Low-dose UV radiation before running wheel access activates brown adipose tissue.

In previous preclinical studies, low (non-burning) doses of UV radiation (UVR) limited weight gain and metabolic dysfunction in mice fed with a high-fat diet. Here, we explored the effects of low-dose UVR on physical activity and food intake and mechanistic pathways in interscapular brown adipose tissue (iBAT). Young adult C57Bl/6J male mice, housed as individuals, were fed a high-fat diet and exposed to low-dose UVR (sub-oedemal, 1 kJ/m2 UVB, twice-a-week) or 'mock' treatment, with or without running wheel access (2 h, for 'moderate' physical activity) immediately after phototherapy. There was no difference in distance run in mice exposed to UVR or mock-treated over 12 weeks of exposure to running wheels (P = 0.14). UVR (alone) did not significantly affect food intake, adiposity, or signs of glucose dysfunction. Access to running wheels increased food intake (after 10 weeks, P ≤ 0.02) and reduced gonadal white adipose tissue and iBAT mass (P ≤ 0.03). Body weight and hepatic steatosis were lowest in mice exposed to UVR with running wheel access. In the iBAT of mice exposed to UVR and running wheels, elevated Atgl, Cd36, Fasn, Igf1, Pparγ, and Ucp1 mRNAs and reduced CD11c on F4-80 + MHC class II+ macrophages were observed, while renal Sglt2 mRNA levels were increased, compared to high-fat diet alone (P ≤ 0.03). Blood levels of 25-hydroxyvitamin D were not increased by exposure to UVR and/or access to running wheels. In conclusion, when combined with physical activity, low-dose UVR may more effectively limit adiposity (specifically, body weight and hepatic steatosis) and modulate metabolic and immune pathways in iBAT.

Characterising nitric oxide-mediated metabolic benefits of low-dose ultraviolet radiation in the mouse: a focus on brown adipose tissue.

AIMS/HYPOTHESIS: Exposure to sunlight has the potential to suppress metabolic dysfunction and obesity. We previously demonstrated that regular exposure to low-doses of ultraviolet radiation (UVR) reduced weight gain and signs of diabetes in male mice fed a high-fat diet, in part via release of nitric oxide from skin. Here, we explore further mechanistic pathways through which low-dose UVR exerts these beneficial effects. METHODS: We fed mice with a luciferase-tagged Ucp1 gene (which encodes uncoupling protein-1 [UCP-1]), referred to here as the Ucp1 luciferase transgenic mouse ('Thermomouse') a high-fat diet and examined the effects of repeated exposure to low-dose UVR on weight gain and development of metabolic dysfunction as well as UCP-1-dependent thermogenesis in interscapular brown adipose tissue (iBAT). RESULTS: Repeated exposure to low-dose UVR suppressed the development of glucose intolerance and hepatic lipid accumulation via dermal release of nitric oxide while also reducing circulating IL-6 (compared with mice fed a high-fat diet only). Dietary nitrate supplementation did not mimic the effects of low-dose UVR. A single low dose of UVR increased UCP-1 expression (by more than twofold) in iBAT of mice fed a low-fat diet, 24 h after exposure. However, in mice fed a high-fat diet, there was no effect of UVR on UCP-1 expression in iBAT (compared with mock-treated mice) when measured at regular intervals over 12 weeks. More extensive circadian studies did not identify any substantial shifts in UCP-1 expression in mice exposed to low-dose UVR, although skin temperature at the interscapular site was reduced in UVR-exposed mice. The appearance of cells with a white adipocyte phenotype ('whitening') in iBAT induced by consuming the high-fat diet was suppressed by exposure to low-dose UVR in a nitric oxide-dependent fashion. Significant shifts in the expression of important core gene regulators of BAT function (Dio2, increased more than twofold), fatty acid transport (increased Fatp2 [also known as Slc27a2]), lipolysis (decreased Atgl [also known as Pnpla2]), lipogenesis (decreased Fasn) and inflammation (decreased Tnf), and proportions of macrophages (increased twofold) were observed in iBAT of mice exposed to low-dose UVR. These effects were independent of nitric oxide released from skin. CONCLUSIONS/INTERPRETATION: Our results suggest that non-burning (low-dose) UVR suppresses the BAT 'whitening', steatotic and pro-diabetic effects of consuming a high-fat diet through skin release of nitric oxide, with some metabolic and immune pathways in iBAT regulated by UVR independently of nitric oxide.

Investigating the Potential for Ultraviolet Light to Modulate Morbidity and Mortality From COVID-19: A Narrative Review and Update.

