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Am J Physiol Endocrinol Metab 293: E794-E801, 2007. First published June 26, 2007; doi:10.1152/ajpendo.00168.2007
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Tissue-dependent loss of phosphofructokinase-M in mice with interrupted activity of the distal promoter: impairment in insulin secretion

Ann-Marie T. Richard,1,2,3 Dominic-Luc Webb,4 Jessie M. Goodman,1,3 Vera Schultz,1,3 John N. Flanagan,3 Lisa Getty-Kaushik,3 Jude T. Deeney,3 Gordon C. Yaney,3 George A. Dunaway,5 Per-Olof Berggren,4 and Keith Tornheim1,3

1Department of Biochemistry; 2Department of Pathology, Boston University School of Medicine; 3Obesity Research Center, Evans Department of Medicine, Boston University Medical Center, Boston, Massachusetts; 4Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and 5Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois

Submitted 14 March 2007 ; accepted in final form 20 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phosphofructokinase is a key enzyme of glycolysis that exists as homo- and heterotetramers of three subunit isoforms: muscle, liver, and C type. Mice with a disrupting tag inserted near the distal promoter of the phosphofructokinase-M gene showed tissue-dependent differences in loss of that isoform: 99% in brain and 95–98% in islets, but only 50–75% in skeletal muscle and little if any loss in heart. This correlated with the continued presence of proximal transcripts specifically in muscle tissues. These data strongly support the proposed two-promoter system of the gene, with ubiquitous use of the distal promoter and additional use of the proximal promoter selectively in muscle. Interestingly, the mice were glucose intolerant and had somewhat elevated fasting and fed blood glucose levels; however, they did not have an abnormal insulin tolerance test, consistent with the less pronounced loss of phosphofructokinase-M in muscle. Isolated perifused islets showed about 50% decreased glucose-stimulated insulin secretion and reduced amplitude and regularity of secretory oscillations. Oscillations in cytoplasmic free Ca2+ and the rise in the ATP/ADP ratio appeared normal. Secretory oscillations still occurred in the presence of diazoxide and high KCl, indicating an oscillation mechanism not requiring dynamic Ca2+ changes. The results suggest the importance of phosphofructokinase-M for insulin secretion, although glucokinase is the overall rate-limiting glucose sensor. Whether the Ca2+ oscillations and residual insulin oscillations in this mouse model are due to the residual 2–5% phosphofructokinase-M or to other phosphofructokinase isoforms present in islets or involve another metabolic oscillator remains to be determined.

OmniBank; calcium; pancreatic islets; adenosine 5'-triphosphate/adenosine 5'-diphosphate ratio

PHOSPHOFRUCTOKINASE (PFK) is a key regulatory enzyme of glycolysis. The three mammalian isoforms differ somewhat in their regulatory properties (13), with a major difference being the strong product activation of the muscle isoform by fructose 1,6-bisphosphate (F16BP) (45, 54). It is this property that we showed was involved in causing spontaneous oscillatory behavior of glycolysis in muscle extracts (1, 46). Active phosphofructokinase is a tetramer. The three subunit isoforms, muscle (M), liver (L), and C type (C), are transcribed from separate genes. Although muscle expresses only PFK-M, other tissues express all three subunit isoforms to different degrees (14, 15). The subunits can associate into heterotetramers as well as homotetramers; it is not clear to what extent heterotetramers exhibit intermediate kinetics or whether there is a degree of independence of the subunits. The expression of PFK-M in different cell types is further complicated by the presence of multiple transcripts. The mouse and human PFK-M genes have both a distal and a proximal promoter, and it has been proposed (16, 28–30, 52) that the proximal promoter is largely muscle specific in function, whereas the distal promoter is used ubiquitously. We report here that mice with a disrupting tag inserted near the distal promoter exhibited tissue-dependent loss of PFK-M, providing strong support for this two-promoter model.

