Defining the In Vivo Role of mTORC1 in Thyrocytes by Studying the TSC2 Conditional Knockout Mouse Model.

Background: The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathway's overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. Methods: To elucidate mTORC1's role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting tuberous sclerosis complex 2 (TSC2), an intracellular inhibitor of mTORC1. Results: The resulting TPO-TSC2KO mice exhibited a 70-80% reduction in TSC2 levels, leading to a sixfold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, with larger follicles and increased colloid and epithelium areas. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone biosynthesis, including thyroglobulin (Tg), thyroid peroxidase (Tpo), and sodium-iodide symporter (Nis), while Tff1, Pax8, and Mct8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although total thyroxine (T4), total triiodothyronine (T3), and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared with littermate control mice. Conclusions: Our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits thyroid hormone (TH) biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation.

Predictors of lack of glycemic control in persons with type 2 diabetes.

BACKGROUND: Professional guidelines recommend an HbA1c < 7% for most people with diabetes and < 8.5% for those with relaxed glycemic goals. However, many people with type 2 diabetes mellitus (T2DM) are unable to achieve the desired HbA1c goal. This study evaluated factors associated with lack of improvement in HbA1c over 3 years. METHODS: All patients with T2DM treated within a major academic healthcare system during 2015-2020, who had at least one HbA1c value > 8.5% within 3 years from their last HbA1c were included in analysis. Patients were grouped as improved glycemic control (last HbA1c ≤ 8.5%) or lack of improvement (last HbA1c > 8.5%). Multivariate logistic regression analysis was performed to assess independent predictors of lack of improvement in glycemic control. RESULTS: Out of 2,232 patients who met the inclusion criteria, 1,383 had an improvement in HbA1c while 849 did not. In the fully adjusted model, independent predictors of lack of improvement included: younger age (odds ratio, 0.89 per 1-SD [12 years]; 95% CI, 0.79-1.00), female gender (1.30, 1.08-1.56), presence of hypertension (1.29, 1.08-1.55), belonging to Black race (1.32, 1.04-1.68, White as reference), living in low income area (1.86,1.28-2.68, high income area as reference), and insurance coverage other than Medicare (1.32, 1.05-1.66). Presence of current smoking was associated with a paradoxical improvement in HbA1c (0.69, 0.47-0.99). In a subgroup analysis, comparing those with all subsequent HbA1c values > 8.5% (N = 444) to those with all subsequent HbA1c values < 8.5% (N = 341), similar factors were associated with lack of improvement, but smoking was no longer significant. CONCLUSION: We conclude that socioeconomic factors like race, type of insurance coverage and living in low-income areas are associated with lack of improvement in HbA1c over a period of 3-years in people with T2DM. Intervention strategies focused on low-income neighborhoods need to be designed to improve diabetes management.

GHRH agonist MR-409 protects ß-cells from streptozotocin-induced diabetes.

Patients with type 1 diabetes (T1D) suffer from insufficient functional ß-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated ß-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and in vivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on ß-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in ß-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in ß-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of ß-cell mass. Increased IRS2 expression in ß-cells in the group treated with MR-409 corroborated the in vitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 in vivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of ß-cells death in T1D.

Mapping the metabolic reprogramming induced by sodium-glucose cotransporter 2 inhibition.

Diabetes is associated with increased risk for kidney disease, heart failure, and mortality. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) prevent these adverse outcomes; however, the mechanisms involved are not clear. We generated a roadmap of the metabolic alterations that occur in different organs in diabetes and in response to SGLT2i. In vivo metabolic labeling with 13C-glucose in normoglycemic and diabetic mice treated with or without dapagliflozin, followed by metabolomics and metabolic flux analyses, showed that, in diabetes, glycolysis and glucose oxidation are impaired in the kidney, liver, and heart. Treatment with dapagliflozin failed to rescue glycolysis. SGLT2 inhibition increased glucose oxidation in all organs; in the kidney, this was associated with modulation of the redox state. Diabetes was associated with altered methionine cycle metabolism, evident by decreased betaine and methionine levels, whereas treatment with SGLT2i increased hepatic betaine along with decreased homocysteine levels. mTORC1 activity was inhibited by SGLT2i along with stimulation of AMPK in both normoglycemic and diabetic animals, possibly explaining the protective effects against kidney, liver, and heart diseases. Collectively, our findings suggest that SGLT2i induces metabolic reprogramming orchestrated by AMPK-mTORC1 signaling with common and distinct effects in various tissues, with implications for diabetes and aging.

