PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans (original) (raw)
Increased expression of PKCδ in liver is a feature of pro-diabetic mice. Compared with 129 mice, B6 mice develop more severe insulin resistance when subjected to either a genetic defect in insulin signaling or environmentally induced insulin resistance following HFD feeding. Genome-wide association analysis reveals several regions linked to increased risk of insulin resistance in the B6 mouse, the strongest of which is around the Prkcd locus on chromosome 14 (6). This genetic background difference was associated with increased expression of PKCδ in B6 mice. Thus, on a normal chow diet, 24-week-old B6 mice had approximately 60% higher levels of Prkcd mRNA in liver than age- and diet-matched 129 mice (1.1 ± 0.1 vs. 0.71 ± 0.04, P < 0.02) (Figure 1A). Moreover, when B6 and 129 mice were placed on a HFD from 6 to 24 weeks of age, hepatic Prkcd expression further increased by 2-fold in B6 mice, but did not increase in 129 mice, leading to 3-fold higher expression of Prkcd in the B6 versus 129 mice (Figure 1A). These differences in mRNA expression led to similar differences in PKCδ expression at the protein level, as assessed by Western blot analysis of liver extracts, which in turn were paralleled by a commensurate increase in PKCδ with phosphorylation on threonine 505 (Figure 1B). This difference in Prkcd expression was also observed at 6 weeks of age (Figure 1C), when 129 mice actually weighed slightly more than the B6 mice (22.1 ± 0.5 vs. 19.8 ± 0.3 g, P < 0.001) and had identical glucose and insulin levels; and more importantly, it was also observed at both mRNA and protein levels in livers of newborn B6 versus 129 mice (Figure 1, C and D). The results suggest that there is a genetically driven differential expression of PKCδ between the insulin resistance–prone B6 and insulin resistance–resistant 129 mice.
Differential hepatic PKCδ expression between diabetes-susceptible B6 and diabetes-resistant 129 mice. (A) Hepatic expression of Prkcd mRNA in 24-week-old chow diet– (CD-) versus 18-week HFD-treated B6 and 129 mice. Results are normalized to Tbp (n = 8 per group, *P < 0.02). (B) Western blot analysis of PKCδ protein expression and phosphorylation on Thr505 in liver of 24-week-old CD- versus 18-week HFD-treated B6 and 129 mice. Lanes were run on the same gel but were noncontiguous. (C) Prkcd mRNA expression in liver of 6-week-old (n = 5 per group, *P < 0.001) and newborn (n = 7 per group, †P < 0.004) B6 and 129 mice. (D) Western blot analysis of PKCδ protein expression in liver of newborn B6 and 129 mice. (E) Schematic of Prkcd, Tkt, Cphx, and Chdh genes localization on mouse chromosome 14. (F) Copy number variation for Prkcd, Tkt, and Cphx genes between B6 and 129 mice lines (n = 6 per group). Results were normalized to Chdh (*P < 0.00003).
Recent studies have revealed that one cause of the differences in gene expression between mouse strains is duplication or deletion of segments of DNA resulting in variation in gene copy number. Copy number variations have been identified in chromosome 14 near the Prkcd locus between commonly used mouse lines, including B6 and 129 (13). To explore whether this could account for the differences in expression of PKCδ, we assessed the relative copy number of the Prkcd gene and the nearby Tkt gene, as well as Cphx and Chdh genes located upstream and downstream of Prkcd on chromosome 14 (Figure 1E). Since the Chdh gene is known not to exhibit copy variations, the results for other genes could be normalized to Chdh to assess copy number variation. Consistent with previous reports indicating duplication in this region (13), we observed a 2-fold increase in copy number for the Cphx gene in B6 mice compared with 129 mice (P < 0.0001). However, genomic PCR revealed no differences on chromosome 14 for Prkcd gene or the nearby Tkt gene (Figure 1F).
