Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity

Animals

All mice in this study were on a 129Sv-C57Bl/6 mixed genetic background. To create the liver double knock-out mice, p110α lox-lox mice⁷ were crossed with p85α lox-lox mice, hemizygous for the Albumin-Cre recombinase transgene¹⁴. Mice were housed on a 12-hour light cycle and fed a standard rodent chow and water ad libitum. All protocols for animal use and euthanasia were approved by the Gothenburg Ethical Committee on Animal Experiments, in accordance with Swedish guidelines and Directive 2010/63/EU for animal experiments, and by the Animal Care Use Committee of the Joslin Diabetes Center and Harvard Medical School in accordance with National Institutes of Health guidelines. All efforts were made to ameliorate any suffering of the mice by reducing stress, hosting mice in small groups with items that stimulate their natural activity, and allowing the mice to recover 1–2 weeks after each procedure (such as measuring blood glucose, glucose tolerance test etc). During the insulin tolerance test, the mice were monitored closely to not fall too low in blood glucose levels.

For each experiment, a group of 5–12 male mice per genotype were used. The mice were studied from 6 weeks of age to 25 weeks of age. During this time weight and fasting blood glucose levels were measured every two weeks. Glucose tolerance test was performed at 8 weeks, 16 weeks and 24 weeks. Pyruvate tolerance test was performed at 15 weeks and insulin tolerance test was performed at 19 weeks.

Metabolic and physiological procedures

Animals were fasted overnight and anesthetized with 2-2-2 tribromoethanol (Sigma-Aldrich, St Louis MO), followed by injection of 5 U of insulin (Actrapid, Novo Nordisk Inc., Plainsboro Township, NJ) or saline via the inferior vena cava. Five minutes after the injection, the liver, muscle and white adipose tissue (WAT) were excised, weighed, and snap-frozen in liquid nitrogen.

Glucose tolerance test was performed by intraperitoneal (i.p.) injection of 2 g glucose/kg BW after an overnight fast. Insulin tolerance test was performed by i.p. injection of 1.25 U insulin/kg BW. Pyruvate tolerance test was performed by i.p. injection of 2 g of pyruvate/kg BW after an over-night fast. Insulin and glucagon was measured with ELISA (Crystal Chem Inc., Downer Grover, IL).

RNA extraction and gene expression analysis

RNA was extracted by homogenization of liver tissue in RLT buffer (Qiagen, Valencia, CA) followed by extraction using the RNeasy kit (Qiagen, Valencia, CA). For gene analysis, cDNA was prepared using a high capacity cDNA archive kit (Applied Biosystems, Foster City, CA) with random hexamer primers. Gene expression was analyzed by real-time reverse transcription-PCR (RT-PCR) on an ABI Prism sequence detection system (Applied Biosystems, Foster City, CA). The cycling conditions used were an initial 95°C 10-minute step followed by 40 cycles of 95°C for 15s and 60°C for 60s. Samples were normalized to the 18S rRNA gene. Primer sequences are available in Table 1.

Protein extraction and analysis

Liver tissue was homogenized in lysis buffer containing 25 mM Tris-HCl, 2 mM Na₃VO4, 10mM Na₄P₂O₇, 1 mM EGTA, 1 mM EDTA, 1% NP-40 and protease inhibitors (Sigma-Aldrich, St Louis MO), then allowed to incubate at 4°C for one hour. Extracts were centrifuged at 55,000 rpm (Beckman 70.1 Ti rotor) for one hour, and the supernatant was stored at -80°C. WAT was homogenized in lysis buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 25 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM orthovanadate, and protease inhibitors (Sigma-Aldrich, St Louis MO) followed by incubation for 2 h at 4°C. The samples were then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and stored at -80°C. Protein analysis was made by SDS-PAGE and subsequent western blot. Briefly, protein samples were loaded onto 4–12% Bis-Tris protein gels (Thermo-Fisher Scientific, Waltham, MA) and subjected to gel electrophoresis using 25mM Tris, 192 mM glycine and 0.1% SDS as running buffer. Samples were transferred onto a nitrocellulose membrane and the membranes were incubated in 5% skim milk solution for 1 h followed by primary antibody incubation according to the manufacturer’s protocol for each antibody. Membranes were washed 2×5 min and 1×15 min in PBS with 0.1% Tween and then incubated with the secondary antibody for 1h followed by another washing procedure. Immunoprecipitation was performed using magnetic beads coated with protein G (Pierce Biotechnology Inc, Rockford, IL). All western blots and immunoprecipitation experiments were performed with a minimum of four replicates (four separate samples).

