Impaired insulin sensitivity in HFD-fed PKCζ-/- mice
As glucose intolerance and insulin resistance are key features of the metabolic syndrome (Semenkovich, 2006
; Shoelson et al., 2006
), and inflammation plays an important role in this phenomenon (Hotamisligil, 2006
; Shoelson et al., 2006
), we have evaluated whether PKCζ deficiency would lead to increased inflammation and the ensuing systemic glucose intolerance and insulin resistance in the context of fat accumulation. To address this question we have used PKCζ-deficient mice. shows the genotype of PKCζ-/-
mice, which lack the PKCζ allele as described previously (Leitges et al., 2001
). Results shown in demonstrate that PKCζ is actually lost, as determined by immunoblotting in different tissues, and that PKCζ is abundantly expressed in WAT, at levels comparable to those of lung and liver, and to a lesser degree in BAT and muscle. Therefore, PKCζ is expressed in metabolically relevant tissues and its genetic ablation results in the expected lack of its protein expression. We then performed glucose tolerance tests (GTT) in WT and PKCζ-/-
mice that had been fed a HFD or regular diet (RD) for 12 weeks. WT mice kept under HFD-fed conditions displayed reduced glucose clearance in response to a bolus of glucose, as compared with WT mice fed in parallel with RD (). PKCζ-/-
mice fed with RD displayed similar glucose clearance to that of identically treated WT mice (), indicating that the loss of PKCζ does not affect glucose metabolism under normal conditions. However, glucose clearance was much more impaired in PKCζ-deficient mice when fed a HFD than in identically treated WT mice or RD-fed WT or PKCζ-/-
mice (). These results indicate that, even though PKCζ does not play a major role in glucose clearance under RD-fed conditions, HFD feeding makes PKCζ-/-
mice highly glucose intolerant. This could indicate a higher level of insulin resistance upon HFD feeding in PKCζ-/-
mice. In keeping with this notion, although glucose clearance in response to insulin injection (ITT) was significantly impaired only at 15 min post injection in HFD-fed WT mice as compared to RD-fed WT mice, it was dramatically worsened at all time points in HFD-fed PKCζ-deficient mice as compared to identically treated WT mice (). This is indicative of insulin resistance in the HFD-fed PKCζ knockout mouse group. In contrast, there were no differences between WT and PKCζ-deficient mice in terms of ITT when they have been fed with RD (). Previous data indicated that the atypical PKCs could be involved in glucose metabolism and insulin action (Farese and Sajan, 2009
). However, our GTT and ITT data demonstrate that, under normal RD feeding conditions, PKCζ does not play such a role in vivo and that, if any atypical PKC is involved in that process, it is more likely to be PKCλ/ι (Farese et al., 2007
Development of insulin resistance in HFD-fed PKCζ-/- mice
These results suggest that, although PKCζ is not required for normal in vivo insulin action in RD-fed mice, its lack in HFD-fed mice leads to enhanced insulin resistance. In keeping with this, blood insulin levels were dramatically increased in HFD-fed PKCζ-deficient mice upon glucose injection, as compared to identically treated WT mice (). Consistently, demonstrates increased basal non-fasting blood insulin levels in HFD-fed PKCζ-/- mice. Fasting glucose levels were increased in HFD-fed PKCζ-/- mice as compared to identically fed WT mice or RD-fed WT and PKCζ-/- mice, although the latter also showed a noticeable, but not statistically significant, increase in levels of glucose as compared to WT controls (). It should be noted that the levels of gluconeogenic genes in WT and PKCζ-deficient livers did not differ when mice were fed a HFD (). Although, the expression of these genes was reduced in PKCζ-/- livers as compared with WT in mice fed a RD (), this alteration did not seem to affect the basal fasting glucose levels (), nor the response to GTT or ITT ().