Novel roles of mTORC2 in regulation of insulin secretion by actin filament remodeling.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in ß-cells has been extensively studied, less is known about mTORC2's function in ß-cells. Here, we show that mice with constitutive and inducible ß-cell-specific deletion of RICTOR (ßRicKO and ißRicKO mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in ßRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in ßRicKO islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.NEW & NOTEWORTHY The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

Medicaid insured persons with diabetes have increased proportion of missed appointments and high HbA1c.

Objective: This study was conducted to evaluate whether the type of insurance coverage is associated with missed appointments and to evaluate the effect of missed appointments on diabetes control. Methods: All patients with diabetes mellitus (DM) managed at a major academic medical center between Jan 2015 and Dec 2020 were included in analysis. Association between insurance coverage and the proportion of missed appointments was evaluated with adjustments for demographic variables and social determinants of health. The relationship between proportion of missed appointments and glycemic control was also evaluated. Results: The dataset included 30,633 patients, out of which 14,064 (46%) reported commercial insurance, 13,376 (44%) reported Medicare and 3,193 (10%) reported Medicaid coverage. Proportion of missed appointments was 18.1 ± 18.1% among Medicaid covered patients,12.1 ± 15.3% among commercially insured and 10.2 ± 14.1% among Medicare covered patients (p < 0.001). Type of insurance was found to be a significant predictor of proportion of missed appointments after adjusting for age, race, language, marital status, smoking, BMI, HbA1c and type of diabetes (p < 0.001) in series regression analysis. Proportion of missed appointments was associated with HbA1c with partial correlation coefficient +0.104 (p < 0.005) after adjusting for age, race, gender, type of insurance coverage, BMI and type of diabetes. Conclusions: Medicaid covered patients with diabetes have higher proportion of missed clinic appointments and higher HbA1c. More research is needed to evaluate the root causes of inability to keep appointments in this population so that strategies for improved healthcare delivery can be designed.

Pancreatic and duodenal homeobox-1 (PDX1) contributes to ß-cell mass expansion and proliferation induced by Akt/PKB pathway.

Maintenance of pancreatic ß-cell mass and function is fundamental to glucose homeostasis and to prevent diabetes. The PI3 K-Akt-mTORC1 pathway is critical for ß-cells mass and function, while PDX1 has been implicated in ß-cell development, maturation, and function. Here we tested whether Akt signaling requires PDX1 expression to regulate ß-cell mass, proliferation, and glucose homeostasis. In order to address that, we crossed a mouse model overexpressing constitutively active Akt mutant in ß-cells (ß-caAkt) with mice lacking one allele of PDX1gene (ß-caAkt/pdx1+/-). While the ß-caAkt mice exhibit higher plasma insulin levels, greater ß-cell mass and improved glucose tolerance compared to control mice, the ß-caAkt/pdx1+/- mice are hyperglycemic and intolerant to glucose. The changes in glucose homeostasis in ß-caAkt/pdx1+/- were associated with a 60% reduction in ß-cell mass compared to ß-caAkt mice. The impaired ß-cell mass in the ß-caAkt/pdx1+/- mice can be explained by a lesser ß-cell proliferation measured by the number of Ki67 positive ß-cells. We did not observe any differences in apoptosis between ß-caAkt/pdx1+/- and ß-caAkt mice. In conclusion, PDX1 contributes to ß-cell mass expansion and glucose metabolism induced by activation of Akt signaling.

The regulatory G protein signaling complex, Gß5-R7, promotes glucose- and extracellular signal-stimulated insulin secretion.