Antibodies

IRS1 (RRID:AB_2127860, rabbit monoclonal, 1:50, Cell Signaling Technology Inc, cat# 3407 for western blot), IRS1 (RRID:AB_631842, rabbit polyclonal, 10μl per reaction, Santa Cruz Biotechnology Inc, cat# sc-559 for immunoprecipitation), p110α (rabbit monoclonal, 1:1000 for western blot and 1:50 for immunoprecipitation, Cell Signaling Technology Inc, cat# 4249), p110β (RRID:AB_2165246, rabbit monoclonal, 1:500 for western blot and 1:50 for immunoprecipitation, Cell Signaling Technology Inc, cat# 3011), p110γ (RRID:AB_10828316, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 5405), p110δ (mouse monoclonal, 1:500, Becton, Dickinson and Company, cat# 611015), p101 (RRID:AB_10829448, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 5569), Vps34 (RRID:AB_2299765, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4263), p150 (rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 14580), Akt/PKB (RRID:AB_329827, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9272), pS-Akt/PKB (RRID:AB_329825, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9271), pT-Akt/PKB (RRID:AB_2255933, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 2965), pT-p70S6K (RRID:AB_330944, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9205), p70S6K (rabbit polyclonal, 1:500, Cell Signaling Technology Inc, cat# 9202), p85α (RRID:AB_2268174, rabbit monoclonal, 1:1000, Abcam, cat# 22653), p85-pan (RRID:AB_10831521, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4257), pT202/Y204-ERK (RRID:AB_2315112, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4370), ERK (RRID:AB_390779, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4695), p55γ (mouse monoclonal, 1:2000, Abcam, cat# ab186612), PIK3C2α (rabbit polyclonal, 1:1000, MyBiosource, cat# MBS9202698), PIK3C2γ (rabbit polyclonal, 1:1000, MyBiosource, cat# MBS820611). Rabbit secondary antibody (RRID:AB_772206, HRP-linked from donkey, 1:1000, GE Healthcare Life Sciences, cat# NA934), mouse secondary antibody (RRID:AB_772210, HRP-linked from sheep, 1:1000, GE Healthcare Life Sciences, cat# NA931).

In vitro kinase assays

Immunoprecipitates, using protein G Dynabeads (Life Technologies), of liver protein lysates were prepared with p110α and p110β antibodies or IRS1 antibody. The PI3K assay was performed as previously described¹³. Briefly, immunoprecipitates were incubated with 5 μg PI substrate (phosphatidylinositol from bovine liver), 20 mM MgCl ₂, 8 μM cold ATP and 0.5 μl radio-labeled [γ- ³²P]-ATP (1.11*10¹⁴ bq/mmol) in PI3K reaction buffer (20 mM Tris-HCl, 100 mM NaCl and 0.5 mM EGTA) for 25 min at room temperature. The resulting radioactively labeled PIP was analyzed with thin layer chromatography and phosphorimaging (FLA-3000, Fujifilm). Prior to these experiments, titration experiments of bead concentration and PI substrate concentration were performed to ensure precipitation of equal amounts of protein, as well as optimal PI concentration to obtain maximal enzyme activity.

Statistics

All data are presented as mean ± standard error of the mean (SEM). Student's t-test was used for statistical analysis between two unpaired groups. A p-value of <0.05 was considered statistically significant. The statistical software used was GraphPad Prism 7.00.