PKCδ has a unique expression profile among all the PKC family members. Since several members of the PKC family have previously been implicated in the regulation of insulin signaling in various tissues and cell models (14), we decided to perform a systematic gene expression analysis of all PKCs in order to establish whether any other PKC isoforms exhibited the unique hepatic expression pattern of PKCδ, i.e., a pattern characterized by (a) an inherited differential expression between B6 and 129 mice, (b) an induction of hepatic expression by HFD in the insulin resistance–prone B6 mice, (c) an absence of induction by HFD in insulin resistance–resistant 129 mice, and (d) a physical location near an SNP marker associated with the hyperinsulinemia observed in B6 versus 129 mice. Hepatic mRNA levels of conventional PKCs (α, β, and γ), novel PKCs (δ, ε, η, and θ) and atypical PKCs (ι/λ, ζ, and Ν1) were determined by quantitative real-time PCR (qPCR).
In 6-month-old chow-fed animals, B6 mice had significantly higher mRNA expression levels for PKCβ, -γ, -δ, -ε, -η, and -ι as compared with 129 mice (Figure 2A). Three of these PKCs (β, δ, η), as well as two others (θ and Ν1), were expressed at significantly greater levels in liver of B6 newborn mice compared with liver of 129 newborn mice, with PKCδ being the most significantly different (Figure 2B). Following HFD feeding, hepatic expression levels of PKCα, -γ, -δ, -ε, -η, -δ, and Ν1 were significantly increased in B6, but not 129, mice (Figure 2A). Interestingly, in insulin-resistant, genetically obese ob/ob mice, a slightly different set of PKCs including PKCα, -β, -δ, -η, and -ι/λ was induced in liver, clearly demonstrating differential regulation of expression among PKC family members in different states of obesity (Figure 2C). More importantly, in the HFD-treated 129 mice, we still observed a significant increase in PKCγ and -η hepatic expression, whereas PKCδ did not exhibit any increase in its hepatic expression in response to high-fat feeding (Figure 2A). Finally, we analyzed the chromosomal locations of all PKCs in the mouse genome and compared them to the chromosomal locations of previously identified SNP markers for QTLs linked to insulin resistance or obesity identified by in our genome-wide scan of an F2 intercross between B6 and 129 mice subjected to HFD feeding (Table 1). Of the 10 chromosomal locations of the different PKC isoforms, only PKCδ was physically associated, i.e., within 2 Mb of an SNP marker (D14Mit52) that has been linked to hyperinsulinemia observed in B6 mice (6). Thus, among all the PKC family members, only Prkcd exhibited the unique characteristics that defined it as one of the major genes that contributed to the differential susceptibility to insulin resistance between B6 and 129 mice.
General expression analysis of PKC mRNA in liver of diabetic or diabetes-resistant mouse models. Expression of all PKCs was measured by qPCR in liver of (A) 24-week-old CD versus 18-week HFD-treated B6 and 129 mice (n = 8 per group, #P < 0.05, B6 CD compared with 129 CD; *P < 0.05, CD compared with HFD); and (B) newborn B6 and 129 mice (n = 7 per group, *P < 0.05, **P < 0.01, ***P < 0.001). (C) ob/+ versus ob/ob mice (n = 9–15 per group, *P < 0.05, **P < 0.01, ***P < 0.001). Results are normalized to Tbp.
Chromosomal location of PKC genes and GWAS SNP markers previously associated with differential hyperinsulinemia or hyperleptinemia between B6 and 129 mice
Hepatic PKCδ expression is influenced by obesity in mice and humans as well as genetic background between B6 and 129 mice. Although the difference in PKCδ expression could be observed between B6 and 129 mice prior to the onset of obesity, other states of obesity and insulin resistance are also associated with regulation of PKCδ. Thus, Prkcd expression in liver was also increased in ob/ob mice versus controls (1.0 ± 0.2 vs. 1.7 ± 0.2, P < 0.05) (Figure 2C), and this was not reversed by short-term infusion of insulin and/or leptin (Supplemental Figure 2A; supplemental material available online with this article; doi:10.1172/JCI46045DS1).
