Abnormalities in microbiota/butyrate/FFAR3 signaling in aging gut impair brain function.

Aging-related abnormalities in gut microbiota are associated with cognitive decline, depression, and anxiety, but underlying mechanisms remain unstudied. Here, our study demonstrated that transplanting old gut microbiota to young mice induced inflammation in the gut and brain coupled with cognitive decline, depression, and anxiety. We observed diminished mucin formation and increased gut permeability ("leaky gut") with a reduction in beneficial metabolites like butyrate because of decline in butyrate-producing bacteria in the aged gut microbiota. This led to suppressed expression of butyrate receptors, free fatty acid receptors 2 and 3 (FFAR2/3). Administering butyrate alleviated inflammation, restored mucin expression and gut barriers, and corrected brain dysfunction. Furthermore, young mice with intestine-specific loss of FFAR2/3 exhibited gut and brain abnormalities akin to those in older mice. Our results demonstrate that reduced butyrate-producing bacteria in aged gut microbiota result in low butyrate levels and reduced FFAR2/3 signaling, leading to suppressed mucin formation that increases gut permeability, inflammation, and brain abnormalities. These findings underscore the significance of butyrate-FFAR2/3 agonism as a potential strategy to mitigate aged gut microbiota-induced detrimental effects on gut and brain health in older adults.

Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting ß cell differentiation.

Expanding ß cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain ß cell number. ß cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of ß cell mass in IRS2-deficient mice. Surprisingly, not only ß cell mass but also ß cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical ß cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that ß cell mass can be expanded without compromising function.

CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism.

Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle-specific deletion of Cdk4's target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber-specification that is of relevance to metabolic and muscular diseases.

A distinct hypothalamus-to-ß cell circuit modulates insulin secretion.

The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic ß cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to ß cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVNOXT), and it reaches the islets of the endocrine pancreas via the sympathetic autonomic branch to innervate ß cells. Stimulation of PVNOXT neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in ß cells and induces hypoglycemia. PVNOXT neuronal activity is triggered by glucoprivation. Our findings reveal that a subset of PVNOXT neurons form functional multisynaptic circuits with ß cells in mice to regulate insulin secretion, and their function is necessary for the ß cell response to hypoglycemia.

CDK2 limits the highly energetic secretory program of mature ß cells by restricting PEP cycle-dependent KATP channel closure.

Hallmarks of mature ß cells are restricted proliferation and a highly energetic secretory state. Paradoxically, cyclin-dependent kinase 2 (CDK2) is synthesized throughout adulthood, its cytosolic localization raising the likelihood of cell cycle-independent functions. In the absence of any changes in ß cell mass, maturity, or proliferation, genetic deletion of Cdk2 in adult ß cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. At the single ß cell level, CDK2 restricts insulin secretion by increasing KATP conductance, raising the set point for membrane depolarization in response to activation of the phosphoenolpyruvate (PEP) cycle with mitochondrial fuels. In parallel with reduced ß cell recruitment, CDK2 restricts oxidative glucose metabolism while promoting glucose-dependent amplification of insulin secretion. This study provides evidence of essential, non-canonical functions of CDK2 in the secretory pathways of quiescent ß cells.

TGF-ß Signaling in Pancreatic Islet ß Cell Development and Function.

Pancreatic islet beta cells (ß-cells) synthesize and secrete insulin in response to rising glucose levels and thus are a prime target in both major forms of diabetes. Type 1 diabetes ensues due to autoimmune destruction of ß-cells. On the other hand, the prevailing insulin resistance and hyperglycemia in type 2 diabetes (T2D) elicits a compensatory response from ß-cells that involves increases in ß-cell mass and function. However, the sustained metabolic stress results in ß-cell failure, characterized by severe ß-cell dysfunction and loss of ß-cell mass. Dynamic changes to ß-cell mass also occur during pancreatic development that involves extensive growth and morphogenesis. These orchestrated events are triggered by multiple signaling pathways, including those representing the transforming growth factor ß (TGF-ß) superfamily. TGF-ß pathway ligands play important roles during endocrine pancreas development, ß-cell proliferation, differentiation, and apoptosis. Furthermore, new findings are suggestive of TGF-ß's role in regulation of adult ß-cell mass and function. Collectively, these findings support the therapeutic utility of targeting TGF-ß in diabetes. Summarizing the role of the various TGF-ß pathway ligands in ß-cell development, growth and function in normal physiology, and during diabetes pathogenesis is the topic of this mini-review.

