Rachel C. Wills

Cytoplasmic-Nuclear Trafficking of G1/S Cell Cycle Molecules and Adult Human β-Cell Replication: A Revised Model of Human β-Cell G1/S Control

Harnessing control of human β-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human β-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human β-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating β-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in β-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein–tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human β-cell. In addition to known obstacles to β-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human β-cell expansion.

Human Pancreatic β-Cell G1/S Molecule Cell Cycle Atlas

Expansion of pancreatic β-cells is a key goal of diabetes research, yet induction of adult human β-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human β-cell. Here, we provide a comprehensive immunocytochemical “atlas” of G1/S control molecules in the human β-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the β-cell. More importantly, and in contrast to anticipated results, the human β-cell G1/S atlas reveals that almost all of the critical G1/S cell cycle control molecules are located in the cytoplasm of the quiescent human β-cell. Indeed, the only nuclear G1/S molecules are the cell cycle inhibitors, pRb, p57, and variably, p21: none of the cyclins or cdks necessary to drive human β-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human β-cells to proliferation. Thus, in addition to known obstacles to human β-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human β-cell represents an unanticipated obstacle to therapeutic human β-cell expansion.

Impact of Reduced ATGL-Mediated Adipocyte Lipolysis on Obesity-Associated Insulin Resistance and Inflammation in Male Mice

Emerging evidence suggests that impaired regulation of adipocyte lipolysis contributes to the proinflammatory immune cell infiltration of metabolic tissues in obesity, a process that is proposed to contribute to the development and exacerbation of insulin resistance. To test this hypothesis in vivo, we generated mice with adipocyte-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme catalyzing triacylglycerol hydrolysis. In contrast to previous models, adiponectin-driven Cre expression was used for targeted ATGL deletion. The resulting adipocyte-specific ATGL knockout (AAKO) mice were then characterized for metabolic and immune phenotypes. Lean and diet-induced obese AAKO mice had reduced adipocyte lipolysis, serum lipids, systemic lipid oxidation, and expression of peroxisome proliferator-activated receptor alpha target genes in adipose tissue (AT) and liver. These changes did not increase overall body weight or fat mass in AAKO mice by 24 weeks of age, in part due to reduced expression of genes involved in lipid uptake, synthesis, and adipogenesis. Systemic glucose and insulin tolerance were improved in AAKO mice, primarily due to enhanced hepatic insulin signaling, which was accompanied by marked reduction in diet-induced hepatic steatosis as well as hepatic immune cell infiltration and activation. In contrast, although adipocyte ATGL deletion reduced AT immune cell infiltration in response to an acute lipolytic stimulus, it was not sufficient to ameliorate, and may even exacerbate, chronic inflammatory changes that occur in AT in response to diet-induced obesity.

Serum Autotaxin/ENPP2 correlates with insulin resistance in older humans with obesity

Autotaxin (ATX) is an adipocyte‐derived lysophospholipase D that generates the lipid signaling molecule lysophosphatidic acid (LPA). The ATX/LPA pathway in adipose tissue has recently been implicated in obesity and insulin resistance in animal models, but the role of circulating ATX in humans remains unclear. The aim of the present study was to determine the relationship between serum ATX and insulin resistance.

Adipose triglyceride lipase deletion from adipocytes, but not skeletal myocytes, impairs acute exercise performance in mice

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triacylglycerol hydrolysis in virtually all cells, including adipocytes and skeletal myocytes, and hence, plays a critical role in mobilizing fatty acids. Global ATGL deficiency promotes skeletal myopathy and exercise intolerance in mice and humans, and yet the tissue-specific contributions to these phenotypes remain unknown. The goal of this study was to determine the relative contribution of ATGL-mediated triacylglycerol hydrolysis in adipocytes vs. skeletal myocytes to acute exercise performance. To achieve this goal, we generated murine models with adipocyte- and skeletal myocyte-specific targeted deletion of ATGL. We then subjected untrained mice to acute peak and submaximal exercise interventions and assessed exercise performance and energy substrate metabolism. Impaired ATGL-mediated lipolysis within adipocytes reduced peak and submaximal exercise performance, reduced peripheral energy substrate availability, shifted energy substrate preference toward carbohydrate oxidation, and decreased HSL Ser(660) phosphorylation and mitochondrial respiration within skeletal muscle. In contrast, impaired ATGL-mediated lipolysis within skeletal myocytes was not sufficient to reduce peak and submaximal exercise performance or peripheral energy substrate availability and instead tended to enhance metabolic flexibility during peak exercise. Furthermore, the expanded intramyocellular triacylglycerol pool in these mice was reduced following exercise in association with preserved HSL phosphorylation, suggesting that HSL may compensate for impaired ATGL action in skeletal muscle during exercise. These data suggest that adipocyte rather than skeletal myocyte ATGL-mediated lipolysis plays a greater role during acute exercise in part because of compensatory mechanisms that maintain lipolysis in muscle, but not adipose tissue, when ATGL is absent.

SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes

Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a ‘cis’ configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in ‘trans’ at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in ‘trans’, unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate ‘cis’ activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER–PM contact sites

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)–resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2. Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2. Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase–mediated conversion to PI(4,5)P2. Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes

Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome–lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.Lysosomal distribution is linked to the role of lysosomes in many cellular functions. Here the authors show that the lysosomal protein TMEM55B is regulated by TFEB and recruits JIP4 to the lysosomal surface inducing dynein-dependent transport of lysosomes toward the cell center in response to stress conditions.

Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells.

Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional β-cell mass. Developmental differences, failure of adaptive expansion and loss of β-cells via β-cell death or de-differentiation have emerged as the possible causes of this reduced β-cell mass. We hypothesized that the proliferative response to mitogens of human β-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human β-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27kip1. Increasing nuclear abundance of p27kip1 by adenoviral delivery decreases the proliferative response of β-cells from non-diabetic donors, mimicking T2D β-cells. However, while both p27kip1 gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human β-cells, only Skp2 was capable of inducing a significant human β-cell expansion. Skp2 was also able to double the proliferative response of T2D β-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D β-cells.

Early and Late G1/S Cyclins and Cdks Act Complementarily to Enhance Authentic Human β-Cell Proliferation and Expansion

β-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the “early” cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the “late” cyclins and cdks (cyclin A/E and cdk1/2). In β-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human β-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human β-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human β-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human β-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human β-cell numbers in vitro. These findings provide additional insight into human β-cell expansion. They also provide a novel tool for assessing β-cell expansion in vitro.