All these results suggest that the loss of PKCζ exacerbates HFD-induced glucose intolerance and insulin resistance, but no differences were observed in body weight, fat content, or blood leptin levels in PKCζ-deficient mice fed a HFD, as compared with identically treated WT mice, although HFD feeding increased all these parameters as compared with RD feeding in WT mice (). The levels of circulating TAG, NEFA, and cholesterol were dramatically induced by HFD in WT mice as compared to RD-fed WT mice, and were significantly higher in HFD-fed PKCζ-deficient mice (Table S1
To determine the effects of PKCζ deficiency on glucose metabolism in individual organs, we performed a 2-hr hyperinsulinemic-euglycemic clamp in conscious PKCζ-/-
mice and WT littermates following HFD feeding (Kim et al., 2004a
) (). The steady-state glucose infusion rates required to maintain euglycemia during the clamps were significantly reduced in HFD-fed PKCζ-/-
mice as compared with HFD-fed WT mice. Insulin-stimulated whole-body glucose turnover rates were reduced by 40% in HFD-fed PKCζ-/-
mice. Basal rates of hepatic glucose production (HGP) were not altered, but HGP rates during insulin clamping tended to be higher in HFD-fed PKCζ-/-
mice, resulting in lower hepatic insulin action in these mice. Insulin-stimulated glucose uptake in skeletal muscle and heart were markedly reduced in HFD-fed PKCζ-/-
mice. Glucose uptake in white adipose tissue was not significantly altered, but brown adipose tissue glucose uptake was reduced by more than 50% in HFD-fed PKCζ-/-
mice. Taken together, these results indicate that PKCζ deficiency exacerbates insulin resistance in peripheral organs and liver following HFD.
Reduced glucose metabolism and insulin signaling in HFD-fed PKCζ-/- mice
To further characterize insulin resistance in these mice, they were injected with insulin for 10 min, after which they were sacrificed and extracts were prepared from liver, WAT, and muscle. Akt activation was determined by immunoblotting with an anti-phospho-Akt antibody. Interestingly, Akt activation was normal in liver and WAT of PKCζ-/-
mice fed a RD (). There was a significant reduction in Akt activation in muscle from RD-fed PKCζ-/-
mice, as compared with identically treated WT mice (), consistent with the evidence that PKCζ plays a role in Akt activation, at least in some tissues (Joshi et al., 2008
). HFD feeding impaired Akt activation in liver and muscle but not in WAT in WT mice (). However, the most striking observation was the dramatic impairment of Akt activation in the liver of HFD-fed PKCζ-/-
mice as compared with identically treated WT mice (). These data are consistent with a model whereby the loss of PKCζ increases insulin resistance in HFD-fed mice especially in the liver.
Role of PKCζ in obesity-induced inflammation
The fact that PKCζ-deficient mice fed a HFD showed enhanced insulin resistance and impaired glucose tolerance suggested that the lack of PKCζ might lead to enhanced inflammation, which has previously been shown to play a major role in obesity-induced insulin resistance and glucose intolerance. Therefore, in the next series of experiments we analyzed whether, in fact, PKCζ deficiency promotes inflammation during HFD-induced obesity. Adipose tissue and liver are the two tissues in which obesity-induced inflammation has been shown to develop and which play a major role in glucose metabolism. Interestingly, morphological analysis of white adipose (WAT, epididymal fat pads) () and brown adipose tissue (BAT, interscapular depots) () from HFD-fed WT and PKCζ-deficient mice demonstrate similar adipose tissue volume in both mouse genotypes, although they were significantly bigger than the same tissues from mice fed a RD (). Similar results were obtained when the weights of these tissues were compared (data not shown). Histological analysis of hematoxylin and eosin (H&E) stained tissue sections revealed increased adipocyte size in WAT from HFD-fed PKCζ-/- mice as compared to identically treated WT controls (), whereas no differences were apparent between the WAT from RD-fed mice of different genotypes (). Although HFD feeding enhanced the adipocyte size of WT mice, as compared with RD feeding, the effect of HFD on PKCζ-deficient mice was greater than in WT mice (). More importantly, WAT from HFD-fed PKCζ-/- mice, but not from HFD-fed WT mice, or from RD-fed mice of either genotype displayed a dramatic increase in macrophage infiltration, as assessed by staining with anti-F4/80 antibody (). This observation was confirmed by RT-PCR analysis of CD68 and F4/80 mRNAs in these tissues (). These results indicate that feeding a HFD to PKCζ-deficient mice leads to an inflammatory response in WAT that is not apparent in identically treated WT mice.
