• adipose tissue;
  • diacylglycerol;
  • insulin resistance;
  • lipid;
  • lipogenesis;
  • lipolysis;
  • liver;
  • metabolism;
  • protein kinase C;
  • skeletal muscle


  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

Upon their discovery almost 40 years ago, isoforms of the lipid-activated protein kinase C (PKC) family were initially regarded only as downstream effectors of the second messengers calcium and diacylglycerol, undergoing activation upon phospholipid hydrolysis in response to acute stimuli. Subsequently, several isoforms were found to be associated with the inhibitory effects of lipid over-supply on glucose homeostasis, especially the negative cross-talk with insulin signal transduction, observed upon accumulation of diacylglycerol in insulin target tissues. The PKC family has therefore attracted much attention in diabetes and obesity research, because intracellular lipid accumulation is strongly correlated with defective insulin action and the development of type 2 diabetes. Causal roles for various isoforms in the generation of insulin resistance have more recently been confirmed using PKC-deficient mice. However, during characterization of these animals, it became increasingly evident that the enzymes play key roles in the modulation of lipid metabolism itself, and may control the supply of lipids between tissues such as adipose and liver. Molecular studies have also demonstrated roles for PKC isoforms in several aspects of lipid metabolism, such as adipocyte differentiation and hepatic lipogenesis. While the precise mechanisms involved, especially the identities of protein substrates, are still unclear, the emerging picture suggests that the currently held view of the contribution of PKC isoforms to metabolism is an over-simplification. Although PKCs may inhibit insulin signal transduction, these enzymes are not merely downstream effectors of lipid accumulation, but in fact control the fate of fatty acids, thus the tail wags the dog.


protein kinase C




endoplasmic reticulum




insulin receptor substrate 1


low-density lipoprotein


3-phosphoinositide-dependent kinase-1


peroxisome proliferator-activated receptor gamma coactivator 1-α


phosphatidylinositide 3-kinase


protein kinase B


protein kinase C


receptor for activated C kinase


sterol regulatory element-binding protein 1c




  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

The protein kinase C (PKC) family has been sub-divided into three groups of isoforms that share similar structure and modes of activation. These aspects have been extensively reviewed previously [1, 2], and are only described briefly here. The conventional (or classical) PKC isoforms PKCα, PKCβI, PKCβII and PKCγ have C1 and C2 domains, and are thus their activity is dependent on both diacylglycerol (DAG) and Ca2+. PKCβI and PKCβII arise through alternative splicing of C-terminal exons of the PKCβ gene [3]. The novel PKC isoforms PKCδ, PKCε, PKCη and PKCθ have C1 and only ‘C2-like’ domains, and are calcium-independent but still sensitive to DAG. Finally, the atypical PKC (aPKC) isoforms PKCζ and PKCι (the mouse ortholog of which is also known as PKCλ) are activated not by DAG but by 3-phosphoinositide-dependent kinase-1(PDK1)-mediated phosphorylation.

DAG is generated acutely upon phospholipase C activation, in response to a wide range of stimuli such as neurotransmitters and growth factors [4] (Fig. 1). Phospholipase C isoforms hydrolyse phosphatidylinositol 4,5-bisphosphate, giving rise to DAG and inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate also contributes to the activation of conventional PKC isoforms by opening a Ca2+ channel in the endoplasmic reticulum (ER) to increase the cytoplasmic concentration of Ca2+. Activated PKC translocates from a cytosolic location and becomes membrane-associated while binding DAG, and this redistribution is frequently used as a surrogate measure of activation. Under these conditions, the elevated DAG and Ca2+ levels are generally short-lived signals, and the kinase returns to its inactive cytosolic state [2]. Longer-term activation, which may be achieved experimentally by treatment with phorbol esters due to their much greater stability compared to DAG, leads to down-regulation of PKC by proteolysis, with the isoforms exhibiting different levels of susceptibility [5, 6].