During the COVID-19 (coronavirus disease of 2019) pandemic, researchers have been seeking low-cost and accessible means of providing protection from its harms, particularly for at-risk individuals such as those with cardiovascular disease, diabetes and obesity. One possible way is via safe sun exposure, and/or dietary supplementation with induced beneficial mediators (e.g., vitamin D). In this narrative review, we provide rationale and updated evidence on the potential benefits and harms of sun exposure and ultraviolet (UV) light that may impact COVID-19. We review recent studies that provide new evidence for any benefits (or otherwise) of UV light, sun exposure, and the induced mediators, vitamin D and nitric oxide, and their potential to modulate morbidity and mortality induced by infection with SARS-CoV-2 (severe acute respiratory disease coronavirus-2). We identified substantial interest in this research area, with many commentaries and reviews already published; however, most of these have focused on vitamin D, with less consideration of UV light (or sun exposure) or other mediators such as nitric oxide. Data collected to-date suggest that ambient levels of both UVA and UVB may be beneficial for reducing severity or mortality due to COVID-19, with some inconsistent findings. Currently unresolved are the nature of the associations between blood 25-hydroxyvitamin D and COVID-19 measures, with more prospective data needed that better consider lifestyle factors, such as physical activity and personal sun exposure levels. Another short-coming has been a lack of measurement of sun exposure, and its potential to influence COVID-19 outcomes. We also discuss possible mechanisms by which sun exposure, UV light and induced mediators could affect COVID-19 morbidity and mortality, by focusing on likely effects on viral pathogenesis, immunity and inflammation, and potential cardiometabolic protective mechanisms. Finally, we explore potential issues including the impacts of exposure to high dose UV radiation on COVID-19 and vaccination, and effective and safe doses for vitamin D supplementation.

Systematic Review of the Effects of Ultraviolet Radiation on Markers of Metabolic Dysfunction.

Emerging findings suggest that exposure to ultraviolet wavelengths of sunlight modulates metabolic function. Here we review the metabolic effects of exposure to ultraviolet radiation (UVR), focusing on the effects of phototherapies (that administer UVR), and advice to increase sun exposure in individuals enrolled in clinical trials and intervention studies. We identified 25 studies in which the effects of UVR on metabolic outcomes were examined, including: narrowband ultraviolet B phototherapy (nbUVB, n = 12); psoralen ultraviolet A phototherapy (n = 4); other types of UVR phototherapy (n = 5); and sun exposure advice (n = 5). Most studies recruited a small number of participants (≤100), who were middle-aged individuals undergoing treatment for psoriasis flare, with phototherapy or sun exposure advice administered for ≤12 weeks. Data obtained at baseline were usually compared with an endpoint following treatment with UVR, for a limited number of outcomes. There were few studies in which markers of glucose metabolism were assessed, with some beneficial effects of sun exposure (but not phototherapy) reported. LDL-cholesterol levels were lower in individuals receiving sun exposure advice, while treatment with nbUVB reduced blood concentrations of inflammatory markers (C-reactive protein and interleukin-6). Future studies should focus on determining whether the effects of these interventions change with time, and if they are dependent on the source of UVR (i.e. phototherapy or sun exposure) and wavelength(s) of light administered. Furthermore, studies need to measure a variety of (clinical) markers of glucose metabolism, adiposity and inflammation, control for factors such as skin type and sex, and stratify participants for metabolic disease diagnosis.

High Dose Vitamin D supplementation alters faecal microbiome and predisposes mice to more severe colitis.

Vitamin D has been suggested as a possible adjunctive treatment to ameliorate disease severity in human inflammatory bowel disease. In this study, the effects of diets containing high (D++, 10,000 IU/kg), moderate (D+, 2,280 IU/kg) or no vitamin D (D-) on the severity of dextran sodium sulphate (DSS) colitis in female C57Bl/6 mice were investigated. The group on high dose vitamin D (D++) developed the most severe colitis as measured by blinded endoscopic (p < 0.001) and histologic (p < 0.05) assessment, weight loss (p < 0.001), drop in serum albumin (p = 0.05) and increased expression of colonic TNF-α (p < 0.05). Microbiota analysis of faecal DNA showed that the microbial composition of D++ control mice was more similar to that of DSS mice. Serum 25(OH)D3 levels reduced by 63% in the D++ group and 23% in the D+ group after 6 days of DSS treatment. Thus, high dose vitamin D supplementation is associated with a shift to a more inflammatory faecal microbiome and increased susceptibility to colitis, with a fall in circulating vitamin D occurring as a secondary event in response to the inflammatory process.

