Jejunal proteins secreted by db/db mice or insulin-resistant humans impair the insulin signaling and determine insulin resistance

Abstract

Background: Two recent studies demonstrated that bariatric surgery induced remission of type 2 diabetes very soon after surgery and far too early to be attributed to weight loss. In this study, we sought to explore the mechanism/s of this phenomenon by testing the effects of proteins from the duodenum-jejunum conditioned-medium (CM) of db/db or Swiss mice on glucose uptake in vivo in Swiss mice and in vitro in both Swiss mice soleus and L6 cells. We studied the effect of sera and CM proteins from insulin resistant (IR) and insulin-sensitive subjects on insulin signaling in human myoblasts.

Methodology/principal findings: db/db proteins induced massive IR either in vivo or in vitro, while Swiss proteins did not. In L6 cells, only db/db proteins produced a noticeable increase in basal (473)Ser-Akt phosphorylation, lack of GSK3β inhibition and a reduced basal (389)Thr-p70-S6K1 phosphorylation. Human IR serum markedly increased basal (473)Ser-Akt phosphorylation in a dose-dependent manner. Human CM IR proteins increased by about twofold both basal and insulin-stimulated (473)Ser-Akt. Basal (9)Ser-GSK3β phosphorylation was increased by IR subjects serum with a smaller potentiating effect of insulin.

Conclusions: These findings show that jejunal proteins either from db/db mice or from insulin resistant subjects impair muscle insulin signaling, thus inducing insulin resistance.

Figure 1. Intraperitoneal insulin tolerance test on Swiss mice and effect of db/db and Swiss conditioned medium (CM) proteins on the in vitro glucose uptake.

Panel A: Glucose concentration profiles during the IPITT in controls (n = 23) and mice injected with 15 (n = 10) or 150 µg (n = 11) of db/db CM proteins. Values are mean ± s.e.m. *P<0.05, ⁺ P<0.01, and ^# P<0.001, db/db 15 or 150 µg vs. control. The lines are the fitting curves obtained by the model of Eqs. (1)-(2) in Methods with the parameter values reported in Table 1. Panel B: Glucose concentration profiles during the IPITT (mean ± s.e.m.) in controls (n = 23) and mice injected with 15 (n = 11) or 150 µg (n = 7) of Swiss CM proteins. Panel C: Transport of 2-deoxyglucose (2-DG) in isolated soleus muscle. Values are mean ± s.d. of n = 7 determinations of 2-DG uptake expressed as µmol per ml intracellular water per 20 min. *P<0.02, db/db 10 µg/ml CM proteins vs. control; ⁺ P<0.0005, db/db 20 µg/ml CM proteins vs. control. Panel D: Rate of 2-DG uptake versus insulin concentration in L6 myoblasts. Insulin mediated glucose uptake was determined in cells exposed to 30 µg/ml of db/db or Swiss CM proteins vs. control. Data points are mean ± s.d. of n = 7 determinations for each insulin concentration expressed as pmol per min per well. P<0.0001 for 2-DG uptake reduction with db/db CM proteins vs. control and Swiss CM at all insulin concentrations. Fitting curves are given by Eq. 3 in Methods with the parameter values reported in Table 2.

Figure 2. Effect of conditioned medium (CM) on ⁴⁷³Ser Akt phosphorylation in differentiated L6 myotubes.

Each bar is the mean of experiments in triplicate with 3 to 5 different preparations of CM. Data are normalized by β-actin amount and expressed as fold change versus control condition (control at 100 nM insulin set at 100). Data are mean ± s.d. Panel A: 15 min incubation of L6 myotubes with 25 µg/ml of CM proteins increased ⁴⁷³Ser Akt phosphorylation level in the absence of insulin, with an effect that tended to be stronger of medium prepared from db/db (*P<0.05 vs. control) than from Swiss mice. The stimulatory effect of 100 nM insulin in control (⁺ P<0.01 vs. no insulin) was preserved in the presence of Swiss CM (^# P<0.05) but did not reach significance in the presence of db/db CM. Panel B: Dose-response profile of insulin action on ⁴⁷³Ser Akt phosphorylation vs. insulin concentration (nM) in logarithmic scale after 15 min co-incubation in the absence (squares) or presence (circles) of 25 µg/ml db/db CM proteins (^# P<0.01 and ^* P<0.05 vs. control). Panel C: Basal (no insulin) ⁴⁷³Ser Akt phosphorylation determined after different incubation times with 25 µg/ml db/db CM proteins. An increased ⁴⁷³Ser Akt phosphorylation was already observed after 5 min of incubation and approached saturation at 15–30 min (^* P<0.05 and ^# P<0.01 vs. control). Panel D: Effect of mTORC inhibitors on basal ⁴⁷³Ser Akt phosphorylation. L6 myotubes were treated with Rapamycin or PP242 for 1 hour before 15 min incubation with 25 µg/ml CM proteins. The stimulatory effect of db/db proteins was inhibited by PP242 in both control (^* P<0.05 vs. no inhibitor) and db/db CM treated cells (^# P<0.01 vs. no inhibitor).

Figure 3. Effect of conditioned medium (CM) on p70 S6kinase and GSK3β phosphorylation in differentiated L6 myotubes.