Increased macrophage infiltration and hepatic steatosis in HFD-fed PKCζ-/- mice
Morphological and histological analysis revealed a significant increase in the size and weight of HFD-fed PKCζ-/-
mouse livers, as compared to identically treated WT mice or RD-fed mice of both genotypes (). This correlated with enhanced lipid accumulation, as determined by histological analysis and triglyceride content, which was more apparent in the HFD-fed PKCζ-deficient mice, although it was also detected to a lesser extent in RD-fed PKCζ-/-
mice (). We did not detect changes in liver triglyceride secretion (Figure S1A
), or in the expression levels of, for example LDLR, (Figures S1B
). Consistent with this fatty liver phenotype, we detected significantly increased hepatic expression levels of the adipogenic/lipogenic genes PPARγ, PGC1β, FAS, C/EBPβ and ACC without changes in the expression levels of SREBP1c or C/EBPβ in PKCζ-/-
mice as compared with WT mice, with both genotypes fed a HFD (Figures S1C-S1I
). These alterations were not apparent when livers of the two genotypes were compared under a RD feeding regime (Figures S1C-S1I
). The increase in the levels of adipogenic/lipogenic enzymes in the liver of HFD-fed PKCζ-deficient mice might contribute to the fatty liver phenotype observed these mice. However, we did not detect macrophage infiltration in these livers (data not shown). These data demonstrate that the targeted deletion of PKCζ leads to selective WAT inflammation when mice are fed a HFD, which is not observed in WT mice. Therefore, the lack of PKCζ sensitizes the adipose tissue to inflammation driven by HFD intake, which correlates with fatty liver, glucose intolerance, and insulin resistance.
Recent data demonstrate that adipocytes secrete type 2 cytokines, such as IL-4, that skew adipose tissue macrophages (ATM) towards an anti-inflammatory type 2 (also known as M2) profile. In contrast to the pro-inflammatory type 1 profile (also known as M1), the M2 profile limits inflammation in vivo via reduced tissular secretion of pro-inflammatory cytokines (Gordon, 2003
; Kang et al., 2008
; Odegaard et al., 2008
; Vats et al., 2006
). This is important because an increased M1/M2 cytokine ratio has been proposed to be responsible for insulin resistance during obesity (Kang et al., 2008
; Odegaard et al., 2008
). Therefore, we tested the hypothesis that PKCζ deficiency could tilt the balance of adipose tissue polarization towards an M1 pro-inflammatory phenotype that could account for the higher insulin resistance and glucose intolerance observed in the HFD-fed PKCζ-deficient mice. To do this, we analyzed the expression of a series of inflammatory genes in WAT from HFD-fed WT and PKCζ-deficient mice. Interestingly, as shown in , WAT from HFD-fed PKCζ-/-
mice displayed increased levels of the pro-inflammatory cytokines IL-6, TNFα, and IFNγ as compared with WAT from identically treated WT mice. In contrast, levels of the Th2 cytokines, IL-4 and IL-13, were unaltered (). HFD feeding did not affect the adipose tissue cytokine signature of WT mice (). Also, no changes, other than a decrease in IL-4 synthesis were detected when this parameter was analyzed in PKCζ-deficient adipose tissue as compared to WT under RD feeding conditions (). Therefore, HFD feeding leads to a hyperinflammatory state in PKCζ-/-
WAT that might account for the glucose intolerance observed in these mice when fed a HFD. Because IL-4 is secreted by adipocytes and T-cells and targets resident macrophages to drive them into an M2 anti-inflammatory phenotype (Kang et al., 2008
; Odegaard et al., 2008
), our results suggest that PKCζ deficiency does not affect the secretion of IL-4 in HFD-fed mice, but that it might be important for its actions. Interestingly, this inflammatory phenotype is not apparent in liver (data not shown), indicating that the major contribution of PKCζ to obesity-induced inflammation is restricted to its function in adipose tissue. This cytokine profile could be consistent with a skewed M1/M2 balance of ATM toward an M1 type of hyperinflammatory response. To test that possibility, we measured NOS (M1 marker), and Arg1 and Mgl2 (M2 markers) transcript levels in WAT from HFD-fed WT and PKCζ-deficient mice. NOS mRNA levels were significantly increased in WAT from HFD-fed knockout mice, in keeping with the pro-inflammatory phenotype of PKCζ-deficient mice under these conditions (). Although little or no alterations were observed in the levels of Arg1 in WAT from HFD-fed knockout mice, as compared with identically treated WT mice, the levels of Mgl2 were significantly lower (), indicating a reduced M2 phenotype in PKCζ-/-
WAT. As no alterations were observed in IL-4 (), these results indicate that IL-4 actions on the adipose tissue from HFD-fed PKCζ-/-
mice are impaired, giving rise to a reduced M2 phenotype and the subsequent appearance of a pro-inflammatory M1 state.