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gß5-R7, a protein complex consisting of the atypical G protein ß subunit Gß5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit ß 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gß5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gß5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Hypusine biosynthesis in ß cells links polyamine metabolism to facultative cellular proliferation to maintain glucose homeostasis.

Deoxyhypusine synthase (DHPS) uses the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet ß cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces ß cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired ß cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in ß cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.

mTORC1 to AMPK switching underlies ß-cell metabolic plasticity during maturation and diabetes.

Pancreatic beta cells (ß-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of ß-cell function, we found that the control of cellular signaling in ß-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult ß-cell phenotype. While forcing constitutive mTORC1 signaling in adult ß-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote ß-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult ß-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate ß-cell metabolism may thus offer new targets to improve ß-cell function in diabetes.

Sexual dimorphism in hypothalamic inflammation in the offspring of dams exposed to a diet rich in high fat and branched-chain amino acids.

Branched-chain amino acid (BCAAs: leucine, isoleucine, and valine) contribute to the development of obesity-associated insulin resistance in the context of consumption of a high-fat diet (HFD) in humans and rodents. Maternal diet is a major determinant of offspring health, and there is strong evidence that maternal HFD alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. In this study, we exposed pregnant and lactating C57BL/6JB female mice to either HFD, HFD with supplemented BCAA (HFD+BCAA), or standard diet (SC), and we studied offspring metabolic phenotypes. Both maternal HFD and HFD supplemented with BCAA had similar effect rendering the offspring metabolic imbalance and impairing their ability to cope with HFD when challenged during aging. The metabolic effects of HFD challenge were more profound in females, worsening female offspring ability to cope with an HFD challenge by activating hypothalamic inflammation in aging. Moreover, the sex differences in hypothalamic estrogen receptor α (ER-α) expression levels were lost in female offspring upon HFD challenge, supporting a link between ER-α levels and hypothalamic inflammation in offspring and highlighting the programming potential of hypothalamic inflammatory responses and maternal nutrition.

Early pancreatic islet fate and maturation is controlled through RBP-Jκ.

Notch signaling is known to control early pancreatic differentiation through Ngn3 repression. In later stages, downstream of Notch, the Presenilins are still required to maintain the endocrine fate allocation. Amongst their multiple targets, it remains unclear which one actually controls the maintenance of the fate of the early islets. Conditional deletions of the Notch effector RBP-Jκ with lineage tracing in Presenilin-deficient endocrine progenitors, demonstrated that this factor is central to the control of the fate through a non-canonical Notch mechanism. RBP-Jκ mice exhibit normal islet morphogenesis and function, however, a fraction of the progenitors fails to differentiate and develop into disorganized masses resembling acinar to ductal metaplasia and chronic pancreatitis. A subsequent deletion of RBP-Jκ in forming ß-cells led to the transdifferentiation into the other endocrine cells types, indicating that this factor still mediates the maintenance of the fate within the endocrine lineage itself. These results highlight the dual importance of Notch signaling for the endocrine lineage. Even after Ngn3 expression, Notch activity is required to maintain both fate and maturation of the Ngn3 progenitors. In a subset of the cells, these alterations of Notch signaling halt their differentiation and leads to acinar to ductal metaplasia.

4E-BP2/SH2B1/IRS2 Are Part of a Novel Feedback Loop That Controls ß-Cell Mass.

The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and ß-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and ß-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of ß-cells.

Disruption of O-linked N-Acetylglucosamine Signaling Induces ER Stress and ß Cell Failure.

Nutrient levels dictate the activity of O-linked N-acetylglucosamine transferase (OGT) to regulate O-GlcNAcylation, a post-translational modification mechanism to "fine-tune" intracellular signaling and metabolic status. However, the requirement of O-GlcNAcylation for maintaining glucose homeostasis by regulating pancreatic ß cell mass and function is unclear. Here, we reveal that mice lacking ß cell OGT (ßOGT-KO) develop diabetes and ß cell failure. ßOGT-KO mice demonstrated increased ER stress and distended ER architecture, and these changes ultimately caused the loss of ß cell mass due to ER-stress-induced apoptosis and decreased proliferation. Akt1/2 signaling was also dampened in ßOGT-KO islets. The mechanistic role of these processes was demonstrated by rescuing the phenotype of ßOGT-KO mice with concomitant Chop gene deletion or genetic reconstitution of Akt2. These findings identify OGT as a regulator of ß cell mass and function and provide a direct link between O-GlcNAcylation and ß cell survival by regulation of ER stress responses and modulation of Akt1/2 signaling.