Each bar is the mean of experiments in triplicate with 3 to 5 different preparations of CM. Data are normalized by β-actin amount and expressed as fold change versus control condition (no insulin nor CM) set at 10. Data are mean ± s.d. Panel A: ³⁸⁹Thr p70 S6K1 phosphorylation. L6 myotubes were treated with Rapamycin or PP242 for 1 hour before 15 min incubation with 25 µg/ml of db/db CM. db/db CM proteins inhibited the basal ³⁸⁹Thr p70 S6K1 phosphorylation (⁺ P<0.05 vs. control no inhibitors). PP242 further reduced the phosphorylation level in both control (^* P<0.01, Rapamycin and PP242 vs. no inhibitor) and db/db CM (^# P<0.05 vs. no inhibitor). Panel B: ⁹Ser GSK3β phosphorylation after 15 min co-incubation with 25 µg/ml CM with or without insulin. db/db CM proteins inhibited the insulin-stimulated increase of ⁹Ser GSK3β phosphorylation (^* P<0.05 vs. insulin-induced phosphorylation in control set at 100). Lower molecular weight bands in the blot suggest proteolysis of phosphorylated form of GSK3β upon db/db CM treatment. Panel C: Time course of the effect of 25 µg/ml db/db CM on ⁹Ser GSK3β phosphorylation and proteolysis. The db/db-derived conditioned medium did not significantly affect basal phosphorylation of GSK3β on ⁹Ser, while it induced a rapid degradation of GSK3β, measurable since 5 min of incubation. After 15 min of pretreatment with db/db CM, a washout for 15 min with medium replacement was able to fully correct the alterations induced by the db/db CM. Panel D: Pretreatment of L6 myotubes with Rapamycin or PP242 for 1 hour before incubation with 25 µg/ml CM from db/db for 15 min in the absence or presence of 100 nM insulin. mTOR inhibitors had no effect on proteolysis of phosphorylated GSK3β induced by db/db CM proteins either in the presence or absence of insulin.

Figure 4. Effect of serum from IR subjects (15 min) on Akt and GSK3β phosphorylation in differentiated human myotubes.

Each bar is the mean of the results with sera from 7 IR subjects. Experiments were performed in triplicate for each individual. Ctr denotes controls. Panel A: Basal ⁴⁷³Ser Akt phosphorylation in the presence of 100 nM insulin or of different serum concentrations (5%, 10% and 20%) from IR subjects. Blots are representative western blots of ⁹Ser Akt phosphorylation and total Akt amounts. Data (mean ± s.d.) are expressed as fold change versus control condition (no insulin nor serum) set at 1 (^* P<0.05 vs. control). Panel B: Basal ⁹Ser GSK3β phosphorylation in the presence of 100 nM insulin or of different serum concentrations (5%, 10% and 20%) from IR subjects. The blot is a representative western blots of ⁹Ser GSK3β phosphorylation. Data (mean ± s.d.) are expressed as fold change versus control condition (no insulin nor serum) set at 1 (^* P<0.05 vs. control).

Figure 5. Effect of conditioned medium (CM) from insulin sensitive subjects and IR subjects on Akt and GSK3β phosphorylation in differentiated human myotubes.

Control denotes phosphorylation in the absence of CM, with phosphorylation level in the absence of insulin set at 1. Data are reported as mean ± s.d. for control subjects (Crohn’s patients denoted as control subjects), while individual values are reported for each IR subject. Each bar is the mean of experiments in triplicate. Panel A: 15 min incubation of human myotubes from insulin sensitive subjects with 25 µg/ml of CM proteins increased ⁴⁷³Ser Akt phosphorylation level in the absence of insulin, with a stronger effect of medium prepared from IR subjects than from insulin sensitive subjects (P<0.05 vs. control subjects). The stimulatory effect of 100 nM insulin in control was increased in the presence of CM from IR subjects (P<0.01 vs. control subjects). Blots are representative western blots of ⁴⁷³Ser Akt phosphorylation and total Akt amounts. Panel B: 15 min incubation of human myotubes from insulin sensitive subjects with 25 µg/ml of CM proteins increased ⁹Ser GSK3β phosphorylation level in the absence of insulin, with a stronger effect of medium prepared from IR subjects than from insulin sensitive subjects (P<0.05 vs. control subjects). The incremental stimulatory effect of 100 nM insulin over basal was reduced mainly in the presence of CM from IR subjects. Blot is a representative western blot of ⁹Ser GSK3β phosphorylation.

Figure 6. Interaction between IRS1−PI3K−Akt signaling pathway and mTOR.

Upon insulin binding insulin receptor activates, through insulin receptor substrate 1 (IRS1), phosphatidylinositol-3-kinase (PI3K), which results in Akt phosphorylation at ³⁰⁸Thr residue via Phosphatidylinositol 4,5-bisphosphate (PIP2) to Phosphatidylinositol 3,4,5-bisphosphate (PIP3) conversion followed by Akt and PDK1 recruitment to plasma membrane (not shown). A factor J present in duodenal-jejunal conditioned medium activates, possibly via the tuberous sclerosis complex TSC1-TSC2, the mammlian target of rapamycin complex 2 (mTORC2). mTORC2 also appears to be regulated by the PI3K pathway and phosphorylates Akt at ⁴⁷³Ser residue. Through TSC1-TSC2 and the GTPase Rheb, Akt activates mTORC1 and its direct substrate S6K1. Akt also inhibits GSK3. S6K1, mTORC1 and GSK3 phosphorylate serine residues on IRS1, thus attenuating insulin signalling. Hypothetical signalling is denoted by dotted lines.