Protection from ß-cell apoptosis by inhibition of TGF-ß/Smad3 signaling.

Prevailing insulin resistance and the resultant hyperglycemia elicits a compensatory response from pancreatic islet beta cells (ß-cells) that involves increases in ß-cell function and ß-cell mass. However, the sustained metabolic stress eventually leads to ß-cell failure characterized by severe ß-cell dysfunction and progressive loss of ß-cell mass. Whereas, ß-cell dysfunction is relatively well understood at the mechanistic level, the avenues leading to loss of ß-cell mass are less clear with reduced proliferation, dedifferentiation, and apoptosis all potential mechanisms. Butler and colleagues documented increased ß-cell apoptosis in pancreas from lean and obese human Type 2 diabetes (T2D) subjects, with no changes in rates of ß-cell replication or neogenesis, strongly suggesting a role for apoptosis in ß-cell failure. Here, we describe a permissive role for TGF-ß/Smad3 in ß-cell apoptosis. Human islets undergoing ß-cell apoptosis release increased levels of TGF-ß1 ligand and phosphorylation levels of TGF-ß's chief transcription factor, Smad3, are increased in human T2D islets suggestive of an autocrine role for TGF-ß/Smad3 signaling in ß-cell apoptosis. Smad3 phosphorylation is similarly increased in diabetic mouse islets undergoing ß-cell apoptosis. In mice, ß-cell-specific activation of Smad3 promotes apoptosis and loss of ß-cell mass in association with ß-cell dysfunction, glucose intolerance, and diabetes. In contrast, inactive Smad3 protects from apoptosis and preserves ß-cell mass while improving ß-cell function and glucose tolerance. At the molecular level, Smad3 associates with Foxo1 to propagate TGF-ß-dependent ß-cell apoptosis. Indeed, genetic or pharmacologic inhibition of TGF-ß/Smad3 signals or knocking down Foxo1 protects from ß-cell apoptosis. These findings reveal the importance of TGF-ß/Smad3 in promoting ß-cell apoptosis and demonstrate the therapeutic potential of TGF-ß/Smad3 antagonism to restore ß-cell mass lost in diabetes.

TGF-ß receptor 1 regulates progenitors that promote browning of white fat.

OBJECTIVE: Beige/brite adipose tissue displays morphological characteristics and beneficial metabolic traits of brown adipose tissue. Previously, we showed that TGF-ß signaling regulates the browning of white adipose tissue. Here, we inquired whether TGF-ß signals regulated presumptive beige progenitors in white fat and investigated the TGF-ß regulated mechanisms involved in beige adipogenesis. METHODS: We deleted TGF-ß receptor 1 (TßRI) in adipose tissue (TßRIAdKO mice) and, using flow-cytometry based assays, identified and isolated presumptive beige progenitors located in the stromal vascular cells of white fat. These cells were molecularly characterized to examine beige/brown marker expression and to investigate TGF-ß dependent mechanisms. Further, the cells were transplanted into athymic nude mice to examine their adipogenesis potential. RESULTS: Deletion of TßRI promotes beige adipogenesis while reducing the detrimental effects of high fat diet feeding. Interaction of TGF-ß signaling with the prostaglandin pathway regulated the appearance of beige adipocytes in white fat. Using flow cytometry techniques and stromal vascular fraction from white fat, we isolated presumptive beige stem/progenitor cells (iBSCs). Upon genetic or pharmacologic inhibition of TGF-ß signaling, these cells express high levels of predominantly beige markers. Transplantation of TßRI-deficient stromal vascular cells or iBSCs into athymic nude mice followed by high fat diet feeding and stimulation of ß-adrenergic signaling via CL316,243 injection or cold exposure promoted robust beige adipogenesis in vivo. CONCLUSIONS: TßRI signals target the prostaglandin network to regulate presumptive beige progenitors in white fat capable of developing into beige adipocytes with functional attributes. Controlled inhibition of TßRI signaling and concomitant PGE2 stimulation has the potential to promote beige adipogenesis and improve metabolism.

Flow Cytometry Assisted Isolation of Adipose Tissue Derived Stem Cells.