Human ß-cell proliferation and intracellular signaling: part 3.

This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic ß-cells. We contrast the large knowledge base in rodent ß-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning ß-cells, there is great urgency to identify therapeutic approaches to expand human ß-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous ß-cells. In these Perspectives, we focus on ß-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin-nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/ß-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal-related kinase, cadherins and integrins, G-protein-coupled receptors, and transforming growth factor ß signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human ß-cell regeneration for diabetes.

Cyclin C stimulates ß-cell proliferation in rat and human pancreatic ß-cells.

Activation of pancreatic ß-cell proliferation has been proposed as an approach to replace reduced functional ß-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic ß-cell proliferation. We hypothesized that cyclin C overexpression would induce ß-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional ß-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [(3)H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected ß-cell death by TUNEL, ß-cell differentiation by RT-PCR, and ß-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human ß-cell proliferation. This augmented proliferation did not induce ß-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing ß-cell regeneration.

Human ß-cell proliferation and intracellular signaling part 2: still driving in the dark without a road map.

Enhancing ß-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of ß-cell intracellular signaling pathways that promote ß-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent ß-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human ß-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human ß-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for ß-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human ß-cell replication. This is a critical aspect in the long-term goal of expanding human ß-cells for the prevention and/or cure of type 1 and type 2 diabetes.

Administration of saccharin to neonatal mice influences body composition of adult males and reduces body weight of females.

Nutritional or pharmacological perturbations during perinatal growth can cause persistent effects on the function of white adipose tissue, altering susceptibility to obesity later in life. Previous studies have established that saccharin, a nonnutritive sweetener, inhibits lipolysis in mature adipocytes and stimulates adipogenesis. Thus, the current study tested whether neonatal exposure to saccharin via maternal lactation increased susceptibility of mice to diet-induced obesity. Saccharin decreased body weight of female mice beginning postnatal week 3. Decreased liver weights on week 14 corroborated this diminished body weight. Initially, saccharin also reduced male mouse body weight. By week 5, weights transiently rebounded above controls, and by week 14, male body weights did not differ. Body composition analysis revealed that saccharin increased lean and decreased fat mass of male mice, the latter due to decreased adipocyte size and epididymal, perirenal, and sc adipose weights. A mild improvement in glucose tolerance without a change in insulin sensitivity or secretion aligned with this leaner phenotype. Interestingly, microcomputed tomography analysis indicated that saccharin also increased cortical and trabecular bone mass of male mice and modified cortical bone alone in female mice. A modest increase in circulating testosterone may contribute to the leaner phenotype in male mice. Accordingly, the current study established a developmental period in which saccharin at high concentrations reduces adiposity and increases lean and bone mass in male mice while decreasing generalized growth in female mice.

mTORC1 signaling and regulation of pancreatic ß-cell mass.

The capacity of ß cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of ß cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for ß-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of ß-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in ß cells. This review will discuss recent advances in the understanding of how this pathway regulates ß-cell mass and present data on the role of TSC1 in modulation of ß-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic ß cells results in improved glucose tolerance, hyperinsulinemia and expansion of ß-cell mass that persists with aging.

Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3ß independent of JNK activation.

BACKGROUND: The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic ß-cell death. This study examines the synergistic effects of glucose and FFA on ß-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined. PRINCIPAL FINDINGS: Increasing glucose (5-25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3ß (Gsk3ß) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3ß. CONCLUSIONS/SIGNIFICANCE: Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3ß activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet ß-cell Gsk3ß as a potential therapeutic target.