Laura C. Alonso

The hair cycle

The hair coat, which keeps most mammals warm, dry and protected from harmful elements, requires a constant supply of new hairs throughout the lifetime of the animal. To produce new hairs, existing follicles undergo cycles of growth (anagen), regression (catagen) and rest (telogen). During each anagen phase, follicles produce an entire hair shaft from tip to root; during catagen and telogen, follicles reset and prepare their stem cells so that they can receive the signal to start the next growth phase and make the new hair shaft. The hair cycle represents a remarkable model for studies of the regulation of stem cell quiescence and activation, as well as transit-amplifying cell proliferation, cell-fate choice, differentiation and apoptosis in a regenerative adult epithelial tissue. Here we summarize the major events of the hair cycle, and touch on known regulators of the transitions. Detailed reviews of the hair cycle and its regulation can be found elsewhere (Lavker et al., 2003; Millar, 2002; Muller-Rover et al., 2001).

Defining BMP functions in the hair follicle by conditional ablation of BMP receptor IA

Using conditional gene targeting in mice, we show that BMP receptor IA is essential for the differentiation of progenitor cells of the inner root sheath and hair shaft. Without BMPRIA activation, GATA-3 is down-regulated and its regulated control of IRS differentiation is compromised. In contrast, Lef1 is up-regulated, but its regulated control of hair differentiation is still blocked, and BMPRIA-null follicles fail to activate Lef1/β-catenin–regulated genes, including keratin genes. Wnt-mediated transcriptional activation can be restored by transfecting BMPRIA-null keratinocytes with a constitutively activated β-catenin. This places the block downstream from Lef1 expression but upstream from β-catenin stabilization. Because mice lacking the BMP inhibitor Noggin fail to express Lef1, our findings support a model, whereby a sequential inhibition and then activation of BMPRIA is necessary to define a band of hair progenitor cells, which possess enough Lef1 and stabilized β-catenin to activate the hair specific keratin genes and generate the hair shaft.

Glucose Infusion in Mice: A New Model to Induce β-Cell Replication

Developing new techniques to induce β-cells to replicate is a major goal in diabetes research. Endogenous β-cells replicate in response to metabolic changes, such as obesity and pregnancy, which increase insulin requirement. Mouse genetic models promise to reveal the pathways responsible for compensatory β-cell replication. However, no simple, short-term, physiological replication stimulus exists to test mouse models for compensatory replication. Here, we present a new tool to induce β-cell replication in living mice. Four-day glucose infusion is well tolerated by mice as measured by hemodynamics, body weight, organ weight, food intake, and corticosterone level. Mild sustained hyperglycemia and hyperinsulinemia induce a robust and significant fivefold increase in β-cell replication. Glucose-induced β-cell replication is dose and time dependent. β-Cell mass, islet number, β-cell size, and β-cell death are not altered by glucose infusion over this time frame. Glucose infusion increases both the total protein abundance and nuclear localization of cyclin D2 in islets, which has not been previously reported. Thus, we have developed a new model to study the regulation of compensatory β-cell replication, and we describe important novel characteristics of mouse β-cell responses to glucose in the living pancreas.

Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic β‐cell replication in mice

Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co‐exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic β‐cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5–6% at 60 cycles h−1 for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic β‐cells from 1.5 ± 0.3 to 4.0 ± 0.8% bromodeoxyuridine (BrdU)‐positive) β‐cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in β‐cells, without any additive effects on β‐cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic β‐cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of β‐cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes.

Glucagon Regulates Hepatic Kisspeptin to Impair Insulin Secretion

Early in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from β cells. However, whether hyperglucagonemia is causally linked to β cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on β cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Lepr(db/db) mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

Disruption of Hepatocyte Growth Factor/c-Met Signaling Enhances Pancreatic β-Cell Death and Accelerates the Onset of Diabetes

OBJECTIVE To determine the role of hepatocyte growth factor (HGF)/c-Met on β-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced β-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in β-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and β-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for β-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced β-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and β-cell apoptosis. c-Met-null β-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human β-cells against cytokines. CONCLUSIONS These results show that HGF/c-Met is critical for β-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing β-cell protection.

Loss of HGF/c-Met Signaling in Pancreatic β-Cells Leads to Incomplete Maternal β-Cell Adaptation and Gestational Diabetes Mellitus

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the β-cell. However, whether HGF/c-Met has a role in maternal β-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and β-cell homeostasis in pregnant mice lacking c-Met in the pancreas (PancMet KO mice). Circulating HGF and islet c-Met and HGF expression were increased in pregnant mice. Importantly, PancMet KO mice displayed decreased β-cell replication and increased β-cell apoptosis at gestational day (GD)15. The decreased β-cell replication was associated with reductions in islet prolactin receptor levels, STAT5 nuclear localization and forkhead box M1 mRNA, and upregulation of p27. Furthermore, PancMet KO mouse β-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human β-cells against dexamethasone in vitro. These detrimental alterations in β-cell proliferation and death led to incomplete maternal β-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased β-cell mass was accompanied by increased blood glucose, decreased plasma insulin, and impaired glucose tolerance. PancMet KO mouse islets failed to upregulate GLUT2 and pancreatic duodenal homeobox-1 mRNA, insulin content, and glucose-stimulated insulin secretion during gestation. These studies indicate that HGF/c-Met signaling is essential for maternal β-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.

ChREBP Mediates Glucose-Stimulated Pancreatic β-Cell Proliferation

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2′-deoxyuridine incorporation, [3H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP−/− mice, in INS-1–derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells.

Molecular Genetic and Endocrine Mechanisms of Hair Growth

The prenatal morphogenesis of hair follicles depends upon a precisely regulated series of molecular genetic processes. Hormones and their receptors play prominent roles in modulating postnatal hair cycling, which recapitulates some aspects of morphogenesis. The responses to androgen are the most obvious of these. The postnatal androgen sensitivity of pilosebaceous units in different skin areas is programmed during prenatal development to permit clinical outcomes such as hirsutism and pattern baldness. Thyroid hormone, glucocorticoids, insulin-like growth factor-I, and prolactin have clinically significant effects on specific aspects of hair growth. The nuclear receptors vitamin D receptor and retinoid X receptor are essential for postnatal hair cycling. Other hormones have less clear effects on hair growth. Advances in research on the interaction of hormone target genes with the biological processes involved in hair morphogenesis and cycling can be expected to improve management of hirsutism and alopecia.

Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced β-cell mass (11, 62). Therapies aimed at expanding β-cell mass may be useful to treat T2D (14). Although feeding rodents a high-fat diet (HFD) for an extended period (3-6 mo) increases β-cell mass by inducing β-cell proliferation (16, 20, 53, 54), evidence suggests that adult human β-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human β-cells, are not known. To develop a model to identify early drivers of β-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The β-cell proliferative response also began within the first week of HFD feeding. Intriguingly, β-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory β-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse β-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human β-cell proliferation.