Figure 1. Contrasting activation of PKC by DAG derived either from phospholipase C (PLC) action at the plasma membrane or synthesized at the ER as a consequence of dietary lipid excess. Acute activation of PLC, in response to stimuli such as neurotransmitters and growth factors, leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate at the plasma membrane, causing release of both DAG and inositol 1,4,5-trisphosphate (IP3), which in turn act as second messengers for activation of conventional and novel PKC isoforms. In contrast, increased fatty acid accumulation in the cell upon dietary lipid excess leads to DAG synthesis at the ER, as an intermediate during formation of intracellular lipid bodies for the storage of TAG. This DAG also activates conventional and novel PKC isoforms, which then participate in negative cross-talk with the insulin signalling pathway, especially by mediating the serine phosphorylation of IRS-1 (P-S, phosphoserine; P-Y, phosphotyrosine). However, it is now becoming evident that several isoforms more directly affect lipid metabolism, potentially acting as sensors for the increased abundance of fatty acids.

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Activation by chronic intracellular DAG accumulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

In contrast to the acute activation of PKC in response to specific stimuli as described above, many studies have reported chronic activation of one or more isoforms in cells or tissues in which DAG levels are elevated over the longer term. In this case, the DAG molecules involved are more likely to be intermediates in lipid metabolism, especially during triacylglycerol (TAG) synthesis, rather than the product of phospholipase C activity (Fig. 1). Thus the lipid over-supply associated with obesity or high-fat feeding in human and animal studies gives rise to elevated DAG levels, which in turn are associated with an increase in membrane-associated PKC. This has been extensively studied in the context of insulin resistance, which is a major aspect of type 2 diabetes. PKC activity may directly interfere with normal insulin signal transduction, providing one mechanistic explanation for the strong association between intracellular lipid accumulation and defective insulin action [7]. Several isoforms have been reported to be chronically activated in this way in insulin-sensitive tissues, with key roles suggested for PKCθ in skeletal muscle and PKCδ and PKCε in liver. In each case, aberrant kinase activity may lead directly or indirectly to increased insulin receptor substrate 1 (IRS-1) serine phosphorylation, which in turn reduces insulin-stimulated tyrosine phosphorylation and subsequent downstream signalling [7].

It is important to consider the intracellular location of the DAG that mediates such long-term activation of PKC. In contrast to acutely released DAG following stimulation of phospholipase C, which tends to be localized at the plasma membrane, de novo synthesis of DAG occurs at the ER, prior to further fatty acid incorporation to generate TAG, which is then stored in lipid bodies. This suggests that the PKC isoforms that are activated upon increased lipid metabolism may at least initially be primarily located at the ER rather than the plasma membrane, with access to a different set of protein substrates. This co-localization with the extensive machinery involved in lipid synthesis, storage and hydrolysis supports the concept that PKC activated in this way can modulate lipid metabolism. Interestingly, recent work has shown that, while newly synthesized DAG at the ER is in the form of the sn-1,2 stereoisomer, which is capable of activating PKC, DAG released from lipid bodies during lipolysis is primarily in the form of sn-1,3 or sn-2,3 isomers, due to the selective action of a key lipase, adipose triglyceride lipase, and is unlikely to participate in PKC activation [8]. However, this does not preclude a role for PKC in regulation of lipolytic processes in response to activation by intermediates of TAG esterification.

Insights from genetically modified mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

Investigation of the effects of PKC deletion in mice has yielded several insights into the specific contributions of individual PKC isoforms in the synthesis, esterification or oxidation of fatty acids by different tissues in vivo, as well as their roles in the response to dietary lipid over-supply. In many cases, these studies were in fact designed to investigate PKC-dependent modulation of glucose homeostasis, but, by comparing and contrasting the effects of kinase deletion on pathophysiological outcomes such as obesity, hepatic steatosis and lipidaemia, it becomes evident that the metabolic effects of PKCs extend beyond the induction of insulin resistance upon chronic activation.

Conventional PKCs

Relatively little work has been performed on the involvement of two of the conventional PKC isoforms in lipid metabolism. PKCγ is expressed mainly in the brain and nervous system, and PKCγ-deficient mice have been generated [9] and extensively studied in terms of behavioural and other neurological effects. However, despite the importance of the brain in regulating many aspects of metabolism [10], no metabolic phenotypes of PKCγ in metabolism have been reported. In the case of mice lacking PKCα, insulin-stimulated glucose uptake by adipocytes as well as skeletal muscle was enhanced, together with upstream insulin signalling, but lipid metabolism itself was not investigated and the mice were not challenged with a high-fat diet [11].

