Two recent studies demonstrated that bariatric surgery induced remission of T2D and significant improvement in metabolic control of the diabetes over and above medical therapy 
. The improvement in glycemia occurs very soon after the surgery and far too early to be attributed to weight loss. In this study, we sought to explore the mechanism/s of this phenomenon. Jejunal conditioned medium proteins from both diabetic, insulin resistant animals and insulin resistant humans impaired insulin signaling in skeletal muscle cell cultures. A similar effect was obtained with the human serum from insulin resistant subjects, suggesting that circulating (and thus by definition endocrine) factors are inducing insulin resistance.
An in vivo and in vitro state of insulin resistance was reproduced in the presence of proteins secreted by the duodenal-jejunal mucosa of db/db mice. Contrary to the conditioned medium proteins from Swiss mice, those secreted by the db/db mice small intestine induced insulin resistance when injected in normal Swiss mice or when added to the incubation medium of skeletal muscle preparations in vitro. Also the experiments with the L6 cells were congruent, as shown by ~65% and ~30% reduction of insulin-dependent (ΔR/K0.5) and insulin-independent (Rb) glucose uptake in the presence of db/db proteins, respectively. Notably, Swiss mice proteins failed to decrease glucose uptake either in vivo or in vitro.
Our study demonstrates that the molecular mechanism through which proteins secreted by the duodenal-jejunal mucosa of db/db mice induce insulin resistance could be mediated by interference with intracellular signaling pathways. We found that db/db proteins robustly induced the phosphorylation of the Akt 473serine residue at zero insulin, leading to a prompt saturation of the insulin dose-response curve. Moreover, db/db CM proteins tended to reduce the insulin-induced phosphorylation of Akt on 308Thr residue. Thus, 100 nM insulin treated L6 cells in the presence of db/db CM exhibited maximal Akt phosphorylation at 473Ser residue but about 50% phosphorylation at 308Thr residue, so full Akt activation could not be attained, consistent with the observed reduction of 2DG uptake in the soleus muscle and L6 cells. CM db/db also determined a marked proteolysis of GSK3β phosphorylated form and inhibited the basal activation of the p70 S6kinase.
Interestingly, when compared with insulin sensitive individuals, both serum and intestinal CM proteins from insulin resistant subjects determined a higher basal 473
Ser Akt and 9
Ser GSK3β phosphorylation in human myotubes. A tendency toward an increased basal phosphorylation of 9
Ser GSK3β was also observed in the presence of CM from insulin sensitive subjects. Furthermore, the incremental 9
Ser GSK3β phosphorylation over basal in the presence of insulin was reduced in IR subjects. Overall, these data suggest that serum and conditioned medium contain factor/s, which are likely in a larger amount in the insulin resistant than in insulin sensitive subjects, that induce insulin resistance. We observe that we could not compare insulin resistant with healthy control subjects because it is near impossible to obtain sufficient duodenal/jejunal mucosa during endoscopy to provide sufficient intestinal secreted proteins for testing in human myoblasts. Our controls were in an inactive phase of their disease with stenosis of the ileum which required elective surgery. We cannot exclude a certain degree of general inflammation, and thus they could not be considered as healthy subjects. However these patients were still insulin sensitive according to a previous observation 
and as assessed by the euglycemic clamp.
The Akt phosphorylation of 473
Ser residue is a target of the mTOR complex 2 
, while the Akt phosphorylation at 308
Thr is operated by PDK1, this last step being essential for the full Akt catalytic activity 
. Fraenkel et al. 
showed that, in the fasting state, basal 473
Ser Akt phosphorylation was higher in the skeletal muscle of diabetic than in normoglycemic Psammomys obesus
), while there was a net reduction of the stimulation by insulin in agreement with the high insulin resistance state of these diabetic animals. Furthermore, it was found that Akt directly mediates Ser/Thr phosphorylation of the insulin receptor substrate 1 (IRS-1), resulting in a negative feedback loop that reduces insulin action 
. Interestingly, this effect was inhibited by Rapamycin 
Akt phosphorylates 9
Ser in GSK3β with subsequent inhibition of the enzymatic function of GSK 
. It was recently demonstrated that GSK activity can also be regulated by calpain-induced proteolysis of its N terminus, which gives way to a short-lived constitutively active form of the enzyme 
. Our data suggest that similar mechanisms could occur in the presence of small intestine db/db
CM. If the products of GSK3β degradation induced by db/db
CM proteins are active, they might contribute to enhance the recognition of GSK substrates. In addition to the well-known effect on glycogen synthase activity 
, GSK3 has been implicated in the phosphorylation of the IRS-1 on serine residues with a consequent impairment of insulin signaling 
. Furthermore, GSK3 overexpression was found in peripheral tissues in a variety of diabetic animals and also in humans 
. It has also been shown that Rapamycin markedly decreased GSK3 phosphorylation in muscle of normoglycemic and diabetic P. obesus
, indicating increased GSK3β activity 
, while diabetes was reversed in obese diabetic mice treated with GSK3 inhibitors 
The mammalian target of rapamycin (mTOR) exists in two forms, mTORC1 and mTORC2. mTORC1 regulates protein synthesis by S6K1 and the eukaryotic initiation factor 4E-binding protein 1 at ribosomal level 
, while mTORC2 phosphorylates Akt at 473
Ser. Rapamycin is an mTORC1 inhibitor, which acts specifically on S6K1, whereas the ATP-competitive inhibitor PP242 completely blocks both mTORCs 
. We found that the effect of db/db
CM proteins on Akt 473
Ser phosphorylation was fully prevented by PP242, but not by Rapamycin, indicating a preferential role of mTORC2 in the response to CM proteins in L6 cells. Furthermore, inhibition of p70S6K1 phosphorylation suggested a negative modulation of mTORC1 inhibitors.