Interestingly, despite the presence of other PFK isoforms in nonmuscle tissues, the PFK-M-deficient mice did show a phenotype of impaired glucose tolerance and somewhat elevated fed and fasting blood glucose levels, and the secretory response to glucose in isolated islets was impaired. Glucose stimulation of insulin secretion requires glucose metabolism and involves a rise in the ATP/ADP ratio to close ATP-sensitive K+ channels and trigger Ca2+ influx. Furthermore, normally there are oscillations in metabolism, cytoplasmic free Ca2+ concentration ([Ca2+]i), and insulin secretion, and we have proposed that these may be due to underlying oscillations in glycolysis generated by PFK-M (9, 44). However, [Ca2+]i and secretory oscillations were still seen in PFK-M-deficient islets/cells. This may indicate that there is another underlying oscillation mechanism; alternatively, because of the great excess of PFK over glucokinase in islets, the 2–5% residual PFK-M seen in the PFK-M-deficient islets may still have been sufficient to generate the oscillations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Heterozygous mice with a disrupting tag inserted in the noncoding region of the PFK-M gene (OmniBank OST no. 56064, 50% C57Bl/6 albino, 50% 129svEvBrd) were received initially from Lexicon Genetics (The Woodlands, TX). OmniBank is a library of more than 200,000 mouse embryonic stem cell clones, each containing a gene trap insertion (created by insertional mutagenesis) in a single gene (55). OST no. 56064 was the only match to the PFK-M gene. The mice were bred in-house at Boston University Medical Center to generate wild-type and homozygous PFK-M-deficient mice. (Note: "PFK-M" has previously been used to refer to the homotetramer M4 or muscle PFK, especially in kinetic studies. In this paper, "PFK-M deficient" technically refers to loss of the subunit isoform as detected by Western analysis; this implies loss of M4 but also of M-containing heterotetramers in tissues expressing other isoforms.) Mice used were bred from heterozygotes to avoid any effects of maternal hyperglycemia, since mild hyperglycemia was noted in homozygous females (see RESULTS). Genotyping (nptII Invader Assay; Third Wave Technologies) of the initial 220 offspring yielded results corresponding with Mendelian inheritance: 25% wild type, 55% heterozygotes, and 20% homozygotes, indicating that the mutation was not acutely detrimental to development. Most litters consisted of 5–8 pups, and there were no deaths among newborns. Mice used for experiments were 3–6 mo of age; no consistent difference in weight was observed between wild-type and homozygous mice of this age. All procedures were approved by the Boston University Medical Center Animal Care and Use Committee.

Intraperitoneal glucose tolerance test. Blood glucose from the tail vein of overnight-fasted female mice was measured with a portable glucometer before and after glucose injection (1 mg/g body wt). For insulin radioimmunoassay, blood was collected in heparinized tubes, placed on ice, centrifuged, and stored at –20°C.

Intraperitoneal insulin tolerance test. Blood glucose levels were measured in fed female mice before and after insulin injection (0.75 U/kg body wt).

Tissue extraction for Western analysis and PFK activity. Tissues (gastrocnemius or quadriceps muscle, heart, and brain) were frozen in liquid nitrogen and extracted as described previously in 6 volumes of extraction buffer (54). Samples were fast-frozen in liquid nitrogen and stored at –80°C.

Western analysis of PFK subunit isoforms. PFK subunit isoforms were separated using SDS-PAGE and blotted essentially as previously described (54). The primary polyclonal antibodies were rabbit anti-dog muscle PFK, which recognizes only the M isoform and rabbit anti-rat brain PFK raised against all three isoforms (27).

PFK activity measurements. PFK activity stimulated by F16BP (20 µM) under inhibitory conditions (0.1 mM fructose 6-phosphate, 2 mM ATP, 0.02 mM AMP, pH 7) was measured in the high-speed supernatant fraction from mouse tissue extracts as described previously (54). Maximal PFK activity was assayed by measuring F16BP production in the presence of 2 mM fructose 6-phosphate and 1 mM ATP, pH 8.2 (2).

