Single-Cell Transcriptional Analyses Identify Lineage-Specific Epithelial Responses to Inflammation and Metaplastic Development in the Gastric Corpus.

BACKGROUND & AIMS: Chronic atrophic gastritis can lead to gastric metaplasia and increase risk of gastric adenocarcinoma. Metaplasia is a precancerous lesion associated with an increased risk for carcinogenesis, but the mechanism(s) by which inflammation induces metaplasia are poorly understood. We investigated transcriptional programs in mucous neck cells and chief cells as they progress to metaplasia mice with chronic gastritis. METHODS: We analyzed previously generated single-cell RNA-sequencing (scRNA-seq) data of gastric corpus epithelium to define transcriptomes of individual epithelial cells from healthy BALB/c mice (controls) and TxA23 mice, which have chronically inflamed stomachs with metaplasia. Chronic gastritis was induced in B6 mice by Helicobacter pylori infection. Gastric tissues from mice and human patients were analyzed by immunofluorescence to verify findings at the protein level. Pseudotime trajectory analysis of scRNA-seq data was used to predict differentiation of normal gastric epithelium to metaplastic epithelium in chronically inflamed stomachs. RESULTS: Analyses of gastric epithelial transcriptomes revealed that gastrokine 3 (Gkn3) mRNA is a specific marker of mouse gastric corpus metaplasia (spasmolytic polypeptide expressing metaplasia, SPEM). Gkn3 mRNA was undetectable in healthy gastric corpus; its expression in chronically inflamed stomachs (from TxA23 mice and mice with Helicobacter pylori infection) identified more metaplastic cells throughout the corpus than previously recognized. Staining of healthy and diseased human gastric tissue samples paralleled these results. Although mucous neck cells and chief cells from healthy stomachs each had distinct transcriptomes, in chronically inflamed stomachs, these cells had distinct transcription patterns that converged upon a pre-metaplastic pattern, which lacked the metaplasia-associated transcripts. Finally, pseudotime trajectory analysis confirmed the convergence of mucous neck cells and chief cells into a pre-metaplastic phenotype that ultimately progressed to metaplasia. CONCLUSIONS: In analyses of tissues from chronically inflamed stomachs of mice and humans, we expanded the definition of gastric metaplasia to include Gkn3 mRNA and GKN3-positive cells in the corpus, allowing a more accurate assessment of SPEM. Under conditions of chronic inflammation, chief cells and mucous neck cells are plastic and converge into a pre-metaplastic cell type that progresses to metaplasia.

Single-cell transcriptional analyses of spasmolytic polypeptide-expressing metaplasia arising from acute drug injury and chronic inflammation in the stomach.

OBJECTIVE: Spasmolytic polypeptide-expressing metaplasia (SPEM) is a regenerative lesion in the gastric mucosa and is a potential precursor to intestinal metaplasia/gastric adenocarcinoma in a chronic inflammatory setting. The goal of these studies was to define the transcriptional changes associated with SPEM at the individual cell level in response to acute drug injury and chronic inflammatory damage in the gastric mucosa. DESIGN: Epithelial cells were isolated from the gastric corpus of healthy stomachs and stomachs with drug-induced and inflammation-induced SPEM lesions. Single cell RNA sequencing (scRNA-seq) was performed on tissue samples from each of these settings. The transcriptomes of individual epithelial cells from healthy, acutely damaged and chronically inflamed stomachs were analysed and compared. RESULTS: scRNA-seq revealed a population Mucin 6 (Muc6)+gastric intrinsic factor (Gif)+ cells in healthy tissue, but these cells did not express transcripts associated with SPEM. Furthermore, analyses of SPEM cells from drug injured and chronically inflamed corpus yielded two major findings: (1) SPEM and neck cell hyperplasia/hypertrophy are nearly identical in the expression of SPEM-associated transcripts and (2) SPEM programmes induced by drug-mediated parietal cell ablation and chronic inflammation are nearly identical, although the induction of transcripts involved in immunomodulation was unique to SPEM cells in the chronic inflammatory setting. CONCLUSIONS: These data necessitate an expansion of the definition of SPEM to include Tff2+Muc6+ cells that do not express mature chief cell transcripts such as Gif. Our data demonstrate that SPEM arises by a highly conserved cellular programme independent of aetiology and develops immunoregulatory capabilities in a setting of chronic inflammation.

Interferon-γ directly induces gastric epithelial cell death and is required for progression to metaplasia.

Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng-/- mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng-/- mouse immune cells were used. Finally, TxA23 × Ifng-/- mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry.

The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells) from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC) I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of chronic inflammation on individual gastric epithelial cells, a critical consideration for the study of gastric cancer.

IL13 Acts Directly on Gastric Epithelial Cells to Promote Metaplasia Development During Chronic Gastritis.

