Jessica A. Hall

The sirtuin family’s role in aging and age-associated pathologies

The 7 mammalian sirtuin proteins compose a protective cavalry of enzymes that can be invoked by cells to aid in the defense against a vast array of stressors. The pathologies associated with aging, such as metabolic syndrome, neurodegeneration, and cancer, are either caused by or exacerbated by a lifetime of chronic stress. As such, the activation of sirtuin proteins could provide a therapeutic approach to buffer against chronic stress and ameliorate age-related decline. Here we review experimental evidence both for and against this proposal, as well as the implications that isoform-specific sirtuin activation may have for healthy aging in humans.

Disruption of Thyroid Hormone Activation in Type 2 Deiodinase Knockout Mice Causes Obesity With Glucose Intolerance and Liver Steatosis Only at Thermoneutrality

OBJECTIVE Thyroid hormone accelerates energy expenditure; thus, hypothyroidism is intuitively associated with obesity. However, studies failed to establish such a connection. In brown adipose tissue (BAT), thyroid hormone activation via type 2 deiodinase (D2) is necessary for adaptive thermogenesis, such that mice lacking D2 (D2KO) exhibit an impaired thermogenic response to cold. Here we investigate whether the impaired thermogenesis of D2KO mice increases their susceptibility to obesity when placed on a high-fat diet. RESEARCH DESIGN AND METHODS To test this, D2KO mice were admitted to a comprehensive monitoring system acclimatized to room temperature (22°C) or thermoneutrality (30°C) and kept either on chow or high-fat diet for 60 days. RESULTS At 22°C, D2KO mice preferentially oxidize fat, have a similar sensitivity to diet-induced obesity, and are supertolerant to glucose. However, when thermal stress is eliminated at thermoneutrality (30°C), an opposite phenotype is encountered, one that includes obesity, glucose intolerance, and exacerbated hepatic steatosis. We suggest that a compensatory increase in BAT sympathetic activation of the D2KO mice masks metabolic repercussions that they would otherwise exhibit. CONCLUSIONS Thus, upon minimization of thermal stress, high-fat feeding reveals the defective capacity of D2KO mice for diet-induced thermogenesis, provoking a paradigm shift in the understanding of the role of the thyroid hormone in metabolism.

Absence of Thyroid Hormone Activation during Development Underlies a Permanent Defect in Adaptive Thermogenesis

Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1alpha and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes.

Cdc2-like Kinase 2 Suppresses Hepatic Fatty Acid Oxidation and Ketogenesis through Disruption of the PGC-1α and MED1 Complex

Hepatic ketogenesis plays an important role in catabolism of fatty acids during fasting along with dietary lipid overload, but the mechanisms regulating this process remain poorly understood. Here, we show that Cdc2-like kinase 2 (Clk2) suppresses fatty acid oxidation and ketone body production during diet-induced obesity. In lean mice, hepatic Clk2 protein is very low during fasting and strongly increased during feeding; however, in diet-induced obese mice, Clk2 protein remains elevated through both fed and fasted states. Liver-specific Clk2 knockout mice fed a high-fat diet exhibit increased fasting levels of blood ketone bodies, reduced respiratory exchange ratio, and increased gene expression of fatty acid oxidation and ketogenic pathways. This effect of Clk2 is cell-autonomous, because manipulation of Clk2 in hepatocytes controls genes and rates of fatty acid utilization. Clk2 phosphorylation of peroxisome proliferator–activated receptor γ coactivator (PGC-1α) disrupts its interaction with Mediator subunit 1, which leads to a suppression of PGC-1α activation of peroxisome proliferator–activated receptor α target genes in fatty acid oxidation and ketogenesis. These data demonstrate the importance of Clk2 in the regulation of fatty acid metabolism in vivo and suggest that inhibition of hepatic Clk2 could provide new therapies in the treatment of fatty liver disease.