Taken together, these results support a possible mechanism of action of the db/db
or IR subjects CM on insulin signaling and action in skeletal muscle cells by which proteins produced by small intestine could act via activation of mTORC2 while inhibiting mTORC1. Activation of mTORC2 may be regulated by activation of TSC1/TSC2 complex, which directly binds to mTORC2 while inhibiting Rheb and thus mTORC1 
. So, the increased 473
Ser Akt phosphorylation is accompanied by decrease in basal (no insulin) p70 S6K1 389
Thr phosphorylation. Accordingly, Rapamycin and PP242 inhibit p70 S6K1 389
Thr phosphorylation (as also observed in the absence of db/db
CM). This is accompanied by the lack of inhibition of GSK3 due to the proteolysis of its phosphorylated form, and may in turn lead to the phosphorylation of IRS-1 on serine/threonine residues that determines inhibition of insulin signaling by reducing IRS-1 tyrosine phosphorylation with consequent insulin resistance 
Interaction between IRS1−PI3K−Akt signaling pathway and mTOR.
Our finding that db/db
CM promotes Akt 473
Ser phosphorylation in L6 cells, likely via mTORC2 or TSC activation and Akt recruitment to plasma membrane, may provide a new insight on the upstream regulation of mTORC2 
. Finally, the reversibility of CM proteins action after washout suggests that these proteins may act through the activation of a membrane receptor. We conclude that, although some difference in the effect of mice and human CM proteins on the insulin signaling does exist, the mechanism of action, as summarized in , likely involves the mTOR pathway.
The strength of the present study is the demonstration that the small intestine of insulin resistant humans and mice secrete a protein factor/s inducing insulin resistance by impairing the insulin signaling.
The limitations of our investigation are that insulin sensitivity in mice was assessed by the IPITT instead of a more sophisticated euglycemic hyperinsulinemic clamp and that the insulin signaling pathway was studied in two different cellular lines, the L6 cells to test the proteins secreted by the mice intestine and human myoblasts to test the proteins secreted by the human intestine. Concerning the first point we note that the steady state conditions of plasma concentration, required by the clamp, could not be assured for the jejunal proteins, whose kinetics is not known. Moreover, the minimal model analysis also provides the estimate of the glucose effectiveness that has been found decreased after the in vivo injection of db/db proteins.
As for the second point, it is well accepted that insulin action is comparable in L6 cells and primary muscle cells. Because the use of human primary muscle cells requires biopsies and time-consuming expansion and differentiation, we restricted the use of these cells to the study of the human secreted proteins. Actually, we found similar results by using the secreted proteins from db/db mice and insulin resistant humans, although the proteolytic degradation of phopsphorylated GSK3, observed in the rodent study, was not present in the human study.
Ideally, the best model to investigate the role of the small intestine in inducing insulin resistance would be that of studying the same insulin resistant subjects and animals before and after bariatric surgery, in particular the bilio-pancreatic diversion that was proven to allow diabetes remission through the normalization of insulin resistance 
. After this operation as well as after Roux-en-Y gastric bypass, however, the biliary limb is excluded from food transit and it is no further explorable endoscopically to obtain mucosal biopsies. To harmonize the experimental design in humans and animals, we have thus chosen to demonstrate that the duodenum/jejunum of insulin resistant humans and mice secrete hormone/s inducing insulin resistance.
The natural evolution our study will be the isolation and identification of the IR hormone/s secreted by the duodenum-jejunum tracts. Their future identification might permit the development of new pharmacological agents for the treatment of type 2 diabetes. Insulin resistance is, in fact, a characteristic feature of type 2 diabetes and plays a central role in the pathogenesis of this disease although the presence of a concomitant β-cell failure is necessary. It is worldwide recognized that the skeletal muscle insulin resistance develops decades before β-cell failure 
. The clinical implications of our study are related to the mechanisms of action of the jejunal hormone/s inducing IR which can explain the immediate metabolic and longer term effects of bariatric surgery in inducing the remission of type 2 diabetes