To study the possible association of hepatic PKCδ levels in humans with obesity and metabolic syndrome, we performed qPCR on mRNA taken from liver samples of 6 lean, 7 obese nondiabetic, and 11 obese type 2 diabetic subjects studied at the Joslin Diabetes Center (15). The clinical data on these groups are presented in Supplemental Table 1. In addition, BMI was greater than 2-fold higher in obese subjects, and subjects with obesity also had fasting hyperinsulinemia and hypertriglyceridemia. By definition, patients with diabetes were also hyperglycemic. qPCR analysis of the liver samples revealed a more than 2-fold increase in PRKCD mRNA levels in livers of obese nondiabetic subjects, as well as a tendency toward increased PRKCD in livers of obese diabetic patients when compared with lean subjects (7.1 ± 2.3 vs. 16.6 ± 3.5 and 11.9 ± 2.0, P < 0.04) (Figure 3A). While there was variability among the samples from obese diabetic patients, most likely related to heterogeneity in diabetes treatment and the poor glycemic control in some individuals, when we considered all diabetic and nondiabetic obese subjects, the increase in PRKCD expression was significant (7.1 ± 2.6 vs. 13.7 ± 1.8, P < 0.04). Repeat gene expression analysis for PRKCD and all other PKCs in the lean versus obese samples confirmed the increase in PRKCD (6.8 ± 0.7 vs. 12 ± 1, P < 0.005) and revealed a significant increase in PRKCE expression (5.6 ± 0.6 vs. 10 ± 1, P < 0.03) in livers of obese nondiabetic patients compared with lean controls (Figure 3B). More importantly, there was a significant correlation between BMI and hepatic PRKCD expression in human subjects, further linking obese states to high PRKCD expression levels in liver (P < 0.035) (Figure 3B). Expression of PRKCB, PRKCH, and PRKCI/L also showed trends toward increases, but these did not reach statistical significance (P = 0.06), possibly due to the small sample size (Figure 3B). To further explore this relationship, we compared mRNA levels for PRKCD and other PKCs using liver samples from 96 obese patients included in the study by Pihlajamaki et al. (16). These levels correlated with multiple physiological parameters including fasting insulin, glucose, age, circulating triglycerides, and homeostatic model assessment of insulin resistance (HOMA-IR) status. While there was a good deal of scatter in the data, there were positive correlations between PRKCD and fasting glycemia (ρ = 0.262, P = 0.01) (Figure 3D), as well as circulating triglycerides levels (ρ = 0.207, P < 0.05) (Figure 3E). None of the other novel PKCs (ε, η, and θ) showed any correlations with these parameters (Table 2).
Hepatic expression of PKCδ is increased in obese subjects and correlates with fasting glucose and circulating triglycerides. Liver mRNA expression was measured by qPCR for (A) PRKCD in lean versus obese or obese diabetic subjects (n = 6–8–11 per group, *P < 0.04); and (B) all PKC isozymes in lean versus obese subjects (*P < 0.05). Correlations between hepatic PRKCD expression and (C) BMI, (D) fasting glucose, and (E) circulating triglycerides in human subjects.
Spearman rank correlations between novel PKCs and characteristics of study subjects
Since obesity is associated with increased inflammation in fat, and B6 and 129 mice differ in the level of basal inflammation (17), we tested whether injection of bacterial LPS, which triggers a massive, generalized inflammatory response, would lead to changes in the expression of PKCδ. While B6 mice again had higher basal levels of Prkcd expression than 129 mice, no significant increase in Prkcd was observed in response to LPS treatment in either strain of mice (Supplemental Figure 3A). ER stress has been shown to be associated with insulin resistance (18), and several studies have suggested a possible association between ER stress and PKCδ (19, 20). However, induction of ER stress by tunicamycin injection also produced no change in PKCδ expression, despite a clear increase in expression of the chaperone protein Bip (Supplemental Figure 3B). Finally, neither hyperglycemia nor insulin itself appeared to directly regulate PKCδ expression, since mice with streptozotocin-induced (STZ-induced), insulin-deficient diabetes showed no alteration in Prkcd expression either in the untreated state or after treatment with insulin or phlorizin (PHZ) to normalize their blood glucose levels (Supplemental Figure 3, C and D). Thus, the genetically programmed difference between B6 and 129 mouse strains appears to be independent of levels of hyperglycemia, insulin, or leptin and independent of the states of inflammation or ER stress that may occur to different levels in these strains when subjected to HFD-induced obesity.
