Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

The local generation of active glucocorticoid by NADPHdependent, 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11␤-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11␤-HSD1 oxoreductase activity and add a new dimension to the investigation of 11␤-HSD1 as a therapeutic target in patients with the metabolic syndrome.Corticosteroid hormone action is modulated in a tissue-specific fashion by the expression and activity of two isozymes of 11␤-hydroxysteroid dehydrogenase (11␤-HSD) 3 that interconvert hormonally active cortisol (or corticosterone in rodents) to their inactive derivatives, cortisone/11-dehydrocorticosterone (11-DHC) (1). 11␤-HSD2 acts as a NAD-dependent dehydrogenase, inactivating glucocorticoids and protecting the mineralocorticoid receptor from illegitimate activation by cortisol in mineralocorticoid receptor-rich tissues such as the kidney and colon (2).By contrast, 11␤-HSD1 is a bidirectional enzyme expressed in liver and adipose tissue. It catalyzes both oxidation and oxoreduction of glucocorticoids but acts in vivo predominantly as a NADPH-dependent oxoreductase (1). Independent of circulating glucocorticoid concentrations, the local generation of cortisol/corticosterone by 11␤-HSD1 has emerged as an important factor in regulating hepatic glucose output (by augmenting gluconeogenesis) and visceral adiposity (by increasing adipocyte differentiation). Transgenic mice overexpressing 11␤-HSD1 in liver and adipose tissue recapitulate features of the metabolic syndrome with visceral obesity, hepatic steatosis, glucose intolerance, insulin resistance, hyperlipidemia, and hypertension (3-5). In contrast, knockout (KO) mice lacking 11␤-HSD1 show improved glucose tolerance, enhanced insulin sensitivity, and reduced weight gain when fed a high fat diet (6, 7).11␤-HSD1 has therefore emerged as a novel therapeutic target to treat patients with obesity and insulin resistance. Indeed, selective 11␤-HSD1 inhibitors improve glucose tolerance in diabetic mice (8 -10).These in vivo findings are dependent upon the oxoreductase activity of 11␤-HSD1, which is puzzling because activity studies performed on the purified 11␤-HSD1 enzyme or on cell/tissue homogenates indicate almost exclusive unidirectional dehydrogenase activity (11). We hypothesized that the enzyme hexose-6-phosphate de...

A new genome-scale metabolic reconstruction of M. pneumonia is used in combination with external metabolite measurement and protein abundance measurements to quantitatively explore the energy metabolism of this genome-reduce human pathogen.

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