Opposing Effects of EGF Receptor Signaling on Proliferation and Differentiation Initiated by EGF or TSH/EGF Receptor Transactivation.

Regulation of thyroid cells by thyrotropin (TSH) and epidermal growth factor (EGF) has been known but different effects of these regulators on proliferation and differentiation have been reported. We studied these responses in primary cultures of human thyroid cells to determine whether TSH receptor (TSHR) signaling may involve EGF receptor (EGFR) transactivation. We confirm that EGF stimulates proliferation and de-differentiation whereas TSH causes differentiation in the absence of other growth factors. We show that TSH/TSHR transactivates EGFR and characterize it as follows: (1) TSH-induced upregulation of thyroid-specific genes is inhibited by 2 inhibitors of EGFR kinase activity, AG1478 and erlotinib; (2) the mechanism of transactivation is independent of an extracellular EGFR ligand by showing that 2 antibodies, cetuximab and panitumumab, that completely inhibited binding of EGFR ligands to EGFR had no effect on transactivation, and by demonstrating that no EGF was detected in media conditioned by thyrocytes incubated with TSH; (3) TSH/TSHR transactivation of EGFR is different than EGFR activation by EGF by showing that EGF led to rapid phosphorylation of EGFR whereas transactivation occurred in the absence of receptor phosphorylation; (4) EGF caused downregulation of EGFR whereas transactivation had no effect on EGFR level; (5) EGF and TSH stimulation converged on the protein kinase B (AKT) pathway, because TSH, like EGF, stimulated phosphorylation of AKT that was inhibited by EGFR inhibitors; and (6) TSH-induced upregulation of thyroid genes was inhibited by the AKT inhibitor MK2206. Thus, TSH/TSHR causes EGFR transactivation that is independent of extracellular EGFR ligand and in part mediates TSH regulation of thyroid hormone biosynthetic genes.

Thyrotropin regulation of differentiated gene transcription in adult human thyrocytes in primary culture.

Thyroid transcription factors (TTFs) - NKX2-1, FOXE1, PAX8 and HHEX - regulate multiple genes involved in thyroid development in mice but little is known about TTF regulation of thyroid-specific genes - thyroglobulin (TG), thyroid peroxidase (TPO), deiodinase type 2 (DIO2), sodium/iodide symporter (NIS) and TSH receptor (TSHR) - in adult, human thyrocytes. Thyrotropin (thyroid-stimulating hormone, TSH) regulation of thyroid-specific gene expression in primary cultures of human thyrocytes is biphasic yielding an inverted U-shaped dose-response curve (IUDRC) with upregulation at low doses and decreases at high doses. Herein we show that NKX2-1, FOXE1 and PAX8 are required for TSH-induced upregulation of the mRNA levels of TG, TPO, DIO2, NIS, and TSHR whereas HHEX has little effect on the levels of these thyroid-specific gene mRNAs. We show that TSH-induced upregulation is mediated by changes in their transcription and not by changes in the degradation of their mRNAs. In contrast to the IUDRC of thyroid-specific genes, TSH effects on the levels of the mRNAs for NKX2-1, FOXE1 and PAX8 exhibit monophasic decreases at high doses of TSH whereas TSH regulation of HHEX mRNA levels exhibits an IUDRC that overlaps the IUDRC of thyroid-specific genes. In contrast to findings during mouse development, TTFs do not have major effects on the levels of other TTF mRNAs in adult, human thyrocytes. Thus, we found similarities and important differences in the regulation of thyroid-specific genes in mouse development and TSH regulation of these genes in adult, human thyrocytes.

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes in vitro.

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro. Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by Gi/Go proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.

Thyrotropin Causes Dose-dependent Biphasic Regulation of cAMP Production Mediated by Gs and Gi/o Proteins.

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.

Evidence That Graves' Ophthalmopathy Immunoglobulins Do Not Directly Activate IGF-1 Receptors.