Adipose tissue dysfunction is typically seen in metabolic diseases, particularly obesity and diabetes. White adipocytes store fat while brown adipocyte dissipates it via thermogenesis. In addition, beige adipocytes develop in white fat depots in response to stimulation of ß-adrenergic pathways. It appears that the three types of adipocytes-white, brown, and beige-can be formed de novo from stem/precursor cells or via transdifferentiation. Identifying the presumptive progenitors that harbor capacity to differentiate to these distinct adipocyte cell types will enable their functional characterization. Moreover, the presence or absence of white/brown/beige adipocytes is correlated with metabolic dysfunction making their study of medical relevance. Robust, reliable, and reproducible methods of identification and isolation of adipocyte progenitors will stimulate further detailed understanding of white, brown, and beige adipogenesis.

Basal Ganglia Dysfunction Contributes to Physical Inactivity in Obesity.

Obesity is associated with physical inactivity, which exacerbates the health consequences of weight gain. However, the mechanisms that mediate this association are unknown. We hypothesized that deficits in dopamine signaling contribute to physical inactivity in obesity. To investigate this, we quantified multiple aspects of dopamine signaling in lean and obese mice. We found that D2-type receptor (D2R) binding in the striatum, but not D1-type receptor binding or dopamine levels, was reduced in obese mice. Genetically removing D2Rs from striatal medium spiny neurons was sufficient to reduce motor activity in lean mice, whereas restoring Gi signaling in these neurons increased activity in obese mice. Surprisingly, although mice with low D2Rs were less active, they were not more vulnerable to diet-induced weight gain than control mice. We conclude that deficits in striatal D2R signaling contribute to physical inactivity in obesity, but inactivity is more a consequence than a cause of obesity.

Loss of Cyclin-dependent Kinase 2 in the Pancreas Links Primary ß-Cell Dysfunction to Progressive Depletion of ß-Cell Mass and Diabetes.

The failure of pancreatic islet ß-cells is a major contributor to the etiology of type 2 diabetes. ß-Cell dysfunction and declining ß-cell mass are two mechanisms that contribute to this failure, although it is unclear whether they are molecularly linked. Here, we show that the cell cycle regulator, cyclin-dependent kinase 2 (CDK2), couples primary ß-cell dysfunction to the progressive deterioration of ß-cell mass in diabetes. Mice with pancreas-specific deletion of Cdk2 are glucose-intolerant, primarily due to defects in glucose-stimulated insulin secretion. Accompanying this loss of secretion are defects in ß-cell metabolism and perturbed mitochondrial structure. Persistent insulin secretion defects culminate in progressive deficits in ß-cell proliferation, reduced ß-cell mass, and diabetes. These outcomes may be mediated directly by the loss of CDK2, which binds to and phosphorylates the transcription factor FOXO1 in a glucose-dependent manner. Further, we identified a requirement for CDK2 in the compensatory increases in ß-cell mass that occur in response to age- and diet-induced stress. Thus, CDK2 serves as an important nexus linking primary ß-cell dysfunction to progressive ß-cell mass deterioration in diabetes.

TGF-ß1/Smad3 Pathway Targets PP2A-AMPK-FoxO1 Signaling to Regulate Hepatic Gluconeogenesis.

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-ß1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-ß1/Smad3 signals suppressed endogenous glucose production. TGF-ß1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-ß1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-ß1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance.

SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) during exercise.

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-ß effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3(-/-)) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-ß, a TGF-ß receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3(-/-) mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-ß suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle.

Telomere dysfunction suppresses multiple endocrine neoplasia in mice.