Loss of Mpzl3 Function Causes Various Skin Abnormalities and Greatly Reduced Adipose Depots

The rough coat (rc) spontaneous mutation causes sebaceous gland hypertrophy, hair loss and extracutaneous abnormalities including growth retardation. The rc mice have a missense mutation in the predicted immunoglobulin protein Mpzl3. In this study, we generated Mpzl3 knockout mice to determine its functions in the skin. Homozygous Mpzl3 knockout mice showed unkempt and greasy hair coat and hair loss soon after birth. Histological analysis revealed severe sebaceous gland hypertrophy and increased dermal thickness, but did not detect significant changes in the hair cycle. Mpzl3 null mice frequently developed inflammatory skin lesions; however, the early onset skin abnormalities were not the results of immune defects. The abnormalities in the Mpzl3 knockout mice resemble closely those observed in the rc/rc mice, as well as mice heterozygous for both the rc and Mpzl3 knockout alleles, indicating that rc and Mpzl3 are allelic. Using a lacZ reporter gene, we detected Mpzl3 promoter activity in the companion layer and inner root sheath of the hair follicle, sebaceous gland, and epidermis. Loss of MPZL3 function also caused a striking reduction in cutaneous and overall adipose tissue. These data reveal a complex role for Mpzl3 in the control of skin development, hair growth and adipose cell functions.

Decreased Genetic Dosage of Hepatic Yin Yang 1 Causes Diabetic-Like Symptoms

Insulin sensitivity in liver is characterized by the ability of insulin to efficiently inhibit glucose production and fatty acid oxidation as well as promote de novo lipid biosynthesis. Specific dysregulation of glucose and lipid metabolism in liver is sufficient to cause insulin resistance and type 2 diabetes; this is seen by a selective inability of insulin to suppress glucose production while remaining insulin-sensitive to de novo lipid biosynthesis. We have previously shown that the transcription factor Yin Yang 1 (YY1) controls diabetic-linked glucose and lipid metabolism gene sets in skeletal muscle, but whether liver YY1-targeted metabolic genes impact a diabetic phenotype is unknown. Here we show that decreased genetic dosage of YY1 in liver causes insulin resistance, hepatic lipid accumulation, and dyslipidemia. Indeed, YY1 liver-specific heterozygous mice exhibit blunted activation of hepatic insulin signaling in response to insulin. Mechanistically, YY1, through direct recruitment to promoters, functions as a suppressor of genes encoding for metabolic enzymes of the gluconeogenic and lipogenic pathways and as an activator of genes linked to fatty acid oxidation. These counterregulatory transcriptional activities make targeting hepatic YY1 an attractive approach for treating insulin-resistant diabetes.

Triumphs of the thyroid despite lesser conversion.

In a seemingly wasteful fashion, the thyroid gland focuses its effort into secreting an inactive version of thyroid hormone T4. In fact, a hormone, from the Greek word for impulse, is a biologically active molecule, so in the case of thyroid hormone, the actual active hormone is that of the triply iodinated derivative, T3. To successfully produce T3, the human thyroid processes about 1000 nmol iodide daily, packaging the iodide into a very large molecule [thyroglobulin (Tg)] that is subsequently hydrolyzed. The resulting iodinated products are sequentially unleashed in a controlled manner so that, eventually, only 10 nmol of the highly potent T3 are secreted. The bulk of T3 production (40 nmol), however, occurs outside of the thyroid parenchyma through the conversion of T4 to T3 by the activating deiodinases, a group of selenoenzymes that can selectively remove iodine moieties from T4 (reviewed in Ref 1). Given that only 10 nmol or less of T3 are directly secreted from the thyroid, one would assume that an absence of the deiodinases should impair the overall daily production of T3, leading to systemic hypothyroidism. In this issue of Endocrinology, Galton et al. (2) challenge the conventional paradigm of thyroid hormone homeostasis by showing that mice lacking both of the activating deiodinases do just fine, because they are still capable of maintaining normal amounts of T3 in serum and do not suffer from systemic hypothyroidism. The low abundance of T3 in the thyroid secretion of humans and rodents is explained by the high T4/T3 ratio in their Tg, with ratios of about 15:1 in humans and 8:1 in rats (1). Even considering that some T4 to T3 conversion occurs in the thyroid parenchyma after Tg hydrolysis, of which very little is known (3), T4 still predominates over T3 in the thyroid secretion. Notably, this varies among species, where the human thyroid produces less than 20% of the body’s T3, whereas the rodent’s thyroid contributes about 40% (1). Still, this leaves the majority of T3 production to extrathyroidal tissues in both species. Conversion of T4 to T3 is catalyzed by the type 1 or type 2 iodothyronine deiodinases (D1 or D2), which activate thyroid hormone through removal of an outer-ring iodine on T4 (known as 5 -deiodination). D2 is the main deiodinase to catalyze local activation of thyroid hormone, being most notable for its expression in pituitary gland, brain, and brown adipose tissue (4). D1, on the other hand, is kinetically inefficient but is expressed at such high levels in kidney and liver that it, too, plays a sizable role in extrathyroidal T3 production, particularly in rodents (5). In fact, it is commonly accepted that D1 and D2 contribute equally to extrathyroidal T3 production in mice and rats. This is unlike the situation in humans, where D2 is argued to play a greater role in the generation of plasma T3 (1). Despite these differences in T3 production between humans and rodents, the generation of mice lacking specific deiodinase activity has allowed for in-depth study of the physiological roles played by D1 and D2 in thyroid hormone homeostasis (6). Remarkably, mice with targeted disruption of Dio1 [D1 knockout (D1KO)] or Dio2 (D2KO) have been found to exhibit a normal serum T3 concentration (7, 8). Even the C3H/D2KO mouse that lacks D2 and expresses only residual D1 activity maintains euthyroid serum T3 levels (9). All three of these mouse models have increased concentrations of serum T4, and both the D2KO and C3H/D2KO have elevated TSH. These adjustments in T4 and TSH indicate that keeping serum T3 concentration within the normal range is the default hypothalamic primary directive for the TRH-TSH-thyroid axis, despite marked elevation in serum T4 concentrations. This innate ability to avoid catastrophic hypothyroidism is fascinating and has only recently been appreciated as a result of these deiodinase-deficient mouse models. To better appreciate the plasticity and adaptive capacity of the T3-generating system, and to rule out the effect of possible compensation by D1 or D2 in the aforementioned mouse models, the present study by Galton et al. (2) evaluates mice lacking both D1 and D2 activity. As with the other mouse models, D1/D2KO mice sustain a normal serum concentration of T3 at the expense of markedly altered thyroidal status. There is a 2-fold greater serum T4 concentration and a 2.6-times higher TSH serum level when compared with wild-type mice. The elevated level of T4 is higher than that seen in either the D1KO or D2KO, and the elevated TSH level is comparable to that measured in D2KO