Vitamin D supplementation of initially vitamin D-deficient mice diminishes lung inflammation with limited effects on pulmonary epithelial integrity.

In disease settings, vitamin D may be important for maintaining optimal lung epithelial integrity and suppressing inflammation, but less is known of its effects prior to disease onset. Female BALB/c dams were fed a vitamin D3-supplemented (2280 IU/kg, VitD+) or nonsupplemented (0 IU/kg, VitD-) diet from 3 weeks of age, and mated at 8 weeks of age. Male offspring were fed the same diet as their mother. Some offspring initially fed the VitD- diet were switched to a VitD+ diet from 8 weeks of age (VitD-/+). At 12 weeks of age, signs of low-level inflammation were observed in the bronchoalveolar lavage fluid (BALF) of VitD- mice (more macrophages and neutrophils), which were suppressed by subsequent supplementation with vitamin D3 There was no difference in the level of expression of the tight junction proteins occludin or claudin-1 in lung epithelial cells of VitD+ mice compared to VitD- mice; however, claudin-1 levels were reduced when initially vitamin D-deficient mice were fed the vitamin D3-containing diet (VitD-/+). Reduced total IgM levels were detected in BALF and serum of VitD-/+ mice compared to VitD+ mice. Lung mRNA levels of the vitamin D receptor (VDR) were greatest in VitD-/+ mice. Total IgG levels in BALF were greater in mice fed the vitamin D3-containing diet, which may be explained by increased activation of B cells in airway-draining lymph nodes. These findings suggest that supplementation of initially vitamin D-deficient mice with vitamin D3 suppresses signs of lung inflammation but has limited effects on the epithelial integrity of the lungs.

High-Dose Intramuscular Vitamin D Provides Long-Lasting Moderate Increases in Serum 25-Hydroxvitamin D Levels and Shorter-Term Changes in Plasma Calcium.

The best management of vitamin D deficiency, defined as a 25-hydroxyvitamin D [(25(OH)D] level <50 nM, is unclear. Intramuscular (IM) injection of a large bolus of vitamin D (≥100 000 IU) is used, but its safety is uncertain. In 10 adults given an IM injection of 600 000IU vitamin D3, we measured at baseline and at 1, 2, 3, and 4 weeks postinjection the serum levels of vitamin D3, 25(OH)D3, 25(OH)D2, total 25(OH)D, 3-epi-25(OH)D3, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] using a standardized LC with tandem MS (MS/MS) assay; serum levels of 25(OH)D using the Abbott ARCHITECT i2000 immunoassay; and markers of bone metabolism. Bone markers and 25(OH)D (immunoassay) were remeasured at 24 weeks. All participants had baseline total 25(OH)D levels >50 nM. Serum 25(OH)D levels increased at 3, 4, and 24 weeks postinjection, peaking at 4 weeks [mean ± SEM of 126 ± 7.9 nM (immunoassay) and 100 ± 5.5 nM (LC-MS/MS)] but generally remained <125 nM, the upper limit recommended by the U.S. Institute of Medicine. Serum 24,25(OH)2D3 levels increased at 3 and 4 weeks postinjection. Serum ionized calcium levels were higher than baseline at 1, 3, and 4 weeks postinjection but remained within the clinically normal range. Other biochemical parameters, including other vitamin D metabolites, plasma alkaline phosphatase, and parathyroid hormone levels, were unchanged. IM injection of a large bolus of vitamin D effectively increases serum 25(OH)D levels without evidence of metabolic abnormality.

Sub-erythemal ultraviolet radiation reduces metabolic dysfunction in already overweight mice.

Exposure to sunlight may limit cardiometabolic risk. In our previous studies, regular exposure to sub-erythemal (non-burning) ultraviolet radiation (UVR) reduced signs of adiposity and cardiometabolic dysfunction in mice fed a high-fat diet. Some of the observed effects were dependent on skin release of nitric oxide after UVR exposure. Here, we examine the effects of sub-erythemal UVR on signs of adiposity and metabolic dysfunction in already overweight mice, comparing the effects of two sunlamps with distinct emitted light spectra. Mice were fed a high-fat diet from 8 weeks of age, with UVR administered twice a week from 14 weeks of age until they were killed at 20 weeks of age. Mice were irradiated with the same dose of UVB radiation (1 kJ/m2) from either FS40 (65% UVB, 35% UVA) or CLEO (4% UVB, 96% UVA) sunlamps, but substantially more UVA from the latter. FS40 UVR (but not CLEO UVR) significantly reduced mouse weights and weight gain, compared to mice fed a high-fat diet (only). These effects were dependent on nitric oxide. Conversely, CLEO UVR (but not FS40 UVR) significantly reduced circulating LDL cholesterol. Both light sources reduced fasting insulin levels, and the extent of hepatic steatosis; the latter was reversed by topical application of cPTIO, suggesting an important role for skin release of nitric oxide in preventing hepatic lipid accumulation. These results suggest that there may be a number of benefits achieved by regular exposure to safe (non-burning) levels of sunlight or UV-containing phototherapy, with effects potentially dependent on the predominance of the wavelengths of UVR administered.

