110, 1869–1874 Frangioudakis, G. et al. (2009) Diverse roles for protein kinase C delta and protein kinase C epsilon in the generation of high-fatdiet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C delta. Diabetologia 52, 2616–2620 Bezy, O. et al. (2011) PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans. J. Clin. Invest. 121, 2504–2517 Considine, R.V. et al. (1995) Protein kinase C is increased in the liver of humans and rats with noninsulin-dependent diabetes mellitus: an alteration not due to hyperglycemia. J. Clin. Invest. 95, 2938–2944 Kumashiro, N. et al. (2011) Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease. Proc. Natl. Acad. Sci. U. S. A. 108, 16381–16385 Magkos, F. et al. (2012) Intrahepatic diacylglycerol content is associated with hepatic insulin resistance in obese subjects. Gastroenterology 142, 1444–1446 Ter Horst, K.W. et al. (2017) Hepatic diacylglycerol-associated protein kinase Cε translocation links hepatic steatosis to hepatic insulin resistance in humans. Cell Rep. 19, 1997–2004 Jayasinghe, S.U. et al. (2019) Reassessing the role of diacylglycerols in insulin resistance. Trends Endocrinol. Metab. 30, 618–635

21. Monetti, M. et al. (2007) Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver. Cell Metab. 6, 69–78 22. Jornayvaz, F.R. et al. (2011) Hepatic insulin resistance in mice with hepatic overexpression of diacylglycerol acyltransferase 2. Proc. Natl. Acad. Sci. U. S. A. 108, 5748–5752 23. Minehira, K. et al. (2008) Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice. J. Lipid Res. 49, 2038–2044 24. Sun, Z. et al. (2012) Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration. Nat. Med. 18, 934–942 25. Schmitz-Peiffer, C. et al. (2007) Inhibition of PKCε improves glucose-stimulated insulin secretion and reduces insulin clearance. Cell Metab. 6, 320–328 26. Raddatz, K. et al. (2011) Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice. Diabetologia 54, 1447–1456 27. Samuel, V.T. et al. (2007) Inhibition of protein kinase Cε prevents hepatic insulin resistance in nonalcoholic fatty liver disease. J. Clin. Invest. 117, 739–745 28. Cantley, J. et al. (2009) Deletion of PKCε selectively enhances the amplifying pathways of glucose-stimulated insulin secretion via increased lipolysis in mouse beta-cells. Diabetes 58, 1826–1834 29. Zick, Y. (2005) Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance. Sci. STKE 2005, pe4 30. Samuel, V.T. et al. (2004) Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J. Biol. Chem. 279, 32345–32353 31. Petersen, M.C. et al. (2016) Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance. J. Clin. Invest. 126, 4361–4371 32. Cohen, P. and Knebel, A. (2006) KESTREL: a powerful method for identifying the physiological substrates of protein kinases. Biochem. J. 393, 1–6 33. Brandon, A.E. et al. (2019) Protein kinase C epsilon deletion in adipose tissue, but not in liver, improves glucose tolerance. Cell Metab. 29, 183–191 34. Gassaway, B.M. et al. (2018) PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling. Proc. Natl. Acad. Sci. U. S. A. 115, E8996–E9005 35. Li, Z. et al. (2018) Global analyses of selective insulin resistance in hepatocytes caused by palmitate lipotoxicity. Mol. Cell. Proteomics 17, 836–849 36. Samuel, V.T. et al. (2019) Considering the links between NAFLD and insulin resistance: revisiting the role of protein kinase c epsilon. Hepatology. Published online July 20, 2019. https://doi. org/10.1002/hep.30829 37. Turner, N. et al. (2013) Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding. Diabetologia 56, 1638–1648 38. Fazakerley, D.J. et al. (2018) Muscle and adipose tissue insulin resistance: malady without mechanism? J. Lipid Res. 60, 1720–1732 39. Okamoto, H. et al. (2005) Restoration of liver insulin signaling in Insr knockout mice fails to normalize hepatic insulin action. J. Clin. Invest. 115, 1314–1322