PKCδ-knockout mice display improved glucose tolerance and insulin sensitivity. To determine how the differences in PKCδ expression could contribute to the differences in insulin sensitivity and metabolic phenotype of B6 versus 129 mice, we studied three different mice models with alterations in PKCδ expression: (a) mice with global inactivation of the Prkcd gene (PKCδKO) created by gene targeting; (b) mice with liver-specific inactivation of the Prkcd gene created using the Cre-lox system of conditional recombination; and (c) mice with overexpression of PKCδ in the liver achieved using adenovirus-mediated gene transfer. For our study, the PKCδKO mice previously described (21) were backcrossed onto a B6 genetic background for 14 generations. Although PKCδKO deficiency led to high rates of mortality when established on the B6 background, surviving PKCδ-deficient mice appeared generally healthy. On a regular chow diet at 20 weeks of age, PKCδKO mice had slightly lower body weights than their WT littermates (30 ± 0.7 and 35 ± 1 g, P < 0.05) (Figure 4A). Intraperitoneal glucose tolerance testing (GTT) revealed that PKCδKO mice had significantly better glucose tolerance than their control littermates (Figure 4B), with a 24% decrease in the AUC for glucose levels following the glucose challenge (Supplemental Figure 1A). Hyperinsulinemic-euglycemic clamp studies confirmed the increased whole body insulin sensitivity, with a striking 7-fold increase in the glucose infusion rates in PKCδKO compared with WT mice (25 ± 6 vs. 3.5 ± 0.5 mg/kg/min, P < 0.05) (Figure 4C), suggesting increased glucose uptake into muscle and/or fat. This increase in peripheral insulin sensitivity was accompanied by increased insulin sensitivity of the liver, with an increased ability of insulin to suppress hepatic glucose production (HGP). Thus, in the basal state, WT and PKCδKO mice had similar levels of HGP, but during the insulin clamp, PKCδKO displayed an 83% ± 6.9% inhibition of HGP versus a 63% ± 8.9% inhibition for the WT mice (Figure 4D). This difference in the ability of insulin to suppress gluconeogenesis was due to differences in insulin’s ability to regulate gluconeogenic gene expression. Thus, the PKCδKO mice had a 33% decrease in phosphoenolpyruvate carboxykinase (Pepck), a 62% decrease in glucose-6-phospatase (G6P; also known as G6pc), and a 48% decrease fructose-1,6-bisphosphatase (F1,6BP; Fbp1) compared with controls (Figure 4G). PKCδ-null mice also had decreased expression of the key lipogenic transcription factors SREBP1c (Srebf1) (70%) and chREBP (Mlxipl) (79%), and this was associated with a major reduction in the expression of their transcriptional targets including the lipogenic enzymes fatty acid synthase (FAS; Fasn) (89%), acetyl-coA-carboxylase (ACC; Acaca) (88%), and stearoyl-CoA desaturase 1 (Scd1) (88%). Glucokinase (Gck), another transcriptional target of SREBP1c, also showed a major reduction (68%) in expression in PKCδ-deficient livers. This reduction in expression of lipogenic enzymes was accompanied by protection against age-related hepatosteatosis, as shown in histological analysis of liver sections from 12-month-old PKCδKO versus control mice (Figure 4E) and a 50% decrease in hepatic triglyceride content (Figure 4F).