BACKGROUND & AIMS: It is well established that chronic inflammation promotes gastric cancer-associated metaplasia, but little is known regarding the mechanisms by which immune cells and cytokines regulate metaplastic cellular changes. The goals of this study were to identify interleukin 13 (IL13)-producing immune cells, determine the gastric epithelial cell response(s) to IL13, and establish the role(s) of IL13 in metaplasia development. METHODS: Experiments used an established mouse model of autoimmune gastritis (TxA23), TxA23×Il4ra-/- mice, which develop gastritis but do not express the IL4/IL13-receptor subunit IL4Rα, and TxA23×Il13-Yfp mice, which express yellow fluorescent protein in IL13-producing cells. Flow cytometry was used to measure IL13 secretion and identify IL13-producing immune cells. Mouse and human gastric organoids were cultured with IL13 to determine epithelial cell response(s) to IL13. Single-cell RNA sequencing was performed on gastric epithelial cells from healthy and inflamed mouse stomachs. Mice with gastritis were administered IL13-neutralizing antibodies and stomachs were analyzed by histopathology and immunofluorescence. RESULTS: We identified 6 unique subsets of IL13-producing immune cells in the inflamed stomach. Organoid cultures showed that IL13 acts directly on gastric epithelium to induce a metaplastic phenotype. IL4Rα-deficient mice did not progress to metaplasia. Single-cell RNA sequencing determined that gastric epithelial cells from IL4Rα-deficient mice up-regulated inflammatory genes but failed to up-regulate metaplasia-associated transcripts. Neutralization of IL13 significantly reduced and reversed metaplasia development in mice with gastritis. CONCLUSIONS: IL13 is made by a variety of immune cell subsets during chronic gastritis and promotes gastric cancer-associated metaplastic epithelial cell changes. Neutralization of IL13 reduces metaplasia severity during chronic gastritis.

Spontaneous development of endoplasmic reticulum stress that can lead to diabetes mellitus is associated with higher calcium-independent phospholipase A2 expression: a role for regulation by SREBP-1.

Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA(2)beta and ER stress, we compared beta-cell lines generated from wild type (WT) and Akita mice. The Akita mouse is a spontaneous model of ER stress that develops hyperglycemia/diabetes due to ER stress-induced beta-cell apoptosis. Consistent with a predisposition to developing ER stress, basal phosphorylated PERK and activated caspase-3 are higher in the Akita cells than WT cells. Interestingly, basal iPLA(2)beta, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (DeltaPsi) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates DeltaPsi loss and apoptosis of Akita cells and is associated with increases in iPLA(2)beta, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA(2)beta small interfering RNA, however, suppresses NSMase message, DeltaPsi loss, and apoptosis. The iPLA(2)beta gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA(2)beta in the Akita cells and suppresses increases in mSREBP-1 and iPLA(2)beta due to thapsigargin. These findings suggest that ER stress leads to generation of mSREBP-1, which can bind to the sterol-regulatory element in the iPLA(2)beta gene to promote its transcription. Consistent with this, SREBP-1, iPLA(2)beta, and NSMase messages in Akita mouse islets are higher than in WT islets.

Xenin-25 potentiates glucose-dependent insulinotropic polypeptide action via a novel cholinergic relay mechanism.

The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, "GIP/DT" animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the beta-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in beta-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation.

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

Interleukin 27 Protects From Gastric Atrophy and Metaplasia During Chronic Autoimmune Gastritis.

BACKGROUND & AIMS: The association between chronic inflammation and gastric carcinogenesis is well established, but it is not clear how immune cells and cytokines regulate this process. We investigated the role of interleukin 27 (IL27) in the development of gastric atrophy, hyperplasia, and metaplasia (preneoplastic lesions associated with inflammation-induced gastric cancer) in mice with autoimmune gastritis. METHODS: We performed studies with TxA23 mice (control mice), which express a T-cell receptor against the H+/K+ adenosine triphosphatase α chain and develop autoimmune gastritis, and TxA23xEbi3-/- mice, which develop gastritis but do not express IL27. In some experiments, mice were given high-dose tamoxifen to induce parietal cell atrophy and spasmolytic polypeptide-expressing metaplasia (SPEM). Recombinant IL27 was administered to mice with mini osmotic pumps. Stomachs were collected and analyzed by histopathology and immunofluorescence; we used flow cytometry to measure IL27 and identify immune cells that secrete IL27 in the gastric mucosa. Single-cell RNA sequencing was performed on immune cells that infiltrated stomach tissues. RESULTS: We identified IL27-secreting macrophages and dendritic cell in the corpus of mice with chronic gastritis (TxA23 mice). Mice deficient in IL27 developed more severe gastritis, atrophy, and SPEM than control mice. Administration of recombinant IL27 significantly reduced the severity of inflammation, atrophy, and SPEM in mice with gastritis. Single-cell RNA sequencing showed that IL27 acted almost exclusively on stomach-infiltrating CD4+ T cells to suppress expression of inflammatory genes. CONCLUSIONS: In studies of mice with autoimmune gastritis, we found that IL27 is an inhibitor of gastritis and SPEM, suppressing CD4+ T-cell-mediated inflammation in the gastric mucosa.

