Supplementary figures
Figure 1. Micro-array of differential gene expression in liver between LD and DD mice using a 6k Unigene
mouse cDNA library. The comparison of Cy 5 to Cy3 signal as plotted by log10 (Cy5/Cy3). The arrow
indicates the gene (mClps) that displays the strongest differential hybridization signal. The cDNA probes were
generated from liver mRNA obtained at ZT 12 by reverse transcriptase labeled with Cy3 (green). Probes from
CT12 were labeled with Cy5 (red). The colipase gene is 04010913 (row 4, column 1, row 9, column 13). An
Excel spread sheet of this analysis with the accession number of the respective gene from this mouse 6K
Unigene mouse cDNA library is available on an accompanying compact disc.
Figure 2. Northern blot analysis of mClps expression on consecutive days. a) Expression of mClps in liver and
adipose tissue of mice. b) Colipase activity in DD mouse livers on consecutive days and in LD animals. Error
bars indicate SEM (n=4). A paired t-test was performed **p<0.01. c) Light inhibition of mClps and mPlrp2
expression in DD mice starting at CT7. A doublet band seen with the mPlrp2 probe could indicate cross
reactivity with its isoform, mPlrp1. Gapdh mRNA was monitored as an internal control.
Figure 3. a) Analysis of HPLC peaks in LD and DD mice. Top panels showed peak #1 and #3 in LD mice. the
bottom panel showed peak #2 and #4 in DD versus LD mice. HPLC peak areas are normalized to their internal
protein concentrations (A260nmarea/mg protein). Error bars indicate SD (n=2). b) HPLC peak areas of peaks #2,
and #4 in LD and DD mice normalized to their internal protein concentrations (A260nmarea/mg protein). Error
bars indicate SEM (n=4). A paired t-test was performed, ** p<0.01, *p<0.05.
Figure 4. The circadian regulated signal is 5’-adenosine monophosphate. a) HPLC retention time of 5’-AMP
and adenosine standards. b) & c) HPLC analysis of a mouse blood extract before and after a 2 min treatment
with affinity purified snake venom 5’nucleotidase. Arrows indicate the corresponding HPLC peaks of 5’-AMP
and adenosine. The retention time of peak #1 matched that of ADP. We excluded the possibility that cyclicAMP, or ATP were associated with peak #2 by demonstrating that their HPLC retention times were
approximately 13 and 3 min, respectively (data not shown).
Figure 5. Northern analysis of mClps induction by adenine nucleotides. a) RT-PCR analysis of mClps
expression in various organs after 5’-AMP injection. b) Concentration gating of adenosine induction of mClps
expression in LD mice. c). Adenosine and 5’-AMP induction of mClps is blocked by dipyridamole. Adenosine
and 5’-AMP were injected at 5 minutes after injection of dipyridamole (500 nmol/gram body weight). d) Effects
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of adenine nucleotides and nucleosides (150nmol/gram body weight) on mClps expression in LD mouse livers.
All Northern blots were probed with 32P labeled mClps and Gapdh cDNAs as described.
Figure 6. Metabolic analysis of DD versus LD mice. a) Food intake over 6 days. b) Water intake over 6 days. c)
Body weight of mice over the corresponding period. d) Free fatty acid levels in serum of DD versus LD mice. e)
Serum glucose levels of DD versus LD mice. All DD mice were kept in constant darkness for 48 h prior to the
start of data sampling. Error bars indicate SEM (n=3).
Figure 7. Northern blot analysis of ecto-5’nucleotidase mRNA expression in livers of LD and DD mice. Ecto5’nucleotidase expression was determined using radiolabeled cDNA probe from pSK-5’NT. Gapdh levels were
used as internal control.
Figure 8. 5’-AMP induction of mClps expression in LD mice is reciprocally linked to blood glucose levels. A)
Blood glucose levels in LD mice injected with saline or 1.5 mol of 5’-AMP/gram body weight. Error bars
indicate SEM (n=3). b) Northern blot analysis of the time course of mClps expression in LD mice after injection
of 5’-AMP. Gapdh mRNA levels were monitored as internal control. c) Model showing the pivotal role of 5’AMP (5’-adenosine monophosphate) in regulation of rate-limiting enzymes fructose 1,6-diphosphatase (FDP),
phosphofructokinase (PFK), glycogen phosphorylase (GP), colipase (CLPS) and pancreatic lipase related
protein 2 (PLRP2) for glucose, glycogen and fat metabolism, respectively.
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