White adipose tissue expression of inflammatory genes in age-matched wild-type (WT) and PKCζ KO mice (6 of each phenotype) fed either a regular diet (RD) for 5 months or an RD for two months followed by a high-fat diet (HFD) for 3months.
Cell-autonomous role of PKCζ in macrophage inflammatory responses
To investigate whether the role of PKCζ in M2 polarization can be detected in ex vivo cultures of macrophages and is thus cell autonomous, we prepared bone marrow-derived macrophages (BMDM) from WT and PKCζ-/-
mice and analyzed their inflammatory gene signature by QRT-PCR. Importantly, we found that PKCζ-deficient macrophages had reduced levels of Arg1 and Mgl2, whereas the levels of the pro-inflammatory genes NOS, TNFα, IL-6, and IL-1β were increased, as compared with the WT controls (Table S2
). These results could be interpreted to mean that PKCζ-deficient macrophages display an intrinsic and cell-autonomous tendency toward an M1-skewed phenotype. Therefore, we next determined whether the lack of PKCζ would impair the expression of bona fide M2 markers in response to IL-4 in ex vivo macrophage cultures. Figures S2A-S2E
shows that exposure of WT BMDM to IL-4 triggered the expression of PPARγ and the M2 markers Arg1, Mgl2, Mgl1, and Mrc1. This activation was significantly and reproducibly inhibited in PKCζ-/-
macrophages (Figure S2A-S2E
), demonstrating that PKCζ plays a major role in IL-4 induced activation of the M2 phenotype. Moreover, exposure to LPS led to enhanced production of pro-inflammatory TNFα and IL-6 in PKCζ-/-
BMDM as compared to identically treated WT cells (Figure S2F and S2G
), consistent with the concept that PKCζ is necessary for a tuned M1/M2 balance. IL-4 is capable of inhibiting LPS-induced production of inflammatory cytokines (Odegaard et al., 2008
). Interestingly, the ability of IL-4 to inhibit LPS induction of TNFα synthesis was likewise dramatically impaired in PKCζ-/-
BMDM as compared with WT controls (Figure S2H
). Figures S3A-S3E
demonstrate that IL-4 signaling through activation of the Arg1 promoter (a marker of M2 macrophages), as well as the Jak1/Stat6 cascade activated by IL-4, is severely impaired in the macrophage cell line Raw267 in which PKCζ has been depleted by shRNAi, and in PKCζ-deficient BMDMs. Collectively, these results indicate that PKCζ is an important intermediary in the acquisition of an anti-inflammatory M2 phenotype by macrophages, acting at the level of IL-4 signaling. According to the prevailing model, impaired M2 differentiation could account for the pro-inflammatory state detected in PKCζ-/-
adipose tissue (), which could, in turn, account for the insulin-resistant state detected in these mutant mice.
Obesity-induced inflammation and insulin resistance in mice lacking PKCζ in the non-hematopoietic compartment
Because the loss of PKCζ leads to increased production of pro-inflammatory cytokines in the adipose tissue of HFD-fed mice, as well as a cell-autonomous skewing of macrophage phenotype towards a more inflammatory state, we sought to determine whether the loss of PKCζ in the hematopoietic system is sufficient to phenocopy the insulin-resistant and glucose-intolerant phenotype of total PKCζ-deficient mice. Thus, we performed adoptive transfer experiments to generate chimeras with PKCζ specifically deleted in the hematopoietic or non-hematopoietic compartments. We reconstituted lethally irradiated PKCζ-deficient and WT mice with either WT or PKCζ-deficient bone marrow cells to generate PKCζ-/- mice with reconstituted WT bone marrow (PKCζ-/-+WT-BM), and WT mice reconstituted with WT bone marrow (WT+WT-BM), or with PKCζ-/- bone marrow (WT+PKCζ-/--BM) (). Flow cytometry showed that white blood cells were efficiently reconstituted and displayed the donor genotype after bone marrow transplantation (). To investigate reconstitution of resident macrophages, we used PCR to genotype different tissues, including BMDMs, 23 weeks after reconstitution. Genotyping of BMDM and parenchymal cells indicated that BMDM cells were almost fully derived from the donor bone marrow, whereas the parenchymal cells were those of the recipient ().
