The physiological regulation and contribution of the multiple phosphorylation sites of insulin receptor substrate 1 (IRS1) to the pathogenesis of insulin resistance is unknown. Our aims were to map the phosphorylated motifs of IRS1 in skeletal muscle from people with normal glucose tolerance (NGT; n = 11) or type 2 diabetes mellitus (T2DM; n = 11).
Skeletal muscle biopsies were obtained under fasted conditions or during a euglycemic clamp and IRS1 phosphorylation sites were identified by mass spectrometry.
We identified 33 phosphorylation sites in biopsies from fasted individuals, including 2 previously unreported sites ([Ser393] and [Thr1017]). In men with NGT and T2DM, insulin increased phosphorylation of 5 peptides covering 10 serine or threonine sites and decreased phosphorylation of 6 peptides covering 9 serine, threonine or tyrosine sites. Insulin-stimulation increased phosphorylation of 2 peptides, and decreased phosphorylation of 2 peptides only in men with NGT. Insulin increased phosphorylation of 2 peptides only in men with T2DM.
Despite severe skeletal muscle insulin resistance, the pattern of IRS1 phosphorylation was not uniformly altered in T2DM. Our results contribute to the evolving understanding of the physiological regulation of insulin signaling and complement the comprehensive map of IRS1 phosphorylation in T2DM.

Autoimmune hypoglycemia includes rare syndromes characterized by the presence of either anti-insulin antibodies (IAA) (Hirata\'s disease) or anti-insulin receptor (anti-ISR) antibodies (Flier\'s syndrome). Diagnosis is usually based on identification of the specific antibodies, in presence of the Whipple triad. However, most of these cases are classified as idiopathic diseases due to the difficulty to define the pathogenic culprit.
Basic research methodologies, including Western Blot and ELISA tests, have been used in this study.
We describe a 21-year-old young woman (PT), non-obese and non-diabetic, with a positive history of autoimmune diseases, admitted to the hospital for recurrent episodes of severe symptomatic hypoglycemia. Counterregulatory response to hypoglycemia was normal as well as the fasting test, so excluding both hormone deficiencies and insulinoma. Since an autoimmune hypoglycemic syndrome was suspected, the hyperactivation of the insulin pathway was experimentally evaluated. At this purpose, human hepatocarcinoma (HepG2) cells were incubated with serum obtained from the patient (PT) and from control individuals. Interestingly, a significant increase of phosphorylation of insulin receptor, Akt, and ERK1/2 was observed in the HepG2 cells incubated with PT serum compared with the controls. ELISA tests revealed significantly increased levels of anti-ISR antibodies in PT serum, while IAA were similar both in PT and in control sera, supporting diagnosis of Flier\'s syndrome.
This study emphasizes the importance to identify new strategies for the differential diagnosis of hypoglycemia, not always possible with the routinely used diagnostic tests.