Deletion of hepatic p110α and p85α results in impaired insulin signaling downstream of PI3K

Mice with a liver-specific deletion of p110α and p85α, termed hereafter liver double knockout (L-DKO) mice, were created by breeding mice carrying homozygous floxed Pik3ca and Pik3r1 alleles^14,26 with transgenic mice carrying the Cre recombinase driven by the albumin promoter (albumin-Cre). Deletion of Pik3ca and Pik3r1 in the liver resulted in markedly reduced gene and protein expression of p110α and p85α (Figures 1A, 1C, 1E), as well as impaired activation of the downstream targets Akt/PKB, with decreased phosphorylation of serine 473 and threonine 308, and p70S6 kinase (Figure 1F–I). p110β gene expression was not affected by the deletion of p110α and p85α (Figure 1B). p85β gene expression was slightly, but significantly, decreased in the L-DKO livers (Figure 1D). As expected, in the floxed control mice, there was an increase in the amount of p110α associated with IRS1 in response to insulin compared to basal conditions, whereas no p110α was associated with IRS1 in the L-DKO mice (Figure 1K and 1L). MAPK signaling, as shown by ERK phosphorylation, was unchanged in the L-DKO mice compared to controls (Figure 1F and 1J).

Figure 1. Gene- and protein-expression of regulatory and catalytic subunits of PI3K and associated insulin signaling mediators.

mRNA expression of (A) Pik3ca, (B) Pik3cb, (C) Pik3r1 and (D) Pik3r2 in livers of flox controls and L-DKO mice. (E) Representative western blot of protein expression of p110α, p110β, p85α, p85-pan (detects both p85 isoforms) and p55γ. Total Akt was used as a loading control. (F, phosphorylated Akt/PKB (Ser 473 and Thr 308), phosphorylated p70S6 kinase, and phosphorylated ERK in livers of flox controls and L-DKO mice. Total Akt, p70S6K and ERK was used as loading controls. (G–J) Quantification measurements of the western blots shown in (F) of pS-Akt, pT-Akt, p-p70S6K and p-ERK respectively. (K) Representative western blot of immunoprecipitation experiments with antibodies for IRS1 and subsequent immunoblotting with antibodies for p110α, p110β, p110δ, p85α, p85-pan (detects both p85 isoforms) and p55γ. The whole-lysate reference sample was from an insulin-treated flox control mouse. (L–P) Quantification measurements of the western blots shown in (K) of p110α, p110β, p85α, p85-pan and p55γ. IP = immunoprecipitation, IB = immunoblot. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight and injection of saline through the vena cava. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 5–8). *, p < 0.05 compared to controls. n.s = non significant. Images containing a vertical line are composites taken from a single original image.

Previous studies have shown that the interaction between the regulatory and catalytic subunits of PI3K to form dimers has a mutual stabilizing effect on both subunits, whereas the monomeric forms are more readily subjected to degradation^19,21,22. We hypothesized that more p85β would bind to IRS1 when p85α was absent, thereby maintaining p110β stabilization. However, only very low levels of p85β protein were detected in the liver of the L-DKO mice, as shown both in assessment of total p85 protein (Figure 1E) and in p85 immunoprecipitates with IRS1 (Figure 1K and 1O), similar to what we have reported earlier in liver-specific p85α knock-out mice¹⁴. The expression of p55γ regulatory isoform protein was not affected by deletion of p110α and p85α (Figure 1E), nor was the amount bound to IRS1 (Figure 1K and 1P). In contrast, total p110β protein expression was decreased in the L-DKO mice compared to controls (Figure 1E), likely due to destabilization of this catalytic isoform in the absence of p85α, supporting an insufficiency for p85β to compensate for the loss of p85α. Interestingly, despite overall decreased protein expression of p110β, similar amounts of p110β were associated with IRS1 in controls and L-DKO mice (Figure 1K and 1M). The third catalytic isoform of class IA PI3Ks, p110δ, has been shown to have a major role in immune cells and the embryonic nervous system, but not in other tissues^10–12. Consistent with this, we found only very small amounts of full length p110δ in whole liver lysates or bound to IRS1 (Figure 1K). However, the antibody picked up a band of about 70 kDa in whole lysates and in IRS1 immunoprecipitates (Figure 1K). The amount of this protein did not appear to be consistently different between controls and L-DKO mice or between basal and insulin-stimulated samples, so is likely non-specific.