Real-time PCR. Primers were designed using Primer Express v2.0 software (PerkinElmer Applied Biosystems, Foster City, CA) to ensure suitability for the ABI Prism 7000 sequence detection system used and the reaction parameters according to the manufacturer's protocol. Three sets of primers were designed, one for total PFK-M message and two to distinguish transcripts derived from the proximal and distal promoters. For total PFK-M (NM_021514.2), the primers were 5'-CAG ATC AGT GCC AAC ATA ACC AA-3' (forward) and 5'-CGG GAT GCA GAG CTC ATC A-3' (reverse), which amplified a fragment from +515 to +670 bp from the mouse cDNA. For proximal (D21868) and distal (D21867) PFK-M promoter transcripts, the forward primers were 5'-ATA CTG CAT CTT AAC CGA CCA TTG T-3' and 5'-CCC TTT CCC CAG AGG ACA A-3', respectively, with a common reverse primer of 5'-TGA CGG CAG CAT TCA TAC CTT-3'. The proximal amplicon corresponded to –114 to +65 bp and the distal amplicon to –121 to +65 bp according to published mouse isoform sequences (29). Total RNA was prepared from mouse tissues using SV Total RNA Isolation System (Promega, Madison, WI). Single-stranded cDNA synthesis was performed using 2 µg of total RNA as template and random hexamer as primer with Superscript II RNase H Reverse Transcriptase (Life Technologies, Rockville, MD) according to the manufacturer's protocol. For each real-time PCR reaction, 20 ng of single stranded cDNA was mixed with 2x SYBR Green PCR Master Mix (ABI) and optimal concentration of sequence-specific primers. To normalize the amount of sample cDNA added to the reaction, Taqman PDAR eukaryotic 18S rRNA (ABI) was used as the endogenous control. Each mouse tissue was tested three times in duplicate for each set of primers.

Islet isolation and perifusion. Pancreatic islets were isolated as previously described (50), hand-picked, and cultured overnight in RPMI 1640 tissue culture medium (GIBCO) with 11.1 mM glucose and 10% (vol/vol) fetal calf serum (HyClone Laboratories). Groups of 50–100 islets were perifused as described previously (10). The effluent fractions were assayed for insulin by radioimmunoassay using the protocol for rat insulin from Linco Research (St. Louis, MO). Data are presented as three-point moving averages (14). Pulses in insulin secretion were detected and analyzed as described previously (10, 11) using Cluster analysis (48) of the raw (nonaveraged) data, with nadir and peak cluster sizes of three points, a minimum t-statistic of 3, and a coefficient of variation of 6%.

Islet PFK purification. Islets were washed three times with phosphate-buffered saline and extracted in 0.4 ml of purification buffer containing 50 mM Tris phosphate (pH 8), 50 mM sodium fluoride, 10 mM dithiothreitol, 0.05 mM F16BP, 0.1 mM disodium EDTA, 0.2 mg/ml leupeptin, and 50 µg/ml aprotinin. The sample was sonicated, centrifuged for 5 min at 16,000 g, and then purified by adding 40 µl of fast-flow cibacron blue 3GA agarose beads (Sigma, St. Louis, MO) and rocking at 4°C for 1 h (14). The beads with bound PFK protein were washed four times with purification buffer, resuspended in 5x SDS-PAGE buffer, heated at 90°C, and centrifuged, and the supernatant was frozen for SDS-PAGE.

[Ca2+]i was measured at the single cell level in small cell clusters or isolated islet cells loaded with fura-2, as described previously, using a microscope-based spectrofluorometer system with an attached CCD camera (20).

ATP/ADP measurements. Islets (8–10/sample) were incubated in HEPES-buffered Krebs salt solution (10) at 3 mM glucose, and then the glucose concentration was raised to 11 mM. Samples were deproteinized with 1% trichloroacetic acid and processed and analyzed by our bioluminometric method as previously described (32, 43).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PFK expression in mouse tissues. There was tissue-dependent loss of the M-type subunit in homozygous animals, which we therefore refer to as PFK-M deficient. Thus, Western analysis using a PFK-M-specific antibody showed 50–75% loss in skeletal muscle and little change in heart but 99% loss in brain (Fig. 1, A and B). Enzymatic activity measured spectrophotometrically in some samples showed similar results. In brain, which normally has ~50% M-type subunit (data not shown) (14, 15, 54), the maximal total activity was decreased 50%, but the activity stimulated by F16BP, which is characteristic of PFK-M, was decreased >95% (sensitivity limited by background ATPase activity).