PKCδKO mice display improved glucose tolerance and hepatic insulin sensitivity. (A) Body weight of WT versus PKCδKO mice (n = 8 per group, *P < 0.02). (B) GTT of WT versus PKCδKO mice (n = 5 per group, *P < 0.03). (C) Glucose infusion rate (GIR) and (D) HGP during euglycemic-hyperinsulinemic clamps of WT versus PKCδKO mice (n = 4 per group, *P < 0.05). (E) H&E-stained sections of liver from 1-year-old WT and PKCδ KO mice (original magnification, ×200). (F) Triglyceride levels in liver of WT and PKCδKO mice (n = 4 per group, *P < 0.05). (G) qPCR analysis of mRNA for gluconeogenic and lipogenic genes in liver at the end of the euglycemic-hyperinsulinemic clamp in control versus PKCδKO mice (n = 4 per group, *P < 0.04). (H) Western blot analysis of proteins in the insulin signaling pathway in liver at the end of the euglycemic-hyperinsulinemic clamp. Each lane represents an individual animal.
PKCδ ablation also improved hepatic insulin signaling as assessed by Western blot analysis of liver extracts from mice taken during the hyperinsulinemic-euglycemic clamp. As shown in Figure 4H, PKCδKO mice had increased insulin stimulation of Akt phosphorylation and phosphorylation of the p42 and p44 MAPKs. PKCδKO mice also had increased insulin-stimulated phosphorylation of p70S6 kinase (p70S6K) on Thr421/Ser424, a site known to inhibit p70S6K activity (22, 23). As a consequence, there was a decrease in the phosphorylation of IRS-1 on Ser307 to almost undetectable levels in PKCδKO mice (Figure 4H). Ser307 is a known site of IRS-1 phosphorylation by p70S6K phosphorylation, which leads to inhibition of insulin action (23). Taken together, these results demonstrate that whole body deletion of PKCδ improves insulin sensitivity in both liver and peripheral tissues and improves insulin action at the level of the liver.
Liver-specific overexpression of PKCδ leads to metabolic syndrome symptoms. To further define the role of increased PKCδ in the liver, we created B6 mice in which we induced overexpression of PKCδ in liver by adenoviral delivery of PKCδ cDNA. This resulted in an 8-fold increase in the levels of PKCδ in liver as determined by Western blot analysis. On intraperitoneal glucose challenge, mice overexpressing PKCδ were glucose intolerant in comparison with GFP-overexpressing control mice created in parallel, with a 33% increase in AUC of the GTT (Figure 5A and Supplemental Figure 1B). Interestingly, when we overexpressed PKCδ in liver of 129 mice, we also observed a subsequent diminution of their glucose tolerance, which became more similar to that of the glucose-intolerant B6 control mice (Supplemental Figure 2B). In contrast to the decreased gluconeogenesis in PKCδKO mice, mice with increased levels of hepatic PKCδ had increased gluconeogenesis as demonstrated by an exaggerated response to a pyruvate tolerance test (PTT), with a 38% increase in AUC compared with control mice (Figure 5B and Supplemental Figure 1D). This also resulted in an approximately 20% increase in plasma glucose in both the fasted and fed states (compare 0 time points in Figure 4, A–C, respectively). Although basal glucose levels were increased, there was no difference in the insulin-mediated reduction of glucose levels during an intraperitoneal insulin tolerance test (ITT), suggesting that there was no difference in insulin sensitivity in muscle between the mice overexpressing PKCδ in liver and the controls (Figure 5C).
Mice with liver-specific overexpression of PKCδ develop features of the metabolic syndrome. (A) GTT in mice overexpressing GFP or PKCδ in the liver (n = 13 per group). (B) PTT in mice overexpressing GFP or PKCδ in the liver (n = 9 per group). (C) ITT in mice overexpressing GFP or PKCδ in the liver (n = 19 per group). (D) Serum insulin and cholesterol levels in mice overexpressing GFP or PKCδ in the liver. (E) Histological pictures of H&E-stained liver sections from mice overexpressing GFP or PKCδ in the liver (original magnification, ×200). (F) Triglyceride levels in blood and liver of 2-hour-fasted mice overexpressing GFP or PKCδ in the liver (n = 8 per group, *P < 0.01).