BACKGROUND: Graves' ophthalmopathy (GO) pathogenesis involves thyrotropin (TSH) receptor (TSHR)-stimulating autoantibodies. Whether there are autoantibodies that directly stimulate insulin-like growth factor 1 receptors (IGF-1Rs), stimulating insulin-like growth factor receptor antibodies (IGFRAbs), remains controversial. This study attempted to determine whether there are stimulating IGFRAbs in patients with GO. METHODS: Immunoglobulins (Igs) were purified from normal volunteers (NV-Igs) and patients with GO (GO-Igs). The effects of TSH, IGF-1, NV-Igs, and GO-Igs on pAKT and pERK1/2, members of pathways used by IGF-1R and TSHR, were compared in orbital fibroblasts from GO patients (GOFs) and U2OS-TSHR cells overexpressing TSHRs, and U2OS cells that express TSHRs at very low endogenous levels. U2OS-TSHR and U2OS cells were used because GOFs are not easily manipulated using molecular techniques such as transfection, and U2OS cells because they express TSHRs at levels that do not measurably stimulate signaling. Thus, comparing U2OS-TSHR and U2OS cells permits specifically distinguishing signaling mediated by the TSHR and IGF-1R. RESULTS: In GOFs, all GO-Igs stimulated pERK1/2 formation and 69% stimulated pAKT. In U2OS-TSHR cells, 15% of NV-IGs and 83% of GO-Igs stimulated increases in pERK1/2, whereas all NV-Igs and GO-Igs stimulated increases in pAKT. In U2OS cells, 70% of GO-Igs stimulated small increases in pAKT. Knockdown of IGF-1R caused a 65 ± 6.3% decrease in IGF-1-stimulated pAKT but had no effect on GO-Igs stimulation of pAKT. Thus, GO-Igs contain factor(s) that stimulate pAKT formation. However, this factor(s) does not directly activate IGF-1R. CONCLUSIONS: Based on the findings analyzing these two signaling pathways, it is concluded there is no evidence of stimulating IGFRAbs in GO patients.

Thyrotropin and Insulin-Like Growth Factor 1 Receptor Crosstalk Upregulates Sodium-Iodide Symporter Expression in Primary Cultures of Human Thyrocytes.

BACKGROUND: Major regulation of thyroid gland function is mediated by thyrotropin (TSH) activating the TSH receptor (TSHR) and inducing upregulation of genes involved in thyroid hormone synthesis. Evidence suggests that the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) may play a role in regulating TSHR functional effects. This study examined the potential role of TSHR/IGF-1R crosstalk in primary cultures of human thyrocytes. RESULTS: TSH/IGF-1 co-treatment elicited additive effects on thyroglobulin (TG), thyroperoxidase (TPO), and deiodinase type 2 (DIO2) mRNA levels but synergistic effects on sodium-iodide symporter (NIS) mRNA. Similar cooperativity was seen on the level of TG protein secretion (additive) and NIS protein expression (synergistic). The IGF-1R tyrosine kinase inhibitor linsitinib inhibited TSH-stimulated upregulation of NIS but not TG, indicating that NIS regulation is in part IGF-1R dependent and occurs via receptor crosstalk. Cooperativity was not seen at the level of cAMP/protein kinase A (PKA) signaling, IGF-1R phosphorylation, or Akt activation. However, TSH and IGF-1 synergistically activated ERK1/2. Pharmacological inhibition of ERK1/2 by the MEK1/2 inhibitor U0126 and of Akt by MK-2206 virtually abolished NIS stimulation by TSH and the synergistic effect of IGF-1. CONCLUSION: As linsitinib inhibited upregulation of NIS stimulated by TSH alone, it is concluded that crosstalk between TSHR and IGF-1R, without agonist activation of IGF-1R, plays a role in NIS regulation in human thyrocytes via a mechanism involving ERK1/2 and/or Akt. Fully understanding the nature of this crosstalk has clinical implications for the treatment of thyroid diseases, including thyroid cancer.

TSH/IGF-1 Receptor Cross Talk in Graves' Ophthalmopathy Pathogenesis.

CONTEXT: The TSH receptor (TSHR) is considered the main target of stimulatory autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO); however, it has been suggested that stimulatory IGF-1 receptor (IGF-1R) autoantibodies also play a role. OBJECTIVE: We previously demonstrated that a monoclonal stimulatory TSHR antibody, M22, activates TSHR/IGF-1R cross talk in orbital fibroblasts/preadipocytes obtained from patients with GO (GO fibroblasts [GOFs]). We show that cross talk between TSHR and IGF-1R, not direct IGF-1R activation, is involved in the mediation of GO pathogenesis stimulated by Graves' autoantibodies. DESIGN/SETTING/PARTICIPANTS: Immunoglobulins were purified from the sera of 57 GO patients (GO-Igs) and tested for their ability to activate TSHR and/or IGF-1R directly and TSHR/IGF-1R cross talk in primary cultures of GOFs. Cells were treated with M22 or GO-Igs with or without IGF-1R inhibitory antibodies or linsitinib, an IGF-1R kinase inhibitor. MAIN OUTCOME MEASURES: Hyaluronan (hyaluronic acid [HA]) secretion was measured as a major biological response for GOF stimulation. IGF-1R autophosphorylation was used as a measure of direct IGF-1R activation. TSHR activation was determined through cAMP production. RESULTS: A total of 42 out of 57 GO-Ig samples stimulated HA secretion. None of the GO-Ig samples exhibited evidence for IGF-1R autophosphorylation. Both anti-IGF-1R antibodies completely inhibited IGF-1 stimulation of HA secretion. By contrast, only 1 IGF-1R antibody partially blocked HA secretion stimulated by M22 or GO-Igs in a manner similar to linsitinib, whereas the other IGF-1R antibody had no effect on M22 or GO-Ig stimulation. These findings show that the IGF-1R is involved in GO-Igs stimulation of HA secretion without direct activation of IGF-1R. CONCLUSIONS: IGF-1R activation by GO-Igs occurs via TSHR/IGF-1R cross talk rather than direct binding to IGF-1R, and this cross talk is important in the pathogenesis of GO.

Thyrotropin Stimulates Differentiation Not Proliferation of Normal Human Thyrocytes in Culture.

Although TSH has been suggested to be a proliferative agent for thyrocytes, the effect of TSH on human thyroid cells remains controversial. In particular, most of the reported studies relied primarily on changes in DNA synthesis but have not included measurement of the number of cells. We argue that only a direct count of cell number, demonstrating classical exponential expansion, serves as a valid measurement of proliferation. Thus, although some data support TSH as a proliferative agent, most do not provide conclusive evidence. To generate conclusive evidence with regard to a proliferative effect of TSH in human thyrocytes, we performed various experiments using primary cultures of human thyrocytes. In contrast to previous reports, TSH [±insulin-like growth factor 1 (IGF-1)] did not induce proliferation of thyrocytes under a variety of different conditions. However, TSH/IGF-1 cotreatment did upregulate thyroid-specific gene expression including thyroglobulin (TG) and TSHR in a manner consistent with cellular differentiation. Evidence for a proliferative effect of TSH has been used to inform the American Thyroid Association's guidelines for the management of thyroid cancer patients, which include TSH suppression. While these recommendations are admittedly based on low- to moderate-quality evidence, TSH suppression is still widely used. We present data that question the consensus view that TSH promotes proliferation of human thyrocytes (upon which the American Thyroid Association's guidelines are based) and suggest that additional studies, including randomized controlled trials, are warranted to address this important clinical question.

Bidirectional TSH and IGF-1 receptor cross talk mediates stimulation of hyaluronan secretion by Graves' disease immunoglobins.

CONTEXT: There is no pathogenetically linked medical therapy for Graves' ophthalmopathy (GO). Lack of animal models and conflicting in vitro studies have hindered the development of such therapy. Recent reports propose that Graves' Igs bind to and activate thyrotropin receptors (TSHRs) and IGF-1 receptors (IGF-1Rs) on cells in orbital fat, stimulating hyaluronan (HA) secretion, a component of GO. OBJECTIVE: The objective of the study was to investigate potential cross talk between TSHRs and IGF-1Rs in the pathogenesis of GO using a sensitive HA assay. DESIGN/SETTING/PARTICIPANTS: Orbital fibroblasts from GO patients were collected in an academic clinical practice and cultured in a research laboratory. Cells were treated with TSH, IGF-1, and a monoclonal Graves' Ig M22. MAIN OUTCOME MEASURES: HA was measured by a modified ELISA. RESULTS: Simultaneous activation by TSH and IGF-1 synergistically increased HA secretion from 320 ± 52 for TSH and 430 ± 65 µg/mL for IGF-1 alone, to 1300 ± 95 µg/mL. IGF-1 shifted the TSH EC50 19-fold to higher potency. The dose response to M22 was biphasic. An IGF-1R antagonist inhibited the higher potency phase but had no effect on the lower potency phase. M22 did not cause IGF-1R autophosphorylation. A TSHR antagonist abolished both phases of M22-stimulated HA secretion. CONCLUSIONS: M22 stimulation of HA secretion by GO fibroblasts/preadipocytes involves cross talk between TSHR and IGF-1R. This cross talk relies on TSHR activation rather than direct activation of IGF-1R and leads to synergistic stimulation of HA secretion. These data propose a model for GO pathogenesis that explains previous contradictory results and argues for TSHR as the primary therapeutic target for GO.

Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization.

Evidence was presented that thyrotropin [thyroid-stimulating hormone (TSH)]-stimulated persistent cAMP signaling is dependent on receptor (with G-protein α subunits and adenylyl cyclase) internalization. Because it is not clear whether G proteins and adenylyl cyclase internalize with receptors, we tested whether persistent cAMP signaling by TSH receptor (TSHR) is dependent on internalization. We measured persistent TSHR signaling as an accumulation of cAMP in HEK-EM293 cells permanently expressing human TSHRs incubated with isobutylmethylxanthine for 30 min after washing the cells to remove unbound TSH, and TSHR internalization by fluorescence microscopy using Alexa-tagged TSH and binding assays using (125)I-TSH. TSHRs, but not the closely related lutropin or follitropin receptors, exhibit persistent cAMP signaling. TSHRs were not internalized by 30 min incubation with unlabeled TSH; however, expression of ß-arrestin-2 promoted TSHR internalization that was inhibited by dynasore, a dynamin inhibitor. Expression of ß-arrestin-2 had no effect on TSHR cAMP signaling, dynasore inhibited TSHR cAMP signaling in the absence or presence of TSHR internalization, and expression of a dominant-negative mutant dynamin, which inhibited internalization, had no effect on persistent cAMP signaling. Persistent cAMP signaling was specifically inhibited by a small molecule TSHR antagonist. We conclude that TSHRs do not have to be internalized to exhibit persistent cAMP signaling.