In contrast, a major role for PKCβ in energy homeostasis and lipid accumulation has emerged. Initial work was limited to mice fed normal chow, and again demonstrated an enhanced uptake of glucose by muscle and fat cells, although this did not translate into improved glucose tolerance in vivo [12]. Subsequently, an investigation of the effects of PKCβ deletion on broader aspects of metabolism revealed a reduction in adipose tissue depots and in the TAG content of liver and skeletal muscle in chow-fed mice [13]. These changes were accompanied by elevated energy consumption and increased fatty acid oxidation in adipose tissue. Several changes in gene expression may have contributed to this phenotype, including the up-regulation of uncoupling protein-2 and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), which was associated with mitochondrial biogenesis [13]. These findings were extended by examining the effects of a high-fat diet on the mice [14]. In this case, there was protection against obesity, hyperlipidaemia and hepatic steatosis, and a preservation of insulin sensitivity and hence glucose tolerance. In high fat-fed wild-type mice and in ob/ob mice, PKCβ mRNA and protein expression was enhanced, further supporting a causative role for this isoform in promoting obesity, and it was suggested that this occurred secondary to hyperinsulinaemia, contributing to a vicious circle between insulin resistance and increased tissue TAG accumulation [14]. Conversely, deletion of PKCβ in ob/ob mice resulted in protection against fat accumulation and insulin resistance [15].

While deletion of the PKCβ gene does not allow selective changes in PKCβI or PKCβII expression, specific over-expression of a constitutively active PKCβII mutant in mouse skeletal muscle demonstrated that this splice variant of PKCβ not only induces insulin resistance, but also affects the levels of several genes involved in lipid metabolism [16]. Thus impairment in the expression of peroxisome proliferator-activated receptor gamma, PGC1α, acyl CoA oxidase and hormone-sensitive lipase, but enhanced expression of the lipogenic transcription factor sterol regulatory element-binding protein 1c (SREBP1c) in skeletal muscle, were associated with decreased lipid oxidation and increased intra-myocellular lipid deposition. In addition to these direct effects in muscle, transgenic over-expression of PKCβII led to indirect defects in insulin action in liver and brain, as well as hepatic lipid accumulation similar to that seen in fat-fed animals. This study is therefore in agreement with those utilizing PKCβ-deficient mice, further supporting a role for this isoform in the regulation of lipogenesis. Evidence that this isoform acts at least in part through the induction of SREBP1c is discussed with other mechanistic investigations further below.

Novel PKCs

In contrast to the conventional isoforms, several investigators have examined the roles of distinct novel PKCs in the regulation of energy homeostasis and metabolism. The motivation for doing so was the strong association of insulin resistance in skeletal muscle and liver with the translocation/activation of these kinases [7]. However, detailed analysis has frequently revealed that, while the kinases do play causative roles in the generation of insulin resistance, their ablation also leads to unexpected effects on hepatic and intramyocellular fat accumulation.

Chronic activation of PKCδ has been observed in skeletal muscle [17] and liver [18] upon fatty acid over-supply, and increased hepatic expression of the kinase has also been linked to obesity in animals and humans [19]. As may be expected from these findings, ablation of PKCδ has beneficial effects on insulin sensitivity and glucose homeostasis in fat-fed mice. However, these studies also indicate that the kinase exerts major effects on the expression of genes involved in hepatic lipid metabolism [19, 20]. PKCδ-deficient mice exhibit down-regulation of genes involved in lipogenesis, associated with reduced liver steatosis, plasma triglyceride levels, muscle TAG accumulation and adipose tissue mass [19, 20]. Interestingly, acute deletion of PKCδ in liver, through adenovirally mediated Cre recombinase expression in PKCδ-floxed mice, reversed insulin resistance in fat-fed animals without improving lipid abnormalities, indicating the independence and also the complexity of these effects of PKCδ [19].

In the case of PKCθ, manipulation of kinase expression or activity by independent investigators, using different models to assess its role in metabolism, has yielded disparate results, which may be reconciled by the conclusion that the phenotypes observed are dependent on the duration of exposure to lipid over-supply in the absence of the enzyme. On the one hand, a short-term elevation in plasma fatty acids induced by lipid infusion in mice showed that PKCθ-deficient mice are protected against the resulting acute whole-body insulin resistance. This was chiefly explained by the preservation of insulin signalling and glucose uptake in skeletal muscle, whereas hepatic glucose output was unaffected [21]. This is in good agreement with the relatively high expression of PKCθ in muscle and its frequently reported translocation in this tissue upon lipid excess [22], and with the inhibition of insulin signalling at the level of IRS-1 mediated by this kinase [23]. Interestingly, glucose uptake into brown but not white adipose tissue was also conserved [21], although the role of PKCθ in this tissue has not been explored.