Interleukin-17A Promotes Parietal Cell Atrophy by Inducing Apoptosis.

BACKGROUND & AIMS: Atrophic gastritis caused by chronic inflammation in the gastric mucosa leads to the loss of gastric glandular cells, including acid-secreting parietal cells. Parietal cell atrophy in a setting of chronic inflammation induces spasmolytic polypeptide expressing metaplasia, a critical step in gastric carcinogenesis. However, the mechanisms by which inflammation causes parietal cell atrophy and spasmolytic polypeptide expressing metaplasia are not well defined. We investigated the role of interleukin-17A (IL-17A) in causing parietal cell atrophy. METHODS: A mouse model of autoimmune atrophic gastritis was used to examine IL-17A production during early and late stages of disease. Organoids derived from corpus glands were used to determine the direct effects of IL-17A on gastric epithelial cells. Immunofluorescent staining was used to examine IL-17A receptors and the direct effect of signaling on parietal cells. Mice were infected with an IL-17A-producing adenovirus to determine the effects of IL-17A on parietal cells in vivo. Finally, IL-17A neutralizing antibodies were administered to mice with active atrophic gastritis to evaluate the effects on parietal cell atrophy and metaplasia. RESULTS: Increased IL-17A correlated with disease severity in mice with chronic atrophic gastritis. IL-17A caused caspase-dependent gastric organoid degeneration, which could not be rescued with a necroptosis inhibitor. Parietal cells expressed IL-17A receptors and IL-17A treatment induced apoptosis in parietal cells. Overexpressing IL-17A in vivo induced caspase-3 activation and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in parietal cells. Finally, IL-17A neutralizing antibody decreased parietal cell atrophy and metaplasia in mice with chronic atrophic gastritis. CONCLUSIONS: These data identify IL-17A as a cytokine that promotes parietal cell apoptosis during atrophic gastritis, a precursor lesion for gastric cancer.

Suppressed insulin signaling and increased apoptosis in CD38-null islets.

CD38 is a multifunctional enzyme capable of generating metabolites that release Ca2+ from intracellular stores, including nicotinic acid adenine dinucleotide phosphate (NAADP). A number of studies have led to the controversial proposal that CD38 mediates an alternate pathway for glucose-stimulated insulin release and contributes to the pathogenesis of diabetes. It has recently been shown that NAADP mediates Ca2+ mobilization by insulin in human pancreatic beta-cells. In the present study, we report altered Ca2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38-/- beta-cells. In keeping with the antiapoptotic role of insulin signaling, Cd38-/- islets were significantly more susceptible to apoptosis compared with islets isolated from littermate controls. This finding correlated with disrupted islet architecture and reduced beta-cell mass in Cd38-/- mice, both in the context of a normal lab diet and a high-fat diet. Nevertheless, we did not find robust differences in glucose homeostasis in vivo or glucose signaling in vitro in Cd38-/- mice on the C57BL/6 genetic background, in contrast to previous studies by others of Cd38 knockout mice on the ICR background. Thus, our results suggest that CD38 plays a role in novel antiapoptotic signaling pathways but does not directly control glucose signaling in pancreatic beta-cells.

Loss of Nix in Pdx1-deficient mice prevents apoptotic and necrotic ß cell death and diabetes.

Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of ß cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined. In this study, we introduced an shRNA into mouse insulinoma MIN6 cells to deplete Pdx1 and found that expression of proapoptotic genes, including NIP3-like protein X (Nix), was increased. Forced Nix expression in MIN6 and pancreatic islet ß cells induced programmed cell death by simultaneously activating apoptotic and mitochondrial permeability transition-dependent necrotic pathways. Preventing Nix upregulation during Pdx1 suppression abrogated apoptotic and necrotic ß cell death in vitro. In Pdx1-haploinsufficient mice, Nix ablation normalized pancreatic islet architecture, ß cell mass, and insulin secretion and eliminated reactive hyperglycemia after glucose challenge. These results establish Nix as a critical mediator of ß cell apoptosis and programmed necrosis in Pdx1-deficient diabetes.

Insulin signaling in alpha cells modulates glucagon secretion in vivo.

Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypoglycemic conditions. In this study, we created and characterized alpha cell-specific insulin receptor knockout (alphaIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male alphaIRKO mice exhibited mild glucose intolerance, hyperglycemia, and hyperglucagonemia in the fed state and enhanced glucagon secretion in response to L-arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in alphaIRKO mice. The mutants also exhibited an age-dependent increase in beta cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intraislet insulin signaling in the regulation of alpha cell function in both normo- and hypoglycemic conditions.

Targeted ablation of glucose-dependent insulinotropic polypeptide-producing cells in transgenic mice reduces obesity and insulin resistance induced by a high fat diet.

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity.

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover.

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.