Access Type

Open Access Dissertation

Date of Award

1-1-2021

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

Zhengping Yi

Abstract

Diabetes is a group of metabolic diseases characterized by hyperglycemia caused by defects in insulin secretion, insulin action, or both. Diabetes is associated with damage, dysfunction, and failure of various organs, such as the eyes, heart, kid-neys, and brain. Diabetes is mainly classified into type 1 (T1DM) and type 2 diabetes (T2D). Diabetes affects more than 34 million people in the USA (about 1 in 10) and more than 90% of diabetic patients have type 2 diabetes (T2D). Insulin resistance is a main characteristic feature of type 2 diabetes. Skeletal muscle insulin resistance is con-sidered to be the primary defect that is evident decades before β-cell failure and overt T2D. Skeletal muscle is the major site of insulin-stimulated glucose uptake (>70%) in the postprandial state in humans. Metformin (N, N-dimethylbiguanide) is an effective oral biguanide antihyperglycemic drug and the most frequently prescribed as a first-line therapy for type 2 diabetes mellitus. It is widely accepted that metformin can reduce glu-cose production by the liver, and increase insulin sensitivity (i.e., decrease insulin re-sistance) in skeletal muscles. Metformin stimulates insulin-mediated glucose uptake in skeletal muscle in T2D patients by increasing Thr172 phosphorylation (pThr172) and activity of AMP-activated protein kinase (AMPK). However, the molecular mechanism of metformin’s action in skeletal muscle is not well understood.Protein phosphorylation, regulated by kinases and phosphatases, plays a key role in many cell signaling events, including insulin signaling. Abnormal protein phos-phorylation has been implicated in the development of skeletal muscle insulin resistance and T2D191,192191,192190,191. However, most studies on protein phosphorylation in insulin resistance and T2D have been focused on kinases and little is known regarding the role of phosphatases. Protein Phosphatase 2A (PP2A) is a ubiquitously expressed ser-ine/threonine phosphatase and plays a pivotal role in cellular processes, such as signal transduction, cell proliferation, and apoptosis, through dephosphorylating key signaling molecules such as AKT, AMPK, etc. Structurally, PP2A is composed of catalytic sub-unit C (PP2Ac), scaffold subunit A and a regulatory subunit B. PP2Ac and scaffold subunit A have two isoforms and the regulatory subunit B has four different families containing different isoforms. Whether PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle remains to be elucidated. Here, we investigated and measured PP2A activity, novel PPAc interaction partners, and novel PP2A substrates in human skeletal muscle cells derived from lean insulin-sensitive and obese insulin-resistant participants. Hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells were cultured from these muscle biopsies that included 8 lean insu-lin-sensitive and 8 obese insulin-resistance participants. The cells were expanded, dif-ferentiated into myotubes, and treated with/without 50µM metformin for 24 hours, oka-daic acid 5nM for 30 minutes, and/or Insulin 100nM for 15 minutes, before harvesting. The PP2A activity was performed and measured according to the manufacturer’s proto-col. The phosphoproteome and proteome were performed according to our protocol us-ing Orbitrap Fusion Lumos UPLC- ESI-MS/MS. We have identified >24,700 phos-phorylation sites in 7,037 proteins, which is one of the largest catalogs of experimental-ly determined phosphorylation sites in primary human skeletal muscle cells. Among all phosphorylation sites identified 1,958 were not reported in human and 1,756 were not reported in any species in the PhosphositePlus database, thus appears to be novel. We identified phosphorylation sites in 291 kinases/kinases subunits and 18 phosphatases subunits of protein phosphatase 2A. Bioinformatics analysis indicated that subcellular localizations, multiple biological processes, molecular functions, and KEGG pathway (e.g., insulin signaling pathway, AMPK signaling, mTOR signaling, MAPK signaling, and ErbB signaling) were significantly enriched for these phosphoproteins. Further-more, we identified proteins that potentially interact with PP2Ac and several proteins previously known to interact with PP2Ac such as CaMK II and GSK3. Many of the newly identified PP2Ac-binding proteins were associated with growth control. We identified 1377 interaction partners in human skeletal muscle cells with 19 partners clas-sified as metformin responsive. Moreover, 450 interaction partners are identified in hu-man skeletal muscle cells in insulin stimulation responsive, and 44 proteins presented a significant difference among the two groups. We observed several proteins associated with insulin signaling as PP2Ac interaction partners in human skeletal muscle cells like Rac1, Akt2, MAPK, and Limk1. We reported that metformin reversed the abnormality in PP2Ac interaction partners in obese insulin-resistant and rendered them similar to lean insulin-sensitive participants.

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