Human islet-derived precursor cells are mesenchymal stromal cells that differentiate and mature to hormone-expressing cells in vivo.

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin-producing cells. Previously, we described proliferative populations of human islet-derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000-fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C-peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105(+)/CD73(+)/CD90(+) cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas-derived MSC that are capable of differentiating into hormone-expressing cells. Their ability to mature into functional insulin-secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet-like cells for replacement therapy in type 1 diabetes. Disclosure of potential conflicts of interest is found at the end of this article.

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells.

Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.

Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells.

Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.

Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates.

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (i) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (ii) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3, and -4; (iii) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (iv) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.

Opposite effects of background genotype on muscle and liver insulin sensitivity of lipoatrophic mice. Role of triglyceride clearance.

The metabolic phenotype of the A-ZIP/F-1 (AZIP) lipoatrophic mouse is different depending on its genetic background. On both the FVB/N (FVB) and C57BL/6J (B6) backgrounds, AZIP mice have a similarly severe lack of white adipose tissue and comparably increased insulin levels and triglyceride secretion rates. However, on the B6 background, the AZIP mice have less hyperglycemia, lower circulating triglyceride and fatty acid levels, and lower mortality. AZIP characteristics that are more severe on the B6 background include increased liver size and liver triglyceride content. A unifying hypothesis is that the B6 strain has higher triglyceride clearance into the liver, with lower triglyceride levels elsewhere. This may account for the observation that the B6 AZIP mice have less insulin-resistant muscles and more insulin-resistant livers, than do the FVB AZIP mice. B6 wild type, as well as B6 AZIP, mice have increased triglyceride clearance relative to FVB, which may be explained in part by higher serum lipase levels and liver CD36/fatty acid translocase mRNA levels. Thus, it is likely that increased triglyceride clearance in B6, as compared with FVB, mice contributes to the strain differences in insulin resistance and lipid metabolism.

Adrenalectomy improves diabetes in A-ZIP/F-1 lipoatrophic mice by increasing both liver and muscle insulin sensitivity.

The virtually fatless A-ZIP/F-1 mouse is profoundly insulin resistant, diabetic, and a good model for humans with severe generalized lipoatrophy. Like a number of other mouse models of diabetes, the A-ZIP/F-1 mouse has elevated serum corticosterone levels. Leptin infusion lowers the corticosterone levels, suggesting that leptin deficiency contributes to the hypercorticosteronemic state. To test the hypothesis that the increased glucocorticoids contribute to the diabetes and insulin resistance, we examined the effect of adrenalectomy on A-ZIP/F-1 mice. Adrenalectomy significantly decreased the blood glucose, serum insulin, and glycated hemoglobin levels. Hyperinsulinemic-euglycemic clamps were performed to characterize the changes in whole-body and tissue insulin sensitivity. The adrenalectomized A-ZIP/F-1 mice displayed a marked improvement in insulin-induced suppression of endogenous glucose production, indicating increased hepatic insulin sensitivity. Adrenalectomy also increased muscle glucose uptake and glycogen synthesis. These results suggest that the chronically increased serum corticosterone levels contribute to the diabetes of the A-ZIP/F-1 mice and that removal of the glucocorticoid excess improves the insulin sensitivity in both muscle and liver.

Characterization of adiposity and metabolism in Lmna-deficient mice.

Dunnigan's Familial Partial Lipodystrophy (FPLD) is an autosomal dominant disease characterized by regional fat loss and insulin resistance. FPLD is caused by mutations in the LMNA gene, which encodes intermediate filaments of the nuclear lamina. Different LMNA mutations cause Emery-Dreifuss muscular dystrophy and/or a dilated cardiomyopathy. It is not known how LMNA mutations cause any of the disease phenotypes. Here we measure physical and metabolic characteristics of Lmna-/- and +/- mice to determine their usefulness as models for FPLD. Lmna-/- mice, which die prematurely of muscular dystrophy, have little fat, but do not show the insulin resistance characteristic of FPLD. Lmna+/- mice, despite treatment with a high fat diet, do not have decreased fat stores or metabolic features of FPLD. We also show, in mice, that Lmna transcripts are expressed at high levels in muscle and adipose tissue, but do not vary by body region or sex. In conclusion, Lmna+/- and -/- mice do not mimic Dunnigan's FPLD, and differential expression of lamins A and C does not appear to contribute to sex- or tissue-specific LMNA phenotypes.