Vitamin D and allergic airway disease shape the murine lung microbiome in a sex-specific manner.

BACKGROUND: Vitamin D is under scrutiny as a potential regulator of the development of respiratory diseases characterised by chronic lung inflammation, including asthma and chronic obstructive pulmonary disease. It has anti-inflammatory effects; however, knowledge around the relationship between dietary vitamin D, inflammation and the microbiome in the lungs is limited. In our previous studies, we observed more inflammatory cells in the bronchoalveolar lavage fluid and increased bacterial load in the lungs of vitamin D-deficient male mice with allergic airway disease, suggesting that vitamin D might modulate the lung microbiome. In the current study, we examined in more depth the effects of vitamin D deficiency initiated early in life, and subsequent supplementation with dietary vitamin D on the composition of the lung microbiome and the extent of respiratory inflammation. METHODS: BALB/c dams were fed a vitamin D-supplemented or -deficient diet throughout gestation and lactation, with offspring continued on this diet post-natally. Some initially deficient offspring were fed a supplemented diet from 8 weeks of age. The lungs of naïve adult male and female offspring were compared prior to the induction of allergic airway disease. In further experiments, offspring were sensitised and boosted with the experimental allergen, ovalbumin (OVA), and T helper type 2-skewing adjuvant, aluminium hydroxide, followed by a single respiratory challenge with OVA. RESULTS: In mice fed a vitamin D-containing diet throughout life, a sex difference in the lung microbial community was observed, with increased levels of an Acinetobacter operational taxonomic unit (OTU) in female lungs compared to male lungs. This effect was not observed in vitamin D-deficient mice or initially deficient mice supplemented with vitamin D from early adulthood. In addition, serum 25-hydroxyvitamin D levels inversely correlated with total bacterial OTUs, and Pseudomonas OTUs in the lungs. Increased levels of the antimicrobial murine ß-defensin-2 were detected in the bronchoalveolar lavage fluid of male and female mice fed a vitamin D-containing diet. The induction of OVA-induced allergic airway disease itself had a profound affect on the OTUs identified in the lung microbiome, which was accompanied by substantially more respiratory inflammation than that induced by vitamin D deficiency alone. CONCLUSION: These data support the notion that maintaining sufficient vitamin D is necessary for optimal lung health, and that vitamin D may modulate the lung microbiome in a sex-specific fashion. Furthermore, our data suggest that the magnitude of the pro-inflammatory and microbiome-modifying effects of vitamin D deficiency were substantially less than that of allergic airway disease, and that there is an important interplay between respiratory inflammation and the lung microbiome.

Can skin exposure to sunlight prevent liver inflammation?

Liver inflammation contributes towards the pathology of non-alcoholic fatty liver disease (NAFLD). Here we discuss how skin exposure to sunlight may suppress liver inflammation and the severity of NAFLD. Following exposure to sunlight-derived ultraviolet radiation (UVR), the skin releases anti-inflammatory mediators such as vitamin D and nitric oxide. Animal modeling studies suggest that exposure to UVR can prevent the development of NAFLD. Association studies also support a negative link between circulating 25-hydroxyvitamin D and NAFLD incidence or severity. Clinical trials are in their infancy and are yet to demonstrate a clear beneficial effect of vitamin D supplementation. There are a number of potentially interdependent mechanisms whereby vitamin D could dampen liver inflammation, by inhibiting hepatocyte apoptosis and liver fibrosis, modulating the gut microbiome and through altered production and transport of bile acids. While there has been a focus on vitamin D, other mediators induced by sun exposure, such as nitric oxide may also play important roles in curtailing liver inflammation.

Comparing the effects of sun exposure and vitamin D supplementation on vitamin D insufficiency, and immune and cardio-metabolic function: the Sun Exposure and Vitamin D Supplementation (SEDS) Study.