Phosphorylation of Beta-3 adrenergic receptor at serine 247 by ERK MAP kinase drives lipolysis in obese adipocytes

Objective The inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity. Methods and Results We provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine. Conclusion Taken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.

Cdkal1, a type 2 diabetes susceptibility gene, regulates mitochondrial function in adipose tissue

Objectives Understanding how loci identified by genome wide association studies (GWAS) contribute to pathogenesis requires new mechanistic insights. Variants within CDKAL1 are strongly linked to an increased risk of developing type 2 diabetes and obesity. Investigations in mouse models have focused on the function of Cdkal1 as a tRNALys modifier and downstream effects of Cdkal1 loss on pro-insulin translational fidelity in pancreatic β−cells. However, Cdkal1 is broadly expressed in other metabolically relevant tissues, including adipose tissue. In addition, the Cdkal1 homolog Cdk5rap1 regulates mitochondrial protein translation and mitochondrial function in skeletal muscle. We tested whether adipocyte-specific Cdkal1 deletion alters systemic glucose homeostasis or adipose mitochondrial function independently of its effects on pro-insulin translation and insulin secretion. Methods We measured mRNA levels of type 2 diabetes GWAS genes, including Cdkal1, in adipose tissue from lean and obese mice. We then established a mouse model with adipocyte-specific Cdkal1 deletion. We examined the effects of adipose Cdkal1 deletion using indirect calorimetry on mice during a cold temperature challenge, as well as by measuring cellular and mitochondrial respiration in vitro. We also examined brown adipose tissue (BAT) mitochondrial morphology by electron microscopy. Utilizing co-immunoprecipitation followed by mass spectrometry, we performed interaction mapping to identify new CDKAL1 binding partners. Furthermore, we tested whether Cdkal1 loss in adipose tissue affects total protein levels or accurate Lys incorporation by tRNALys using quantitative mass spectrometry. Results We found that Cdkal1 mRNA levels are reduced in adipose tissue of obese mice. Using adipose-specific Cdkal1 KO mice (A-KO), we demonstrated that mitochondrial function is impaired in primary differentiated brown adipocytes and in isolated mitochondria from A-KO brown adipose tissue. A-KO mice displayed decreased energy expenditure during 4 °C cold challenge. Furthermore, mitochondrial morphology was highly abnormal in A-KO BAT. Surprisingly, we found that lysine codon representation was unchanged in Cdkal1 A-KO adipose tissue. We identified novel protein interactors of CDKAL1, including SLC25A4/ANT1, an inner mitochondrial membrane ADP/ATP translocator. ANT proteins can account for the UCP1-independent basal proton leak in BAT mitochondria. Cdkal1 A-KO mice had increased ANT1 protein levels in their white adipose tissue. Conclusions Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity.