Divergent opioid-mediated suppression of inhibition between hippocampus and neocortex across species and development.

Opioid receptors within the CNS regulate pain sensation and mood and are key targets for drugs of abuse. Within the adult rodent hippocampus (HPC), µ-opioid receptor agonists suppress inhibitory parvalbumin-expressing interneurons (PV-INs), thus disinhibiting the circuit. However, it is uncertain if this disinhibitory motif is conserved in other cortical regions, species, or across development. We observed that PV-IN mediated inhibition is robustly suppressed by opioids in HPC but not neocortex in mice and nonhuman primates, with spontaneous inhibitory tone in resected human tissue also following a consistent dichotomy. This hippocampal disinhibitory motif was established in early development when immature PV-INs and opioids already influence primordial network rhythmogenesis. Acute opioid-mediated modulation was partially occluded with morphine pretreatment, with implications for the effects of opioids on hippocampal network activity during circuit maturation as well as learning and memory. Together, these findings demonstrate that PV-INs exhibit a divergence in opioid sensitivity across brain regions that is remarkably conserved across evolution and highlights the underappreciated role of opioids acting through immature PV-INs in shaping hippocampal development.

High-frequency variants in PKA signaling-related genes within a large pediatric cohort with obesity or metabolic abnormalities.

Introduction: Pediatric obesity has steadily increased in recent decades. Large-scale genome-wide association studies (GWAS) conducted primarily in Eurocentric adult populations have identified approximately 100 loci that predispose to obesity and type II diabetes. GWAS in children and individuals of non-European descent, both disproportionately affected by obesity, are fewer. Rare syndromic and monogenic obesities account for only a small portion of childhood obesity, so understanding the role of other genetic variants and their combinations in heritable obesities is key to developing targeted and personalized therapies. Tight and responsive regulation of the cAMP-dependent protein kinase (PKA) signaling pathway is crucial to maintaining healthy energy metabolism, and mutations in PKA-linked genes represent the most common cause of monogenic obesity. Methods: For this study, we performed targeted exome sequencing of 53 PKA signaling-related genes to identify variants in genomic DNA from a large, ethnically diverse cohort of obese or metabolically challenged youth. Results: We confirmed 49 high-frequency variants, including a novel variant in the PDE11A gene (c.152C>T). Several other variants were associated with metabolic characteristics within ethnic groups. Discussion: We conclude that a PKA pathway-specific variant search led to the identification of several new genetic associations with obesity in an ethnically diverse population.

The regulation of PKA signaling in obesity and in the maintenance of metabolic health.

The cAMP-dependent protein kinase (PKA) system represents a primary cell-signaling pathway throughout systems and across species. PKA facilitates the actions of hormones, neurotransmitters and other signaling molecules that bind G-protein coupled receptors (GPCR) to modulate cAMP levels. Through its control of synaptic events, exocytosis, transcriptional regulation, and more, PKA signaling regulates cellular metabolism and emotional and stress responses making it integral in the maintenance and dysregulation of energy homeostasis. Neural PKA signaling is regulated by afferent and peripheral efferent signals that link specific neural cell populations to the regulation of metabolic processes in adipose tissue, liver, pancreas, adrenal, skeletal muscle, and gut. Mouse models have provided invaluable information on the roles for PKA subunits in brain and key metabolic organs. While limited, human studies infer differential regulation of the PKA system in obese compared to lean individuals. Variants identified in PKA subunit genes cause Cushing syndrome that is characterized by metabolic dysregulation associated with endogenous glucocorticoid excess. Under healthy physiologic conditions, the PKA system is exquisitely regulated by stimuli that activate GPCRs to alter intracellular cAMP concentrations, and by PKA cellular localization and holoenzyme stability. Adenylate cyclase activity generates cAMP while phosphodiesterase-mediated cAMP degradation to AMP decreases cAMP levels downstream of GPCRs. Chronic perturbations in PKA signaling appear to be capable of resetting PKA regulation at several levels; in addition, sex differences in PKA signaling regulation, while not well understood, impact the physiologic consequences of metabolic dysregulation and obesity. This review explores the roles for PKA signaling in the pathogenesis of metabolic diseases including obesity, type 2 diabetes mellitus and associated co-morbidities through neural-peripheral crosstalk and cAMP/PKA signaling pathway targets that hold therapeutic potential.

The PRKAR1B p.R115K Variant is Associated with Lipoprotein Profile in African American Youth with Metabolic Challenges.

CONTEXT: High childhood obesity rates coincide with increased incidence of nonalcoholic fatty liver disease (NAFLD) and other comorbidities. Understanding the genetics of susceptibility to obesity and its comorbidities could guide intervention. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) signaling pathway regulates energy balance, glucose homeostasis, and lipid metabolism. OBJECTIVE: We hypothesized that PKA-related gene variants may be associated with obesity or associated metabolic conditions. METHODS: We included 457 youths from the Yale Obesity Clinic into the Pathogenesis of Youth-Onset Diabetes cohort (NCT01967849); a variety of clinical tests were performed to characterize NAFLD. Exon sequencing of 54 PKA pathway genes was performed. Variants were confirmed by Sanger sequencing. Clinical data were analyzed, correcting for NAFLD status and body mass index z-score with adjustments for multiple comparisons. Fluorescence resonance energy transfer (FRET) and PKA enzymatic assays were performed in HEK293 cells transfected with the PRKAR1B p.R115K construct. In silico structural analysis for this variant was done. RESULTS: We identified the variant PRKAR1B p.R115K in 4 unrelated, African American patients. Analyses compared this variant group to other African American patients in the cohort. PRKAR1B p.R115K was associated with favorable circulating lipoprotein levels. Analysis of FRET and PKA enzymatic assay showed stronger interaction between the R1ß mutant and PKA catalytic subunit Cα and decreased basal PKA activity compared with the wildtype (P < .0001). Structural analysis revealed that p.R115K may hinder conformational changes resulting from cAMP binding at cAMP binding domain A. CONCLUSION: Data suggest PRKAR1B p.R115K affects cAMP signaling and may favorably modulate lipoprotein profile in African American youth, protecting them from some adverse metabolic outcomes.

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain.

PURPOSE: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1ß subunit of the cyclic AMP-dependent protein kinase A (PKA). METHODS: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development. RESULTS: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs. CONCLUSION: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.

Cushing Syndrome in a Pediatric Patient With a KCNJ5 Variant and Successful Treatment With Low-dose Ketoconazole.

CONTEXT: Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS). PATIENT: We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS. RESULTS: Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance. CONCLUSION: We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.

Genomic and sequence variants of protein kinase A regulatory subunit type 1ß (PRKAR1B) in patients with adrenocortical disease and Cushing syndrome.

PURPOSE: Protein kinase A (PKA) subunit defects (in PRKAR1A and PRKACA) are known to contribute to adrenal tumor pathogenesis. We studied the PRKAR1B gene for any genetic changes in bilateral adrenocortical hyperplasia (BAH) and cortisol-producing adrenal adenomas (CPA). METHODS: Exome sequencing and PRKAR1B copy-number variant (CNV) analysis were performed in 74 patients with BAH and 21 with CPA. PKA activity was studied in tumors with defects; sequence variants were investigated in vitro. RESULTS: Three PRKAR1B germline variants (p.I40V, p.A67V, p.A300T) were identified among 74 patients with BAH. PRKAR1B copy-number gains (CNG) were found in 3 of 21 CPAs, one in a tumor carrying a somatic PRKACA "hotspot" pathogenic variant p.L206R. CPAs bearing PRKAR1B CNGs showed higher PRKAR1B messenger RNA (mRNA) levels and reduced PKA activity. Baseline PKA activity was also decreased for p.A67V and p.A300T in vitro, and mutant PRKAR1ß bound PRKACα in fluorescence resonance energy transfer (FRET) recordings of cotransfected HEK293 cells stronger than normal. CONCLUSION: PRKAR1B is yet another PKA subunit that may potentially contribute to adrenal tumor formation. Its involvement in adrenocortical disease may be different from that of other subunits, because PRKAR1B variants and PRKAR1B CNGs were associated with decreased (rather than increased) overall PKA activity in vitro.

Phosphodiesterase 2A and 3B variants are associated with primary aldosteronism.

Familial primary aldosteronism (PA) is rare and mostly diagnosed in early-onset hypertension (HT). However, 'sporadic' bilateral adrenal hyperplasia (BAH) is the most frequent cause of PA and remains without genetic etiology in most cases. Our aim was to investigate new genetic defects associated with BAH and PA. We performed whole-exome sequencing (paired blood and adrenal tissue) in six patients with PA caused by BAH that underwent unilateral adrenalectomy. Additionally, we conducted functional studies in adrenal hyperplastic tissue and transfected cells to confirm the pathogenicity of the identified genetic variants. Rare germline variants in phosphodiesterase 2A (PDE2A) and 3B (PDE3B) genes were identified in three patients. The PDE2A heterozygous variant (p.Ile629Val) was identified in a patient with BAH and early-onset HT at 13 years of age. Two PDE3B heterozygous variants (p.Arg217Gln and p.Gly392Val) were identified in patients with BAH and HT diagnosed at 18 and 33 years of age, respectively. A strong PDE2A staining was found in all cases of BAH in zona glomerulosa and/or micronodules (that were also positive for CYP11B2). PKA activity in frozen tissue was significantly higher in BAH from patients harboring PDE2A and PDE3B variants. PDE2A and PDE3B variants significantly reduced protein expression in mutant transfected cells compared to WT. Interestingly, PDE2A and PDE3B variants increased SGK1 and SCNN1G/ENaCg at mRNA or protein levels. In conclusion, PDE2A and PDE3B variants were associated with PA caused by BAH. These novel genetic findings expand the spectrum of genetic etiologies of PA.

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation.

PDE8B, PRKAR1A and the Wnt/ß-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b-/-); Pde8b haploinsufficient (Pde8b+/-) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b+/-;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b+/-;Prkar1a+/-). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b-/- male mice were infertile with down-regulation of the Wnt/ß-catenin pathway which did not change significantly in the Pde8b+/-;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.

Loss of habenular Prkar2a reduces hedonic eating and increases exercise motivation.

The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression, addiction, rewards processing, and motivation. Of its 2 major subdivisions, the medial Hb (MHb) and lateral Hb (LHb), MHb circuitry and function are poorly understood relative to those of the LHb. Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIß) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb. We previously showed that RIIα-knockout (RIIα-KO) mice resist diet-induced obesity. In the present study, we report that RIIα-KO mice have decreased consumption of palatable, "rewarding" foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Reexpression of Prkar2a in the Hb rescued this phenotype, confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for palatable foods, while enhancing the motivation for exercise, a desirable combination of behaviors.

PRKACB variants in skeletal disease or adrenocortical hyperplasia: effects on protein kinase A.

Genetic variants in components of the protein kinase A (PKA) enzyme have been associated with various defects and neoplasms in the context of Carney complex (CNC) and in isolated cases, such as in primary pigmented nodular adrenocortical disease (PPNAD), cortisol-producing adrenal adenomas (CPAs), and various cancers. PRKAR1A mutations have been found in subjects with impaired cAMP-dependent signaling and skeletal defects; bone tumors also develop in both humans and mice with PKA abnormalities. We studied the PRKACB gene in 148 subjects with PPNAD and related disorders, who did not have other PKA-related defects and identified two subjects with possibly pathogenic PRKACB gene variants and unusual bone and endocrine phenotypes. The first presented with bone and other abnormalities and carried a de novo c.858_860GAA (p.K286del) variant. The second subject carried the c.899C>T (p.T300M or p.T347M in another isoform) variant and had a PPNAD-like phenotype. Both variants are highly conserved in the PRKACB gene. In functional studies, the p.K286del variant affected PRKACB protein stability and led to increased PKA signaling. The p.T300M variant did not affect protein stability or response to cAMP and its pathogenicity remains uncertain. We conclude that PRKACB germline variants are uncommon but may be associated with phenotypes that resemble those of other PKA-related defects. However, detailed investigation of each variant is needed as PRKACB appears to be only rarely affected in these conditions, and variants such as p.T300M maybe proven to be clinically insignificant, whereas others (such as p.K286del) are clearly pathogenic and may be responsible for a novel syndrome, associated with endocrine and skeletal abnormalities.

PKA functions in metabolism and resistance to obesity: lessons from mouse and human studies.

Both direct and indirect evidence demonstrate a central role for the cAMP-dependent protein kinase (PKA) signaling pathway in the regulation of energy balance and metabolism across multiple systems. However, the ubiquitous pattern of PKA expression across cell types poses a challenge in pinpointing its tissue-specific regulatory functions and further characterizing its many downstream effects in certain organs or cells. Mouse models of PKA deficiency and over-expression and studies in living cells have helped clarify PKA function in adipose tissue (AT), liver, adrenal, pancreas, and specific brain nuclei, as they pertain to energy balance and metabolic dysregulation. Limited studies in humans suggest differential regulation of PKA in AT of obese compared to lean individuals and an overall dysregulation of PKA signaling in obesity. Despite its complexity, under normal physiologic conditions, the PKA system is tightly regulated by changes in cAMP concentrations upstream via adenylate cyclase and downstream by phosphodiesterase-mediated cAMP degradation to AMP and by changes in PKA holoenzyme stability. Adjustments in the PKA system appear to be important to the development and maintenance of the obese state and its associated metabolic perturbations. In this review we discuss the important role of PKA in obesity and its involvement in resistance to obesity, through studies in humans and in mouse models, with a focus on the regulation of PKA in energy expenditure, intake behavior, and lipid and glucose metabolism.

Acute Statin Administration Reduces Levels of Steroid Hormone Precursors.

Cholesterol-lowering statin drugs are used by approximately 25% of US adults 45 years of age and older and frequency of use is even higher among the elderly. Cholesterol provides the substrate for steroid hormone synthesis and its intracellular concentrations are tightly regulated. Our aim was to evaluate whether statin use acutely changes the circulating levels of cortisol, other glucocorticoid precursor molecules and their metabolites. Fourteen subjects not taking statins were administered a single oral dose (2 mg) of pitavastatin. Blood samples collected at baseline and 24 h post-treatment were analyzed for plasma cholesterol and steroid hormone profile. A parallel study in mice entailed the administration of atorvastatin (10 mg/kg) via orogastric delivery for three consecutive days. Cholesterol and corticosterone levels were quantified at baseline and at 1-day and 1-week post-treatment. Several precursor molecules in the steroidogenic pathway (corticosterone, cortisone, and 11-deoxycortisol) were significantly decreased 24 h after administration of a single dose of pitavastatin in human study subjects. Their circulating cholesterol concentrations were unchanged. In mice, there were no significant differences in serum cholesterol or corticosterone at 1-day or 1-week post-treatment compared to both pre-treatment baseline levels and control group levels. We conclude that acute dysregulation of the production of certain glucocorticoid precursor molecules was observed after a single treatment with a lipophilic statin drug. This may be of clinical relevance for individuals with underlying or subclinical adrenal insufficiency.

The Catalytic Subunit ß of PKA Affects Energy Balance and Catecholaminergic Activity.

The protein kinase A (PKA) signaling system mediates the effects of numerous hormones, neurotransmitters, and other molecules to regulate metabolism, cardiac function, and more. PKA defects may lead to diverse phenotypes that largely depend on the unique expression profile of the affected subunit. Deletion of the Prkarcb gene, which codes for PKA catalytic subunit ß (Cß), protects against diet-induced obesity (DIO), yet the mechanism for this phenotype remains unclear. We hypothesized that metabolic rate would be increased in Cß knockout (KO) mice, which could explain DIO resistance. Male, but not female, CßKO mice had increased energy expenditure, and female but not male CßKO mice had increased subcutaneous temperature and increased locomotor activity compared with wild-type (WT) littermates. Urinary norepinephrine (NE) and normetanephrine were elevated in female CßKO mice. CßKO mice had increased heart rate (HR); blocking central NE release normalized HR to that of untreated WT mice. Basal and stimulated PKA enzymatic activities were unchanged in adipose tissue and heart and varied in different brain regions, suggesting that Prkacb deletion may mediate signaling changes in specific brain nuclei and may be less important in the peripheral regulation of PKA expression and activity. This is a demonstration of a distinct effect of the PKA Cß catalytic subunit on catecholamines and sympathetic nerve signaling. The data provide an unexpected explanation for the metabolic phenotype of CßKO mice. Finally, the sexual dimorphism is consistent with mouse models of other PKA subunits and adds to the importance of these findings regarding the PKA system in human metabolism.

Harvey Cushing Treated the First Known Patient With Carney Complex.

CONTEXT: Carney complex (CNC) is a syndrome characterized by hyperplasia of endocrine organs and may present with clinical features of Cushing syndrome and acromegaly due to functional adrenal and pituitary gland tumors. CNC has been linked to mutations in the regulatory subunit of protein kinase A type I-alpha (PRKAR1A) gene. DESIGN: Tissue samples were taken from the hypothalamus or thalamus or tumors of patients with pituitary adenomas seen and operated on by neurosurgeon Harvey Cushing between 1913 and 1932. Following DNA extraction, sequencing for genes of interest was attempted, including PRKAR1A, AIP, USP8, GNAS1, and GPR101, to explore the possibility that these mutations associated with acromegaly, CNC, and Cushing syndrome have been conserved over time. RESULTS: We report a patient described by Dr. Cushing in 1914 with a clinical presentation and postmortem findings suggestive of CNC. Genetic sequencing of the hypothalamus and pituitary adenoma revealed a germline heterozygous p.Arg74His mutation in the PRKAR1A gene, a codon previously described as mutated in CNC, but with a novel amino acid change. CONCLUSIONS: This patient is, to our knowledge, the first molecularly confirmed individual with CNC. This case demonstrates the power of modern genetics in studying archived tissues and the importance of recording detailed clinical notes in the diagnosis of disease.

Acute vs chronic exposure to high fat diet leads to distinct regulation of PKA.

The cAMP-dependent protein kinase (PKA) is an essential regulator of lipid and glucose metabolism that plays a critical role in energy homeostasis. The impact of diet on PKA signaling has not been defined, although perturbations in individual PKA subunits are associated with changes in adiposity, physical activity and energy intake in mice and humans. We hypothesized that a high fat diet (HFD) would elicit peripheral and central alterations in the PKA system that would differ depending on length of exposure to HFD; these differences could protect against or promote diet-induced obesity (DIO). 12-week-old C57Bl/6J mice were randomly assigned to a regular diet or HFD and weighed weekly throughout the feeding studies (4 days, 14 weeks; respectively), and during killing. PKA activity and subunit expression were measured in liver, gonadal adipose tissue (AT) and brain. Acute HFD-feeding suppressed basal hepatic PKA activity. In contrast, hepatic and hypothalamic PKA activities were significantly increased after chronic HFD-feeding. Changes in AT were more subtle, and overall, altered PKA regulation in response to chronic HFD exposure was more profound in female mice. The suppression of hepatic PKA activity after 4 day HFD-feeding was indicative of a protective peripheral effect against obesity in the context of overnutrition. In response to chronic HFD-feeding, and with the development of DIO, dysregulated hepatic and hypothalamic PKA signaling was a signature of obesity that is likely to promote further metabolic dysfunction in mice.

Prkar1a gene knockout in the pancreas leads to neuroendocrine tumorigenesis.

Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predisposition to pancreatic tumors; it is caused most frequently by the inactivation of the PRKAR1A gene, a regulator of the cyclic AMP (cAMP)-dependent kinase (PKA). The method used was to create null alleles of prkar1a in mouse cells expressing pdx1 (Δ-Prkar1a). We found that these mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetrance by the age of 4-5 months. Malignant behavior of the tumors was seen as evidenced by stromal invasion and metastasis to locoregional lymph nodes. Histologically, most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Biochemically, the lesions exhibited high PKA activity, as one would expect from deleting prkar1a The primary neuroendocrine nature of these tumor cells was confirmed by immunohistochemical staining and electron microscopy, the latter revealing the characteristic granules. Although the Δ-Prkar1a mice developed hypoglycemia after overnight fasting, insulin and glucagon levels in the plasma were normal. Negative immunohistochemical staining for the most commonly produced peptides (insulin, c-peptide, glucagon, gastrin and somatostatin) suggested that these tumors were non-functioning. We hypothesize that the recently identified multipotent pdx1+/insulin- cell in adult pancreas, gives rise to endocrine or mixed endocrine/acinar pancreatic malignancies with complete prkar1a deficiency. In conclusion, this mouse model supports the role of prkar1a as a tumor suppressor gene in the pancreas and points to the PKA pathway as a possible therapeutic target for these lesions.

Cholesterol Biosynthesis and Trafficking in Cortisol-Producing Lesions of the Adrenal Cortex.

CONTEXT: Cortisol-producing adenomas (CPAs), primary pigmented nodular adrenocortical disease (PPNAD), and primary macronodular adrenocortical hyperplasia (PMAH) cause ACTH-independent Cushing syndrome (CS). Investigation of their pathogenesis has demonstrated their integral link to the cAMP-dependent protein kinase signaling pathway. OBJECTIVE: The aim of this study was to identify differences in cholesterol biosynthesis among different CS-causing adrenocortical tumors. Because of the concomitant associations of cAMP levels with cholesterol and with steroid biosynthesis, we hypothesized that benign cortisol-producing tumors would display aberration of these pathways. DESIGN AND SETTING: Twenty-three patients with CPA, PPNAD, or PMAH who underwent adrenalectomy for CS were included in the study. Preoperative biochemical analyses were performed, and excised adrenal tissues were studied. MAIN OUTCOME MEASURES: Serum, urinary hormone levels, serum lipid profiles, and anthropometric data were obtained preoperatively. Adrenal tissues were analyzed for total protein, cholesterol, and neutral sterol content by mass spectrometry and expression of HMGCR, LDLR, ABCA1, DHCR24, and STAR genes. RESULTS: There were differences in cholesterol content and markers of cholesterol biosynthesis and metabolism that distinguished CPAs from PMAH and PPNAD; cholesterol, lathosterol, and lathosterol/cholesterol ratio were significantly higher in CPAs. ABCA1 mRNA was lower among CPAs compared to tissues from bilateral adrenocortical hyperplasia (PMAH and PPNAD), and mRNA expression of LDL-R, DCHR24, and HMGCR tended to be higher in CPA tumor tissues. CONCLUSION: CPAs displayed characteristics of "cholesterol-starved" tissues when compared to PPNAD and PMAH and appeared to have increased intrinsic cholesterol production and uptake from the periphery, as well as decreased cholesterol efflux. This has implications for a potential new way of treating these tumors.

Differentially regulated protein kinase A (PKA) activity in adipose tissue and liver is associated with resistance to diet-induced obesity and glucose intolerance in mice that lack PKA regulatory subunit type IIα.

The cAMP-dependent protein kinase A (PKA) signaling system is widely expressed and has a central role in regulating cellular metabolism in all organ systems affected by obesity. PKA has four regulatory (RIα, RIIα, RIß, RIIß) and four catalytic (Cα, Cß, Cγ, Prkx) subunit isoforms that have tissue-specific expression profiles. In mice, knockout (KO) of RIIß, the primary PKA regulatory subunit in adipose tissue or knockout of the catalytic subunit Cß resulted in a lean phenotype that resists diet-induced obesity and associated metabolic complications. Here we report that the disruption of the ubiquitously expressed PKA RIIα subunit in mice (RIIαKO) confers resistance to diet-induced obesity, glucose intolerance, and hepatic steatosis. After 2-week high-fat diet exposure, RIIαKO mice weighed less than wild-type littermates. Over time this effect was more pronounced in female mice that were also leaner than their wild-type counterparts, regardless of the diet. Decreased intake of a high-fat diet contributed to the attenuated weight gain in RIIαKO mice. Additionally, RIIα deficiency caused differential regulation of PKA in key metabolic organs: cAMP-stimulated PKA activity was decreased in liver and increased in gonadal adipose tissue. We conclude that RIIα represents a potential target for therapeutic interventions in obesity, glucose intolerance, and nonalcoholic fatty liver disease.