IRS1-associated phosphatidylinositol kinase activity is intact in the L-DKO mice

As expected, p110α kinase activity, as assessed by the ability to add the 3’-phosphate group to phosphoinositides in p110α immunoprecipitates, was markedly decreased both in the basal- and insulin-stimulated states of the L-DKO mice compared to controls (Figure 2A). Overall p110β kinase activity was much lower than p110α kinase activity in control mice, consistent with previous studies^7,27, and was not changed in the basal state between controls and L-DKO mice (Figure 2B). However, p110β kinase activity was significantly decreased in the insulin-stimulated state of the L-DKO mice (Figure 2B), even though similar amounts of p110β protein were associated with IRS1 in controls and L-DKO mice (Figure 1K and 1M). Surprisingly, IRS1-associated phosphatidylinositol kinase activity in response to insulin was intact in the L-DKO, despite lack of p110α kinase activity and decreased p110β activity (Figure 2C).

Figure 2. PI3K activity and assessment of the association of IRS1 with class IB-, class II- and class III-members of phosphoinositide 3-kinases.

(A) p110α lipid kinase activity, (B) p110β lipid kinase activity, and (C) IRS1-associated phosphatidylinositol (PI) kinase activity in L-DKO mice and controls were assessed by immunoprecipitation. (D) Representative western blot of immunoprecipitation experiments with antibodies for IRS1 and subsequent immunoblotting with antibodies for p110γ, p101, Vps34, p150, PIK3-C2α and PIK3-C2γ. The whole-lysate reference sample was from an insulin-treated flox control mouse. IP = immunoprecipitation, IB = immunoblot. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight and injection of saline through the vena cava. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 4). *, p < 0.05 compared to controls.

The intact IRS1-associated kinase activity in the L-DKO mice cannot be explained by compensatory upregulation of other classes of PI3K

To explore if other classes of phosphoinositide 3-kinases were responsible for the intact IRS1-associated activity, we investigated the association of IRS1 with class IB-, class II and class III members of phosphoinositide 3-kinases in liver lysates from controls and L-DKO mice. Class IB PI3K consists of the p110γ catalytic subunit and the p101 regulatory subunit. The amount of p101 was low in whole lysates and no p101 was associated with IRS1 in either control mice or L-DKO mice (Figure 2D). p110γ was associated with IRS1, but the amounts were similar between controls and L-DKO mice (Figure 2D). Class III PI3K catalytic subunit Vps34 and regulatory subunit p150 were present in whole lysates, but not associated with IRS1 in either control mice or L-DKO mice (Figure 2D). Class II PI3K consists of only one subunit, but exists in several isoforms. The PI3K-C2α isoform is ubiquitously expressed, whereas PI3K-C2γ has been reported to have a more limited tissue distribution, including hepatocytes^28,29. However, we detected only little or no PI3K-C2α or PI3K-C2γ in whole liver lysates or associated with IRS1 in controls or L-DKO mice (Figure 2D). Thus, other classes of phosphoinositide 3-kinases did not compensate for the loss of p110α and p85α in the L-DKO mice and could not explain the intact IRS1-associated phosphatidylinositol kinase activity in the absence of p110α and p85α.

L-DKO mice have decreased liver weight and WAT weight compared to controls

On standard chow (4% fat content by weight, 12% by calories), L-DKO mice had similar body weights to the flox control mice until 10–12 weeks of age, after which the L-DKO mice showed slower weight gain compared to controls (Figure 3A). At least part of this decrease was due to a decrease in liver weight. Thus, by 10 weeks of age, the ratio of liver weight to body weight in the L-DKO mice was decreased by 13% compared to control mice (Figure 3B), whereas there was no difference in WAT weight or muscle weight (Figures 3C and 3D). At 24 weeks of age, liver weight remained decreased (18%), and there was also a significant decreased WAT weight (22%) for the L-DKO mice compared to controls (Figures 3B and 3C). To assess a possible change in insulin sensitivity in WAT, associated with the decreased WAT mass, we investigated insulin signaling mediators in this tissue. WAT p110α- and p85α expression was similar in controls and L-DKO mice as was Akt/PKB phosphorylation in response to insulin (Figure 3E). There was no difference in insulin-stimulated Akt/PKB-activation at 10w compared to 25w in either controls or L-DKO mice (Figure 3E).