On the other hand, longer-term studies have revealed additional roles for PKCθ in metabolic control that are essential for normal glucose and lipid homeostasis. Transgenic over-expression of a dominant-negative PKCθ mutant in skeletal muscle resulted in obesity in older mice, associated with impaired glucose tolerance and diminished insulin sensitivity [24]. It was speculated that PKCθ may participate in cross-talk between muscle and fat, resulting in the observed accumulation of visceral fat. Similarly, the same line of PKCθ-deficient mice that exhibited protection of glucose homeostasis when subjected to acute lipid infusion [21] was found to become obese with increasing age, associated with reduced energy expenditure, defective fat oxidation and muscle insulin resistance [25]. These phenotypes were exacerbated by high-fat feeding, leading to greater insulin resistance in muscle, fat and liver, although glucose intolerance was averted by compensatory hyperinsulinaemia [25]. Additional effects of PKCθ on adipose tissue function were suggested by adverse changes in adiponectin expression and plasma fatty acid levels in fat-fed mice. Overall, it is clear that, while acute activation of PKCθ by fatty acid excess may inhibit insulin signalling and subsequent glucose disposal, especially in skeletal muscle, the normal function of the enzyme is important in the maintenance of body composition and energy homeostasis in the longer term.

PKCε is another novel PKC isoform that has been extensively linked to lipid-induced insulin resistance, both in skeletal muscle [17, 26-29] and liver [30, 31]. Its activation in models of hepatic insulin resistance is now frequently used as a indicator of the underlying mechanism causing defective insulin action [32-37]. A number of studies have examined the effect of its deletion or ablation not only directly on insulin signalling, where it plays an inhibitory role as expected, but also on its broader metabolic role in several tissues and under various nutritional conditions. While these have indicated more complex interactions, a unifying theme is the promotion of lipid partitioning away from β-oxidation and towards TAG storage.

Knockdown of PKCε using specific oligonucleotides reduced expression of the kinase in certain tissues, including liver and adipose tissue but not skeletal muscle [38]. This protected mice against the insulin resistance caused by a 3-day high-fat diet, with a major effect on suppression of glucose output by the liver as assessed by the euglycaemic clamp technique, and this was associated with improved insulin receptor signalling [38]. This indicates that, as for several other PKC isoforms, an important acute action of PKCε is the inhibition of insulin signal transduction upon lipid over-supply. A similar beneficial effect was observed in mice in which the PKCε gene had been deleted, when fed a high-fat diet for up to 3 weeks [39]. In these mice, glucose tolerance was unimpaired by the diet, whereas wild-type mice became glucose-intolerant. Insulin levels were not elevated under these conditions by PKCε deletion, consistent with the maintenance of insulin sensitivity, and pyruvate tolerance tests again demonstrated an effect at the level of liver insulin action [39].

While these findings support a direct role of PKCε in the inhibition of glucose disposal in response to insulin, additional studies indicated that this enzyme also contributes to the early response of the liver to dietary lipid excess. First, the sustained insulin action observed in the absence of PKCε during the early phase of a high-fat diet was unexpectedly associated with an increase in hepatic TAG content and a defect in lipid oxidation [39, 40]. Thus, although liver lipid content was still much lower than in long term fat-fed mice, it was up to 50% greater in the PKCε-deficient mice compared to wild-type mice, and this was associated with reduced energy consumption despite normal switching to fat as the major oxidative fuel [39]. Second, PKCε-deficient mice fed high-fat diets for over 6 weeks no longer exhibited protection against insulin resistance [39, 41]. These studies suggest that when activated, PKCε functions as a early lipid sensor, promoting β-oxidation in the liver when dietary fat is more prevalent. Although insulin resistance may also result in the shorter term, either through inhibition of insulin signalling [38] or through mechanisms related to accelerated β-oxidation such as increased production of reactive oxygen species [39], the overall effect may be beneficial when the nutritional excess is temporary.

Interestingly, the lipid partitioning observed in the liver that results from the absence of PKCε is not confined to this tissue. PKCε-deficient mice fed a high-fat diet for longer periods are still protected against glucose tolerance despite exhibiting insulin resistance, which is explained by an enhancement of insulin release that fully compensates for the impairment in insulin action [39, 41, 42]. This is associated with altered lipid partitioning in pancreatic islets upon chronic lipid exposure [41], and an enhancement of the production of esterification products that may act as signals to promote the amplification pathway of glucose-stimulated insulin secretion [42, 43]. Importantly, insulin secretion is not increased in an unregulated manner in PKCε-deficient mice, but only upon demand when glucose levels become elevated, avoiding β-cell exhaustion. This phenotype shows a different temporal dependence to the protection against insulin resistance, in that it is only observed under conditions of longer-term dietary lipid excess [39], suggesting that it depends on accumulation of specific lipids or other factors in the pancreatic islets. Thus islets from mice fed a high-fat diet for only 1 week secrete insulin in a similar manner to islets from wild-type mice, and glucose tolerance is protected only at the level of insulin sensitivity [39].

As well as modulating lipid partitioning during over-nutrition, PKCε also affects the supply of lipid from adipose tissue to the liver in mice upon chronic fasting. Normally, lipolytic signals such as glucagon and catecholamines promote the release of free fatty acids from adipocytes into the circulation under these conditions, which may be taken up by the liver to generate ketone bodies such as β-hydroxybutyrate, for use as alternative fuels by muscle and the brain when glucose levels are reduced [44]. In PKCε-deficient mice, fat pad mass is not reduced to the same extent as in wild-type mice after a 48 h fast, plasma free fatty acids are not as elevated, and liver lipid stores are not supplemented to the same extent. This reduced supply of ketogenic precursors to the liver results in plasma β-hydroxybutyrate levels that are almost 50% lower than in wild-type mice [40]. This prevention of fatty acid release by adipose tissue under fasting conditions, together with the alterations in lipid flux exhibited by the liver and pancreatic islets in the fed state, contributes to the emerging theme of lipid partitioning towards TAG storage in the absence of PKCε.

Atypical PKC

Studies of aPKC function in vivo have also helped to establish a major contribution of this sub-group in the regulation of lipid metabolism. The aPKC isoforms PKCζ and PKCι (referred to as PKCλ in mouse) appear to be the key PKCs involved in normal insulin signalling, activated by IRS-1- or IRS-2-dependent phosphatidylinositide 3-kinase (PI3K) activity and contributing to insulin-stimulated glucose transport in muscle and lipogenesis in liver [45]. From over-expression studies, they also appear to be functionally interchangeable, and their importance in specific tissues may be a reflection of their relative levels. Thus, in mouse skeletal muscle, PKCλ levels are dominant and muscle-specific deletion of PKCλ in mice causes muscle insulin resistance and impaired glucose tolerance, whereas PKCζ deletion in this tissue has no effect [46]. Although aPKC activation by insulin in muscle through IRS-1/PI3K signalling is defective in obesity and diabetes, activation in the liver is preserved and in fact increased due to intact IRS-2/PI3K signalling and hyperinsulinaemia. This promotes lipid synthesis, further lipid abnormalities and insulin resistance [47]. Whole-body insulin sensitivity in several animal models can thus be improved by liver-specific knockdown or functional inhibition of aPKC [48, 49]. The mechanism by which aPKC activity promotes lipogenesis may be similar to that downstream of PKCβ, involving SREBP1c as discussed below. These and other studies suggest that aPKC activity plays a dominant role in insulin-stimulated lipogenesis in the liver, while the other PDK1-dependent kinase protein kinase B (PKB)/Akt is more important for the inhibitory effects of the hormone on gluconeogenesis [50, 51]. However, it should be noted that other investigators have suggested that Akt activity is critical for hepatic lipid accumulation [52].

Cell-based and molecular studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

It may be argued in some of the cases discussed above that the alterations observed in lipid metabolism upon manipulation of PKC expression are secondary to the changes in insulin sensitivity, especially as the cross-talk between obesity, ectopic lipid accumulation and insulin resistance is multi-directional [53]. However, when taken together with studies examining the molecular effects of PKC isoforms on metabolism in various cell types and tissues, it is clear that these kinases act directly to control the fate of fatty acids.