In addition to its effect on glucose metabolism, overexpression of PKCδ in liver resulted in a significant increase in hepatosteatosis, which was easily observed in H&E-stained histological sections of liver (Figure 5E). This correlated with a 33% increase in hepatic triglyceride content in mice overexpressing PKCδ compared with controls (Figure 5F). In 2-hour-fasted animals, overexpression of PKCδ in the liver also resulted in mild hyperglycemia (85 ± 3.6 vs. 69 ± 3.4 mg/dl in controls), hyperinsulinemia (0.33 ± 0.04 vs. 0.18 ± 0.03 ng/ml), and hypertriglyceridemia (101 ± 3 vs. 66 ± 4 mg/dl) (Figure 5, D–F). Thus, overexpression of PKCδ in liver reproduced the differential glucose tolerance differentiating B6 from 129 mice as well as many of the signs of the metabolic syndrome, even in lean mice on regular chow diet.
PKCδ overexpression alters hepatic insulin signaling. The physiological alterations induced by PKCδ overexpression were associated with hepatic insulin resistance at the level of the insulin signaling pathway. While PKCδ overexpression did not alter tyrosine phosphorylation of the insulin receptor itself (data not shown), overexpression of PKCδ resulted in a significant decrease in insulin-stimulated phosphorylation of IRS-1 on Tyr612 and decreased phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) on Ser473 and Ser9, respectively. As opposed to what had been observed in PKCδKO mice, p70S6K phosphorylation on Thr389 in response to insulin was greatly enhanced by PKCδ overexpression, and this correlated with the increased phosphorylation of IRS-1 on Ser307 (Figure 6A). As a consequence of the repression of insulin signaling, Foxo1 nuclear localization was enhanced about 2-fold in livers of mice overexpressing PKCδ, as demonstrated by Western blotting of nuclear extracts (Figure 6D). Consistent with the role of Foxo1 as a regulator of hepatic gluconeogenesis (24), mice overexpressing PKCδ had 1.2- to 2.5-fold increases in the levels of mRNA for the gluconeogenic enzymes Pepck, G6pc, and Fbp1 (Figure 6B).
High hepatic PKCδ expression levels lead to hepatic insulin resistance. (A) Western blot analysis of insulin signaling pathway in liver of mice overexpressing GFP or PKCδ in the liver 5 minutes after intraperitoneal injection of insulin or vehicle as described in Methods. (B) qPCR analysis in liver overexpressing GFP or PKCδ of lipogenic gene expression (n = 6 per group, **P < 0.01) and gluconeogenic gene expression (n = 6 per group, *P < 0.05). Results are normalized to Tbp. (C) Western blot analysis of PKCδ and SREBP1c expression in liver overexpressing GFP or PKCδ. (D) Western blot analysis of Foxo1 and lamin A/C expression in nuclear extracts of liver overexpressing GFP or PKCδ. Lanes in D were run on the same gel but were noncontiguous.
Overexpression of PKCδ also increased the levels of SREBP1c 2-fold at both mRNA and protein levels (Figure 6, B and C), as well as Mlxipl 2-fold at the mRNA level. This was accompanied by a 2-fold increase in the expression of the three key lipogenic enzymes Fasn, Acaca, and Scd1, as well as Gck, all transcriptional targets of Srebf1 (Figure 6B). Thus, increasing the expression of PKCδ by only a few fold was sufficient to induce a significant liver insulin resistance and stimulate basal hepatic lipogenesis.
Liver-specific reduction of PKCδ restores glucose tolerance and insulin signaling in HFD-induced diabetic animals. To determine whether decreasing PKCδ specifically in the liver could improve the hepatic insulin sensitivity in obese diabetic mice, we created a liver-specific PKCδ-knockout mouse. To this end, exon 2 of the Prkcd gene was flanked with two loxP sites and introduced in mice by homologous recombination. Loss of exon 2 of the floxed Prkcd gene results in a deletion and frameshifts the PKCδ transcript, leading to the production of a truncated, inactive PKCδ protein (Supplemental Figure 2C). Intravenous administration of an adenoviral vector containing a Cre recombinase expression cassette resulted in recombination of the Prkcd transgene in liver. At the dose of adenovirus used, 5 days after injection, there was a reproducible 40% reduction in PKCδ mRNA and protein compared with control mice injected with empty adenoviral vector (Figure 7A).