Impaired glucose tolerance in mice with PKCζ-/- deficiency in the non-hematopoietic compartment
The chimeras were fed a HFD for 20 weeks starting at 3 weeks after transplantation, after which the three chimeric lines displayed comparable body weights (). We next performed GTT in these mice and found that the lack of PKCζ in the non-hematopoietic compartment, but not in the hematopoietic compartment, was sufficient to induce glucose intolerance upon HFD feeding (). These were unexpected observations demonstrating that PKCζ deficiency does not need to be manifested in the hematopoietic system to promote glucose intolerance in HFD-fed mice, despite the cell-autonomous role of PKCζ in M2 inflammation in macrophages and the inflammatory response detected in PKCζ-deficient WAT from HFD-fed PKCζ knockout mice (Table S2 and Figures S2 and S3
). When ITTs were performed with these chimeras, it was apparent that the lack of PKCζ in the hematopoietic compartment was not sufficient to trigger insulin resistance upon HFD feeding, whereas mice with deficiency of PKCζ in the non-hematopoietic compartment displayed a defective insulin response (). Morphological analysis of WAT demonstrated similar adipose tissue volume in the three chimeras (). Interestingly, mice with PKCζ deficiency in the non-hematopoietic compartment, but not in the hematopoietic system, show macrophage infiltration, as determined histologically () and by RT-PCR (). These results indicate that the simple deletion of PKCζ in the non-hematopoietic system is sufficient to induce WAT inflammation. Consistent with this, these mice also had fatty liver (). Of note, deletion of PKCζ just in the hematopoietic compartment not only did not favor fatty liver induction upon HFD feeding, but even slightly reduced HFD-induced fatty liver ().
To identify the underlying cause of this intriguing observation, we determined the cytokine inflammatory profile of WAT from the HFD-fed PKCζ-/-
chimeras. For this, we analyzed the mRNA levels of different inflammatory genes as described above. Interestingly, WAT from mice with WT stroma and PKCζ-deficient hematopoietic systems (WT+PKCζ-/-
-BM) showed no significant alterations in any of the inflammatory cytokines, although Arg1 levels were reduced and NOS levels were increased (Table S3
). These results can be interpreted to mean that the loss of PKCζ in the hematopoietic compartment is not sufficient to trigger an inflammatory phenotype in WAT in obese mice, which correlates with normal glucose tolerance and insulin responsiveness in these chimeric mice (). The alterations in Arg1 and NOS levels observed in these mice suggest that the loss of PKCζ in the hematopoietic compartment of HFD-fed mice is sufficient to at least partially skew the macrophage linage towards M1, but that this is not enough to induce an inflammatory response that would result in glucose intolerance and insulin resistance.
The lack of PKCζ in the non-hematopoietic compartment (PKCζ-/-
+WT-BM) led to increased IL-6 levels in HFD-fed chimeras with no changes in any of the other cytokines or macrophage differentiation markers (Table S3
). This indicates that PKCζ in the non-hematopoietic compartment exerts an anti-inflammatory role in WAT during HFD-induced obesity. This increase in IL-6 mRNA levels in the adipose tissue correlated with significantly enhanced levels of circulating IL-6 in the chimeras with PKCζ deleted in the non-hematopoietic compartment (). As the chimeric mice lacking PKCζ in the non-hematopoietic compartment are glucose intolerant, these results suggest that IL-6 overproduction due to the lack of PKCζ in the non-hematopoietic compartment may be the conducive agent in the glucose intolerance manifested in these HFD-fed mice, and probably the only cytokine required to induce glucose intolerance in obese PKCζ-deficient mice.