A number of cell-based investigations have highlighted various roles for PKC activity in the differentiation and function of adipocytes. Initially, non-isoform-specific PKC activators and inhibitors, such as phorbol esters and bisindomaleimides, were employed. More detailed insights have been generated by analysis of PKC isoform expression during and after adipogenesis, as well as by isoform over-expression and knockdown, which have indicated that different isoforms may play opposing roles. Thus total PKCα, PKCγ and PKCδ levels are transiently elevated during the differentiation of both 3T3-L1 and 3T3-F442A adipocytes [54-56]. While this may be interpreted as indicating a positive role for these isoforms in adipogenesis, interventive experiments have shown that PKCα and PKCδ knockdown or inhibition promote early differentiation [57, 58], and inhibition of PKCα also blocks the anti-adipogenic effect of platelet-derived growth factor [59], suggesting negative effects of these kinases. In contrast, membrane translocation/activation of PKCβI, rather than changes in total levels of this isoform, is observed during the first 2 days of 3T3-L1 adipocyte differentiation, and over-expression of a dominant-negative PKCβI mutant blocks adipogenesis, consistent with a positive role [58]. PKCβ was among the most strongly induced genes observed by cDNA microarray analysis of 3T3-LI adipocyte differentiation [60], while investigation of PKCβ splicing during differentiation showed that PKCβI levels were down-regulated and PKCβII levels were increased [61]. Evidence of a positive role for PKCε has also been reported by several groups. Three independent studies have shown that PKCε levels are increased in cultured fat cells in the fully differentiated state [55, 56, 62]. Knockdown of PKCε inhibited 3T3-F442A cell differentiation, and it was suggested that, while PKCε may not be required for clonal expansion of differentiating cells, it is probably necessary in the later stages of differentiation for attainment and maintenance of the adipocyte phenotype [56]. Similarly, over-expression of PKCε, but not other isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers, and PKCε inhibitory peptides reduced differentiation [62]. Finally, PKCε levels are also diminished in primary adipocytes from a mouse model of insulin resistance, suggesting that a decrease in PKCε expression may contribute to metabolic alterations in adipocytes associated with insulin resistance and obesity [55].


Extensive evidence indicates that PKC activity is also involved in the control of lipolysis in mature adipocytes, through more than one signalling pathway [63, 64], although isoform-specific effects have not been well-characterized, and both stimulatory and inhibitory effects of PKC activators and inhibitors have been reported. For example, phorbol ester-mediated PKC activation in rat adipocytes led to inhibition of the major pathway that promotes lipolysis, namely the cAMP-dependent activation of protein kinase A and phosphorylation of hormone-sensitive lipase and perilipin 1, possibly through negative effects on proximal β-adrenergic receptor signalling [65-67]. However, PKC may also promote lipolysis independently of the protein kinase A pathway [68, 69]; this may involve hormone-sensitive lipase phosphorylation upon mitogen-activated protein kinase activation [63]. PKCε is able to activate the mitogen-activated protein kinase pathway [70] and may participate in the promotion of lipolysis in this way, and this isoform has been proposed to mediate the phorbol ester-dependent inhibition of the anti-lipolytic effect of insulin [71]. The recent finding that PKCε is localized to lipid droplets, in a manner that correlates with the DAG content of these bodies and that may affect its ability to affect insulin sensitivity [72, 73], increases the importance of clarification of the role of this isoform.


The mechanisms by which PKC affects lipogenesis have been addressed mainly at the level of the key lipogenic transcription factor SREBP1c. The lipogenic effects of PKCβ observed in mice involve alterations in activation of the SREBP1c promoter in the liver [14, 74], and, using hepatocytes, it was shown that the kinase modulated Sp1 and Sp3 protein binding to a sterol regulatory element complex of the SREBP1c promoter to increase transcription [74]. On the other hand, PKCβ deficiency in mice was accompanied by up-regulation of β1- and β3-adrenergic receptor expression and p38 mitogen-activated protein kinase signalling in white adipose tissue, which was proposed to underlie the beneficial remodelling observed in this study, including the up-regulation of PGC1α and UCP-1 [15].

As in the case of PKCβ depletion, mice deficient in PKCδ also exhibited decreased hepatic expression of SREBP1c, as well as of its downstream targets such as fatty acid synthase and acetyl CoA carboxylase [19, 20]. Conversely, adenovirally mediated over-expression of PKCδ in liver resulted in enhanced expression of these proteins, consistent with the lipogenic actions of PKCδ reported previously [19]. In contrast, PKCε-deficient mice did not exhibit changes in the hepatic mRNA or protein levels of a broad range of lipogenic genes, as well as others associated with lipid metabolism, despite the enhanced TAG accumulation observed in the early phase of dietary lipid excess, consistent with the hypothesis that lipid supply to the liver is more important than direct effects in this tissue [39].

Insulin also stimulates the induction of SREBP1c in the liver, which, as described above has been linked to PI3K-mediated activation of aPKC isoforms by the hormone, especially PKCι [50]. The mechanism by which aPKC activity promotes lipogenesis may indeed be similar to that downstream of PKCβ, as PKCι deletion specifically in the liver led to reduced expression of SREBP1c and fatty acid synthase, as well as lower hepatic TAG levels in mice [50]. This phenotype was reversed by adenovirally mediated re-expression of hepatic PKCι, while a dominant-negative mutant of PKCι expressed in primary hepatocytes attenuated the effects of insulin on the induction of SREBP1c and fatty acid synthase [50]. Similarly, over-expression of PKCι in livers of mice lacking functional PI3K activity rescued SREBP1c expression [51].

LDL receptor expression

Certain PKC isoforms may also regulate lipid uptake into cells, especially hepatocytes, through modulation of LDL receptor expression [75-78]. PKC inhibitors or PKCβ knockdown blocked induction of the low-density lipoprotein (LDL) receptor in HepG2 cells, while over-expression of PKCβ but not PKCα increased LDL receptor promoter activity several fold [75]. PKCβ appears to act in a complex with protein phosphatise 2A, modulating histone 3 phosphorylation and in turn induction of the LDL receptor [76, 77].

In addition, the same investigators showed that over-expression of PKCε in HepG2 cells greatly induced LDL receptor promoter activity, whereas PKCα, PKCγ, PKCδ or PKCζ failed to do so. Conversely, knockdown of endogenous PKCε blocked induction of LDL receptor transcription following sterol depletion. Interestingly, a specific interaction between PKCε and sterols was also reported, and it was suggested that sterol depletion promoted PKCε activity and hence the induction of LDL receptor transcription in liver cells [78].

PKC substrates and binding partners

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

The protein targets of individual PKC isoforms that mediate the effects of the activated kinases on lipid metabolism after direct phosphorylation are poorly characterized. Ideally, several criteria must be met in order to define a protein as a bona fide PKC substrate, as proposed for other kinases such as PKB [79]. Although putative substrates may initially be identified through phosphorylation by purified or recombinant PKC in vitro [80], other supporting evidence is required, such as demonstration of the expected changes in phosphorylation upon PKC over-expression or knockdown in intact cells, identification of the phosphorylation site and the demonstration that this affects downstream functionality. Although several PKC isoforms clearly affect glucose and lipid metabolism as discussed above, the important phosphorylation events mediated by PKCs remain to be determined. The best-characterized target of PKC activity in this respect is IRS-1, and several studies have identified serine phosphorylation sites on this docking protein that may be directly or indirectly modulated by PKC isoforms to inhibit insulin receptor binding or recruitment of downstream effectors [7].

A major determinant of the specificity of PKCs for their substrates is likely to be binding of the enzymes to particular scaffold proteins. These binding partners presumably ensure localization of the kinases to distinct intracellular locations, in close proximity to their targets [81, 82]. For example, PKCβ and PKCε bind receptors for activated C kinase (RACK1 and RACK2, respectively), and disruption of the interactions using competing peptides specifically and potently inhibits the function of these kinases [83]. The contribution of receptors for activated C kinase and other anchoring proteins to PKC function has been widely investigated [81, 82, 84], and may explain, at least in part, why there is apparently little functional redundancy within the PKC family: although each isoform contains highly conserved domains for activation and catalytic activity, additional variable domains determine protein–protein interactions contributing to localization and substrate specificity. This has been only been addressed in a limited fashion in terms of metabolism. Thus, the protection of insulin secretion and hence glucose tolerance in fat-fed mice observed upon deletion of PKCε may be reproduced by treatment of diabetic animals with cell-permeant peptide inhibitors of the interaction of the kinase with RACK2 [41]. However, the involvement of RACK2 and other PKC-anchoring proteins in lipid metabolism remains to be determined.

Summary and perspective

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References

Over the past two decades, several lines of evidence have established isoforms of the PKC family as important agents that mediate the inhibitory effects of lipid over-supply on insulin target tissues such as liver and skeletal muscle. Early studies demonstrated strong correlations between PKC translocation and insulin resistance, while more recent work, performed using mice in which specific isoforms have been genetically deleted, has confirmed roles for several of these kinases in the inhibition of insulin signal transduction and hence its downstream effect on glucose metabolism. It is now important to recognize that the actions of these enzymes are not limited to simple inhibitory cross-talk with insulin signalling, and that several members of the PKC family have direct effects on lipid metabolism (summarized in Table 1). The isoforms PKCβ, PKCδ and PKCι/λ are somewhat similar in this respect as they promote lipogenesis, especially in liver. Meanwhile, PKCε and PKCθ may be important for the normal response to dietary fat over-supply, albeit over different time frames, promoting oxidation to avoid intracellular lipid accumulation and obesity. In the case of PKCε, the modulation of lipid partitioning is exerted in various tissues and under diverse nutritional states. A model illustrating these isoform-specific roles is presented in Fig. 2. With hindsight, the control of lipid metabolism by PKCs may have been expected, at least in the case of the conventional and novel isoforms, because these enzymes are lipid-activated, and therefore ideally suited to act as sensors for intracellular lipid accumulation, and to modulate the subsequent fate of fatty acids.

Table 1. Summary of the reported effects of PKC isoforms on lipid metabolism
PKC isoformEffect of deletion in vivoFindings from in vitro studiesReferences
PKCαNo effects on lipid metabolism reportedAnti-adipogenic [57, 59]
PKCβReduced adipose tissue accumulation; reduced muscle and liver TAG

Pro-adipogenic; SREBP1c promoter activation; β-adrenergic receptor up-regulation

LDL receptor induction.

[13-15, 60, 61, 74-77]
PKCγNo effects on lipid metabolism reported  
PKCδReduced hepatic steatosis, plasma lipids, muscle TAG, adipose tissue massAnti-adipogenic [19, 20, 58]
PKCεShort-term hepatic lipid accumulation; β-cell lipid partitioning; reduced release of fatty acids by adipose tissue on fastingPro-adipogenic; opposes anti-lipolytic effect of insulin; LDL receptor induction [39-42, 55-57, 64, 73]
PKCθVisceral adipose tissue accumulation; reduced fatty acid oxidation  [24, 25]
PKCιReduced hepatic steatosisSREBP1c activation [48-51]

Figure 2. Effects of PKC isoforms on lipid metabolism in specific tissues. Various roles for individual PKC isoforms have been identified by studying the deletion of specific kinases in vivo, as well as manipulating their expression in cultured cells. PKCβ and PKCε have been most widely implicated in adipogenesis [55-58, 60, 61, 64, 73] and fat cell metabolism [13-15, 39-42]. In the liver, PKCβ, PKCδ and PKCι/λ have been associated with lipogenesis [19, 20, 48-51, 74], while PKCε appears to play a role in the acute response to fat over-supply [39], promoting β-oxidation in the initial phase of a high-fat diet. This enzyme also affects ketogenesis in the liver under fasting conditions, most likely by regulating fatty acid (FA) release from adipose tissue [40]. In skeletal muscle, increased PKCβ activity is associated with enhanced lipogenesis [16], but the immediate effects of PKCθ in this tissue on lipid metabolism are unclear, but influence whole-body energy expenditure [24, 25]. In the β-cell, PKCε affects the partitioning of fatty acids between β-oxidation and esterification, which in turn modulates the amplification pathway of insulin secretion through the lipolysis/esterification cycle. Specific molecular targets for most of these functions have been poorly characterized, and in several cases it remains to be determined whether the effects seen in one tissue are direct or are dependent on PKC action in another tissue.

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Although the effects of individual isoforms are becoming clearer, the direct targets that undergo phosphorylation, and the pathways that link them with metabolic events, remain poorly understood. With developments in quantitative phosphoproteomic analysis of cells and tissues, it is likely that major advances will be made in this area of PKC function, most likely through studies combining such analysis with the use of existing research tools such as global and tissue-specific PKC-deficient mice.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation by chronic intracellular DAG accumulation
  5. Insights from genetically modified mice
  6. Cell-based and molecular studies
  7. PKC substrates and binding partners
  8. Summary and perspective
  9. References
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