Figure 3. Body weight, tissue weights and adipose insulin signaling.

(A) whole body weight, (B) liver weight, (C) white adipose tissue (WAT) weight and (D) muscle weight of 10 and 24 week old male L-DKO mice and controls. (E) Representative western blot of p110α, p85α and phosphorylated Akt/PKB (Ser 473) in WAT of 10 and 25 week old male flox controls and L-DKO mice. Total Akt/PKB was used as a loading control. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight and injection of saline through the vena cava. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 8–17). *, p < 0.05 compared to controls. w = weeks.

L-DKO mice have impaired glucose tolerance, but normal insulin tolerance and normal blood glucose levels

Despite similar body weight (Figure 3A), L-DKO mice were severely glucose intolerant as early as at 8 weeks of age (Figure 4A), and remained similarly glucose intolerant throughout the 24 week study (Figures 4B and 4C). This was associated with markedly increased fasting insulin levels (Figure 4D). However, somewhat surprisingly, the L-DKO mice showed a normal response to exogenous insulin during an intraperitoneal insulin tolerance test (Figure 4E). Despite the markedly impaired glucose tolerance and marked hyperinsulinemia, fasting and random fed glucose levels in the L-DKO mice remained similar between controls and L-DKO mice (Figures 4F and 4G). Circulating glucagon levels were also similar between L-DKO mice and controls both at 10 weeks of age and 24 weeks of age (Figure 4H).

Figure 4. Glucose homeostasis.

Glucose tolerance test at (A) 8 weeks, (B) 16 weeks and (C) 24 weeks of age; (D) fasting insulin levels at 10 weeks of age; (E) insulin tolerance test at 19 weeks of age; (F) fasting glucose levels; (G) random fed glucose levels; (H) fasting glucagon levels; (I) pyruvate tolerance test at 15 weeks of age; (J–L) hepatic mRNA expression of the gluconeogenic markers glucose 6-phosphatase (G6pc), phosphoenolpyruvate carboxykinase (Pck1) or fructose 1,6-bisphosphatase (Fbp1). L-DKO mice and controls were given 2 g glucose/kg body weight intraperitoneally (i.p.) for the glucose tolerance test or 1.25 U insulin/kg body weight i.p. for the insulin tolerance test or 2 g pyruvate/kg body weight i.p. for the pyruvate tolerance test. Blood glucose was measured at 0, 15, 30, 60, and 120 min. Error bars indicate SEM (n =6–12). *, p < 0.05. The insets in the glucose tolerance test graphs and the pyruvate tolerance test graph show the area under the curve (AUC) with subtracted basal glucose values.

L-DKO mice display normal hepatic glucose production

Increased gluconeogenesis is one of the hallmarks of hepatic insulin resistance in type 2 diabetes. To determine whether the impaired glucose tolerance in L-DKO mice was due to increased hepatic glucose output, we subjected these animals to a challenge with pyruvate, the major gluconeogenic substrate. Over the 120 min period following administration of pyruvate, there was a trend toward increased glucose levels in L-DKO mice compared to controls, but this was not statistically significant (Figure 4I). This was associated with a significant change in hepatic gene expression of the gluconeogenic enzyme glucose 6-phosphatase (G6pc) (Figure 4J), whereas gene expression of the other key mediators of gluconeogenesis, phosphoenolpyruvate carboxykinase (Pck1) and fructose 1,6-bisphosphatase (Fbp1), remained similar between control mice and L-DKO mice (Figures 4K and 4L).