IL-6 overproduction in HFD-fed PKCζ-deficient mice is necessary for glucose intolerance
Our data show that WAT from HFD-fed PKCζ-deficient mice have higher IL-6 mRNA levels than identically treated WT mice (). Consistent with this, PKCζ-/- mice fed a HFD, but not a RD, displayed increased levels of circulating IL-6 (). To address the potential role of PKCζ-controlled IL-6 production in glucose intolerance during obesity, we crossed PKCζ-/- mice with IL-6-/- mice to generate a double-knockout mouse line. These mice were fed a HFD, as above, and GTTs and ITTs were performed. Although IL-6-/- mice showed reduced body weight and a lower percentage change in fat weight (), WT, PKCζ-/-, and double-knockout mice displayed very similar values for both parameters (). Interestingly, IL-6 ablation inhibited the glucose intolerance of PKCζ-deficient mice in the double-knockout setting (). ITT experiments confirm that ablation of IL-6 in the PKCζ-deficient mouse line restores the glucose clearance in response to insulin to normal values (). Therefore, the enhanced IL-6 levels in HFD-fed PKCζ-/- mice are necessary and likely sufficient to induce glucose intolerance in obese mice, and that PKCζ is instrumental for the basal repression of IL-6 production by the non-hematopoietic system under conditions of obesity-induced inflammation. Consistent with the role of IL-6 in PKCζ-/--enhanced inflammation during obesity, we found that the loss of IL-6 in PKCζ-deficient WAT completely ablated the recruitment of macrophages (). Interestingly, IL-6 ablation also reduced fatty liver induction by HFD feeding of PKCζ-deficient mice (). These results indicate that IL-6 regulation by PKCζ is a key event in HFD-induced glucose intolerance and insulin resistance.
Restored glucose tolerance in HFD-fed PKCζ/IL-6 double-knockout mice
Cell-autonomous effect of PKCζ on IL-4 repressed inflammation in adipocytes
To determine the mechanism whereby PKCζ regulates IL-6 production in the adipose tissue, we exposed WT and PKCζ-deficient primary adipocytes to lipopolysaccharide (LPS) in the presence or absence of IL-4, after which IL-6 levels were determined by RT-PCR. It is well established that IL-4 inhibits inflammatory functions, which might be the basis for the induction of the type 2 anti-inflammatory state during obesity (Kang et al., 2008
; Odegaard et al., 2008
). Results in demonstrate that the ability of IL-4 to inhibit LPS-induced IL-6 production was severely impaired in PKCζ-deficient cell cultures. These results are in keeping with a model in which PKCζ is required for IL-4 signaling in adipocytes to dampen proinflammatory activation during obesity. Interestingly, like in macrophages (Figure S3
), the loss of PKCζ in adipocytes results in the inhibition of Stat6 activation by IL-4 (). This negative role of PKCζ in the synthesis of IL-6 was not only detected in primary adipocyte cultures activated by IL-4, but also in fat pads (kept in culture for 3 days) from HFD-fed PKCζ-deficient mice. Thus, as shown in , IL-6 secretion was significantly higher in cultures of epididymal and retroperitoneal fat from HFD-fed PKCζ-/-
mice than in those of WT fat. IL-6-/-
and double knockout fat pads produced no IL-6, as expected (). To further demonstrate that this is due to the lack of PKCζ, we cultured 3T3-L1 fully differentiated adipocytes, controls or cells with PKCζ levels reduced by siRNA for 3 days, after which the levels of IL-6 secretion were determined. As shown in , depletion of PKCζ levels resulted in increased production of IL-6. On the other hand, it has recently been shown that fatty acids, which are abundant lipids in adipocytes, can activate inflammation in these cells (Shi et al., 2006
). The results in demonstrate that PKCζ-deficient primary adipocytes secrete more IL-6 than their WT controls when exposed in culture to myristic or palmitic acid. Collectively, these results demonstrate that PKCζ exerts a negative role on IL-6 secretion by adipose tissue and isolated adipocyte cultures. Results in show that PKCζ-/-
precursors differentiate into adipocytes more efficiently than do WT controls. The reasons for these observations still need to be explored, as well as the potential role of increased lipid accumulation in PKCζ-/-
adipocytes in the enhanced secretion of IL-6. Future studies will be designed to address these questions. Interestingly, PKCζ levels were severely reduced in WAT from HFD-fed mice (). This strongly suggests that obesity leads to reduction of PKCζ levels, which results in increased IL-6 production and the subsequent enhancement in fatty liver induction, glucose intolerance, and insulin resistance. In keeping with this notion, WAT from obese Ob/Ob mice also displayed reduced levels of PKCζ ().
Role of PKCζ in IL-6 production in adipocytes