Trends in Endocrinology & Metabolism, Month 2020, Vol. xx, No. xx

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40. Taniguchi, C.M. et al. (2006) Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKC lambda/zeta. Cell Metab. 3, 343–353 41. Lu, M. et al. (2012) Insulin regulates liver metabolism in vivo in the absence of hepatic Akt and Foxo1. Nat. Med. 18, 388–395 42. Jaiswal, N. et al. (2019) The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis. Mol Metab. 28, 1–13 43. Czech, M.P. (2017) Insulin action and resistance in obesity and type 2 diabetes. Nat. Med. 23, 804–814 44. Hammad, E.S. et al. (1982) Morphological and biochemical observations on hepatic glycogen metabolism in mice on a controlled feeding schedule. II. Streptozotocin-diabetic mice. Dig. Dis. Sci. 27, 692–700 45. Wang, Z.V. et al. (2010) Identification and characterization of a promoter cassette conferring adipocyte-specific gene expression. Endocrinology 151, 2933–2939 46. Kraegen, E.W. et al. (1991) Development of muscle insulin resistance after liver insulin resistance in high-fat fed rats. Diabetes 40, 1397–1403 47. Best, J.D. et al. (1996) Role of glucose effectiveness in the determination of glucose tolerance. Diabetes Care 19, 1018–1030 48. Alford, F.P. et al. (2018) Glucose effectiveness is a critical pathogenic factor leading to the emergence of glucose intolerance and type 2 diabetes mellitus: an ignored hypothesis. Diabetes Metab. Res. Rev. 34, e2989 49. Dube, S. et al. (2015) The forgotten role of glucose effectiveness in the regulation of glucose tolerance. Curr. Diab. Rep. 15, 605 50. Raddatz, K. et al. (2012) Deletion of protein kinase Cε in mice has limited effects on liver metabolite levels but alters fasting ketogenesis and gluconeogenesis. Diabetologia 55, 2789–2793 51. Schmitz-Peiffer, C. (2013) The tail wagging the dog - regulation of lipid metabolism by protein kinase C. FEBS J. 280, 5371–5383 52. Deevska, G.M. and Nikolova-Karakashian, M.N. (2017) The expanding role of sphingolipids in lipid droplet biogenesis. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1862, 1155–1165 53. Dasgupta, S. et al. (2011) Mechanism of lipid induced insulin resistance: activated PKCε is a key regulator. Biochim. Biophys. Acta 1812, 495–506 54. Ryu, D. et al. (2009) TORC2 regulates hepatic insulin signaling via a mammalian phosphatidic acid phosphatase, LIPIN1. Cell Metab. 9, 240–251 55. Steinberg, S.F. (2008) Structural basis of protein kinase C isoform function. Physiol. Rev. 88, 1341–1378 56. Parekh, D. et al. (1999) Mammalian TOR controls one of two kinase pathways acting upon nPKC delta and nPKC epsilon. J. Biol. Chem. 274, 34758–34764 57. Cenni, V. et al. (2002) Regulation of novel protein kinase C epsilon by phosphorylation. Biochem. J. 363, 537–545

58. Ikenoue, T. et al. (2008) Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J. 27, 2270 59. Xu, T.R. et al. (2007) Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCepsilon) in 3T3 fibroblasts. Cell. Signal. 19, 1986–1995 60. Kajimoto, T. et al. (2010) Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases. J. Biol. Chem. 285, 41896–41910 61. Braun, D.C. et al. (2005) Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell. J. Biol. Chem. 280, 8164–8171 62. Conway, J.R.W. et al. (2017) Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors. Methods 128, 78–94 63. Vogt, M.C. and Bruning, J.C. (2013) CNS insulin signaling in the control of energy homeostasis and glucose metabolism - from embryo to old age. Trends Endocrinol. Metab. 24, 76–84 64. Borg, M.L. et al. (2012) Consumption of a high-fat diet, but not regular endurance exercise training, regulates hypothalamic lipid accumulation in mice. J. Physiol. 590, 4377–4389 65. Benoit, S.C. et al. (2009) Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents. J. Clin. Invest. 119, 2577–2589 66. Schechtman, D. and Mochly-Rosen, D. (2001) Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene 20, 6339–6347 67. Kraft, A.S. and Anderson, W.B. (1983) Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301, 621–623 68. Titchenell, P.M. et al. (2017) Unraveling the regulation of hepatic metabolism by insulin. Trends Endocrinol. Metab. 28, 497–505 69. Brown, M.S. and Goldstein, J.L. (2008) Selective versus total insulin resistance: a pathogenic paradox. Cell Metab. 7, 95–96 70. Taniguchi, C.M. et al. (2006) Critical nodes in signalling pathways: insights into insulin action. Nat. Rev. Mol. Cell Biol. 7, 85–96 71. Cherrington, A.D. et al. (2007) Insulin action on the liver in vivo. Biochem. Soc. Trans. 35, 1171–1174 72. Perry, R.J. et al. (2015) Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes. Cell 160, 745–758 73. Titchenell, P.M. et al. (2015) Hepatic insulin signalling is dispensable for suppression of glucose output by insulin in vivo. Nat. Commun. 6, 7078 74. Titchenell, P.M. et al. (2016) Direct hepatocyte insulin signaling is required for lipogenesis but is dispensable for the suppression of glucose production. Cell Metab. 23, 1154–1166 75. Sargsyan, A. and Herman, M.A. (2019) Regulation of glucose production in the pathogenesis of type 2 diabetes. Curr. Diab. Rep. 19, 77 76. Sharabi, K. et al. (2019) Regulation of hepatic metabolism, recent advances, and future perspectives. Curr. Diab. Rep. 19, 98

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