BACKGROUND: Adults living in the sunny Australian climate are at high risk of skin cancer, but vitamin D deficiency (defined here as a serum 25-hydroxyvitamin D (25(OH)D) concentration of less than 50 nmol/L) is also common. Vitamin D deficiency may be a risk factor for a range of diseases. However, the optimal strategies to achieve and maintain vitamin D adequacy (sun exposure, vitamin D supplementation or both), and whether sun exposure itself has benefits over and above initiating synthesis of vitamin D, remain unclear. The Sun Exposure and Vitamin D Supplementation (SEDS) Study aims to compare the effectiveness of sun exposure and vitamin D supplementation for the management of vitamin D insufficiency, and to test whether these management strategies differentially affect markers of immune and cardio-metabolic function. METHODS/DESIGN: The SEDS Study is a multi-centre, randomised controlled trial of two different daily doses of vitamin D supplementation, and placebo, in conjunction with guidance on two different patterns of sun exposure. Participants recruited from across Australia are aged 18-64 years and have a recent vitamin D test result showing a serum 25(OH)D level of 40-60 nmol/L. DISCUSSION: This paper discusses the rationale behind the study design, and considers the challenges but necessity of data collection within a non-institutionalised adult population, in order to address the study aims. We also discuss the challenges of participant recruitment and retention, ongoing engagement of referring medical practitioners and address issues of compliance and participant retention. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry: ACTRN12613000290796 Registered 14 March 2013.

Ultraviolet radiation suppresses obesity and symptoms of metabolic syndrome independently of vitamin D in mice fed a high-fat diet.

The role of vitamin D in curtailing the development of obesity and comorbidities such as the metabolic syndrome (MetS) and type 2 diabetes has received much attention recently. However, clinical trials have failed to conclusively demonstrate the benefits of vitamin D supplementation. In most studies, serum 25-hydroxyvitamin D [25(OH)D] decreases with increasing BMI above normal weight. These low 25(OH)D levels may also be a proxy for reduced exposure to sunlight-derived ultraviolet radiation (UVR). Here we investigate whether UVR and/or vitamin D supplementation modifies the development of obesity and type 2 diabetes in a murine model of obesity. Long-term suberythemal and erythemal UVR significantly suppressed weight gain, glucose intolerance, insulin resistance, nonalcoholic fatty liver disease measures; and serum levels of fasting insulin, glucose, and cholesterol in C57BL/6 male mice fed a high-fat diet. However, many of the benefits of UVR were not reproduced by vitamin D supplementation. In further mechanistic studies, skin induction of the UVR-induced mediator nitric oxide (NO) reproduced many of the effects of UVR. These studies suggest that UVR (sunlight exposure) may be an effective means of suppressing the development of obesity and MetS, through mechanisms that are independent of vitamin D but dependent on other UVR-induced mediators such as NO.

Reversible control by vitamin D of granulocytes and bacteria in the lungs of mice: an ovalbumin-induced model of allergic airway disease.

Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We have developed a 'low dose' model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 µg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.

Exposure to UV Wavelengths in Sunlight Suppresses Immunity. To What Extent is UV-induced Vitamin D3 the Mediator Responsible?

Reduced immunity following exposure of skin to UV radiation (UVR) may explain the positive latitude gradient measured for a number of autoimmune diseases (greater incidence of disease with residence at higher latitudes), including multiple sclerosis, allergic asthma and diabetes. Humans obtain >80% of their vitamin D3 by exposure of skin to UVR in sunlight. In experimental models, both vitamin D3-dependent and vitamin D3-independent pathways have been implicated in the mechanisms of UVR-induced systemic suppression of immunity. However, where does the balance of control lie? How important is vitamin D3 other than providing a biomarker of sun exposure? Are other molecules/pathways activated by UVR more important? Murine and human studies suggest many molecules may play a role and their participation may vary with different diseases and the time of UVR exposure or vitamin D3 sufficiency/deficiency. Although low vitamin D3 levels have been associated with increased prevalence and progression of human autoimmune diseases, the benefits of supplementation with vitamin D3 have not been definitive. Vitamin D3 levels are a measure of past sun exposure but vitamin D3-dependent and vitamin D3-independent immunosuppressive effects of UVR may play a role in control of autoimmune diseases.

Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes.

The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D. In this study, topically applied 1,25(OH)(2)D(3), enhanced the suppressive capacity of CD4(+)CD25(+) cells from the draining lymph nodes. The effects of topical 1,25(OH)(2)D(3) were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)(2)D(3) production in skin. CD4(+) cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)(2)D(3)-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4(+)CD25(+) cells. Furthermore, purified CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated or UVB-irradiated mice compared with equal numbers of CD4(+)CD25(+) cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4(+)Foxp3(+) cells of donor origin significantly increased in recipients of CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)(2)D(3) may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.