Liver-specific reduction of PKCδ expression improves HFD-induced hepatic insulin resistance. (A) Validation of effective DNA recombination by PCR analysis of genomic DNA, qPCR measurement of Prkcd mRNA expression (n = 6 per group, *P < 0.005), and Western blot analysis of PKCδ protein in livers of PKCδ-floxed mice after administration of empty or Cre recombinase–expressing adenovirus. (B) GTT of PKCδ-floxed mice injected with empty (white squares) or Cre recombinase–expressing adenovirus (black circles) following 10 weeks of CD (dashed lines) or HFD (plain lines) feeding (n = 6 per group). (C) Western blot analysis of insulin signaling pathway in liver of PKCδ-floxed mice injected with empty or Cre recombinase–expressing adenovirus following 10 weeks of CD or HFD feeding and 5 minutes after injection of insulin or vehicle as described in Methods. (D) qPCR analysis in liver overexpressing GFP or PKCδ of lipogenic and gluconeogenic gene expression in liver of PKCδ-floxed mice injected with empty or Cre recombinase–expressing adenovirus following 10 weeks of HFD (n = 14 per group, *P < 0.05). Results are normalized to Tbp.
To assess the consequences of reduced levels of PKCδ on hepatic insulin resistance, we injected empty or Cre recombinase–expressing adenoviruses into 3-month-old PKCδ-floxed mice that had previously been subjected to 10 weeks of either a HFD (60% fat by calories) or a regular chow diet (23% fat by calories). Five days after adenovirus injection, there were no differences in body weight between floxed mice and mice with liver-specific knockdown of PKCδ on either the chow diet or HFD (data not shown). As expected, however, mice that had been on HFD for 10 weeks were glucose intolerant compared with the chow diet–fed control animals. Furthermore, while the modest reduction of PKCδ had no effects on glucose tolerance in lean mice, liver-specific reduction of PKCδ in the HFD obese mice was sufficient to significantly improve the glucose tolerance as compared with the obese empty adenovirus–treated controls, as demonstrated by a significant 20%–25% reduction in the AUC for glucose (Figure 7B and Supplemental Figure 1E).
This improvement in metabolism was secondary to an improvement in insulin signaling. Thus, in the HFD obese control mice, insulin signaling was markedly blunted, with no detectable increase in Akt phosphorylation following insulin stimulation (Figure 7C). This was due to high levels of inhibitory phosphorylation of IRS-1 on Ser307, which was secondary to high levels of p70S6K activation as indicated by p70S6K phosphorylation. Reducing PKCδ reversed many of these changes. Thus, there was a reversal of p70S6K phosphorylation/activation, leading to a reduction in IRS-1 Ser307 phosphorylation levels and a rescue of Akt/GSK3 phosphorylation/activation by insulin (Figure 7C). Parallel to this improvement in insulin signaling, we also observed reduction in expression of gluconeogenic genes such as G6pc and Fbp1 (Figure 7D). Despite the improvement in glucose tolerance, however, this short-term reduction in PKCδ did not improve the hepatosteatosis present in HFD obese mice as assessed by histological examination of liver sections (Supplemental Figure 2E) and measurement of liver triglyceride content (Supplemental Figure 2F). Likewise, there was no change in gene expression levels for the lipogenic genes Srebf1, Mlxipl, Fasn, Acaca, or Scd1 (Figure 7D and data not shown). Finally, we did not observe any modulation of the hyperinsulinemia, hypertriglyceridemia, or the high levels of free fatty acids induced by HFD (Supplemental Figure 2D). Therefore, a short-term reduction of hepatic PKCδ was sufficient to rescue glucose tolerance and insulin signaling in the liver of HFD-fed mice, but not sufficient to reverse the secondary hyperinsulinemia or the lipid abnormalities in these mice.