Multiple endocrine neoplasia (MEN) syndrome is typified by the occurrence of tumors in two or more hormonal tissues. Whereas the genetics of MEN syndrome is relatively well understood, the tumorigenic mechanisms for these cancers remain relatively obscure. The Cdk4 (R24C) mouse model develops highly penetrant pituitary tumors and endocrine pancreas adenomas, and, as such, this model is appropriate to gain insight into mechanisms underlying MEN. Using this model, here we provide evidence supporting an important role for telomerase in the pathogenesis of MEN. We observed increased aneuploidy in Cdk4 (R/R) fibroblasts along with significantly elevated telomerase activity and telomere length in Cdk4 (R/R) islets and embryonic fibroblasts. To better understand the role of telomerase, we generated Cdk4 (R24C) mice with inactivation of the mTERC locus, which codes for the essential RNA component of the enzyme telomerase (mTERC (-/-) Cdk4 (R/R) mice). Embryonic fibroblasts and islets derived from mTERC (-/-) Cdk4 (R/R) mice exhibit reduced telomere length and proliferative capacity. Further, mTERC (-/-) Cdk4 (R/R) fibroblasts display reduced transformation potential. Importantly, mTERC (-/-) Cdk4 (R/R) mice display significantly reduced spontaneous tumorigenesis. Strikingly, we observed dramatic suppression of pituitary tumors and endocrine pancreas adenomas in mTERC (-/-) Cdk4 (R/R) mice. Telomere dysfunction suppressed tumor initiation and increased latency of tumor development while not affecting the progression of established tumors. In summary, these results are suggestive of an important role for telomerase in tumor development in the Cdk4 (R24C) mouse model, specifically in the genesis of tumors in the pituitary and the endocrine pancreas.

Transforming growth factor-ß3 (TGF-ß3) knock-in ameliorates inflammation due to TGF-ß1 deficiency while promoting glucose tolerance.

Three homologues of TGF-ß exist in mammals as follows: TGF-ß1, TGF-ß2, and TGF-ß3. All three proteins share high homology in their amino acid sequence, yet each TGF-ß isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-ß proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-ß knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-ß1 ligand with a sequence from TGF-ß3 using targeted recombination to create chimeric TGF-ß1/3 knock-in mice (TGF-ß1(Lß3/Lß3)). In the TGF-ß1(Lß3/Lß3) mouse, localization and activation still occur through the TGF-ß1 latent associated peptide, but cell signaling is triggered through the TGF-ß3 ligand that binds to TGF-ß receptors. Unlike TGF-ß1(-/-) mice, the TGF-ß1(Lß3/Lß3) mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-ß3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-ß1 deficiency. However, the TGF-ß1(Lß3/Lß3) mice have a shortened life span and display tooth and bone defects, indicating that the TGF-ß homologues are not completely interchangeable. Remarkably, the TGF-ß1(Lß3/Lß3) mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-ß isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-ß pathway in human disease.

Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion.

Obesity and diabetes are associated with excess caloric intake and reduced energy expenditure resulting in a negative energy balance. The incidence of diabetes has reached epidemic proportions, and childhood diabetes and obesity are increasing alarmingly. Therefore, it is important to develop safe, easily deliverable, and economically viable treatment alternatives for these diseases. Here, we provide data supporting the candidacy of probiotics as such a therapeutic modality against obesity and diabetes. Probiotics are live bacteria that colonize the gastrointestinal tract and impart beneficial effects for health. However, their widespread prescription as medical therapies is limited primarily because of the paucity of our understanding of their mechanism of action. Here, we demonstrate that the administration of a probiotic, VSL#3, prevented and treated obesity and diabetes in several mouse models. VSL#3 suppressed body weight gain and insulin resistance via modulation of the gut flora composition. VSL#3 promoted the release of the hormone GLP-1, resulting in reduced food intake and improved glucose tolerance. The VSL#3-induced changes were associated with an increase in the levels of a short chain fatty acid (SCFA), butyrate. Using a cell culture system, we demonstrate that butyrate stimulated the release of GLP-1 from intestinal L-cells, thereby providing a plausible mechanism for VSL#3 action. These findings suggest that probiotics such as VSL#3 can modulate the gut microbiota-SCFA-hormone axis. Moreover, our results indicate that probiotics are of potential therapeutic utility to counter obesity and diabetes.

TGF-ß/Smad3 Signaling Regulates Brown Adipocyte Induction in White Adipose Tissue.

Recent identification of active brown fat reserves in adult humans has re-stimulated interest in the role of brown adipocytes in energy homeostasis. In addition, there is accumulating evidence to support the concept of an alteration in energy balance through acquisition of brown fat features in traditional white fat depots. We recently described an important role played by the TGF-ß/Smad3 signaling pathway in modulating the appearance of brown adipocytes in traditional white fat, and its implications to thermogenesis, mitochondrial energetics, energy expenditure, and protection from diabetes and obesity. Here we review the data supporting this phenomenon and put into perspective the promise of conversion of white fat to a brown fat state as a potential therapeutic option for obesity and diabetes.