Figure 1
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  		Fig. 1. Western blots of phosphofructokinase (PFK)-M in mouse tissues. Blots were probed with a PFK-M-specific antibody (A–C and D, top) or anti-brain PFK antibody (D, bottom). Results shown are representative of 3 sample sets for brain, 4 for skeletal muscle, and 2 for heart. WT, wild type; Def, PFK-M deficient; Mus std, muscle standard; C, M, and L, C-type, muscle, and liver PFK subunit isoforms, respectively.

 
The likely explanation for the large tissue-dependent differences in loss of PFK-M is disruption of the distal promoter. The distal transcription start site is normally used ubiquitously, whereas the proximal transcription initiation site was reported to be used primarily in muscle in mice (9). In agreement with this, skeletal muscle, heart, and brain from wild-type mice all contained distal promoter transcript; skeletal muscle and heart also had detectable levels of proximal promoter transcript, whereas the brain did not (Table 1). Levels of the distal promoter transcript were greatly decreased (>99%) in all of these tissues from the PFK-M-deficient mice. Levels of the proximal promoter transcript were decreased somewhat (50%) in skeletal muscle and increased in heart and remained essentially undetectable in brain. Total PFK-M transcript levels were decreased perhaps 30–40% in skeletal muscle and heart but 99% in brain.


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  		Table 1. Expression of PFK-M transcripts in tissues from wild-type and PFK-M-deficient mice

 
In initial Western blotting studies in pancreatic islets from one or two PFK-M-deficient mice, the M-type subunit could not be detected (>85–90% reduction). To better quantitate the loss, it was necessary to pool islets from seven deficient mice and purify and concentrate the islet extracts (see MATERIALS AND METHODS) to detect any M-type subunit. This revealed a level that was a small percentage (2–5%) of the level in wild-type islets (Fig. 1C), in other words, a 95–98% reduction. Western blot analysis using an anti-brain PFK antibody that binds to all three PFK isoforms showed increased amounts of the other two subunit isoforms, C and L (n = 3; Fig. 1D), perhaps as a form of compensation. This pattern was also observed in brain (data not shown).

In vivo measurements: glucose and insulin tolerance tests. Female PFK-M-deficient mice had elevated fasting blood glucose levels [7.6 ± 0.3 mM (means ± SE), n = 6, vs. 4.6 ± 0.6 mM, n = 3, in wild-type controls; P = 0.001] and exhibited impaired glucose tolerance (Fig. 2). Basal insulin values were not significantly different (0.44 ± 0.07 vs. 0.34 ± 0.08 ng/ml, PFK-M deficient vs. wild type, n = 4 and 5, respectively). Glucose-stimulated insulin levels (15 min after injection) were not significantly lower in the PFK-M-deficient mice (0.64 ± 0.14 vs. 0.78 ± 0.16 ng/ml in wild type), but the percentage increase in insulin levels was significantly lower in the PFK-M-deficient mice (42 ± 15 vs. 140 ± 32% in wild type, P < 0.02). Insulin tolerance tests on fed female mice showed a similar drop in blood glucose in response to the insulin in the two groups (Fig. 3), indicating that the PFK-M-deficient mice were not grossly insulin resistant. Basal blood glucose levels were also elevated in these nonfasted animals (11.2 ± 0.3 mM, n = 5, vs. 8.2 ± 0.2 mM, n = 7, for wild type; P < 0.0001). The absence of insulin resistance argues that the glucose intolerance is due largely to a defect in insulin secretion.


Figure 2
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  		Fig. 2. Intraperitoneal glucose tolerance test. Blood glucose was measured in fasted female mice before and after glucose injection (1 mg/g body wt) at the times indicated. Results are means ± SE for 6 PFK-M-deficient and 3 WT mice.

 

Figure 3
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  		Fig. 3. Intraperitoneal insulin tolerance test. Blood glucose was measured in fed female mice before and after injection of insulin (0.75 U/kg body wt) at the times indicated. Results are means ± SE for 5 PFK-M-deficient and 7 WT mice.

 
Insulin secretion from isolated islets. Islets isolated from wild-type and PFK-M-deficient mice were perifused with glucose in a column. Although both groups of islets responded to stimulatory concentrations of glucose (11 or 16.7 mM), there was ~50% reduction in average secretion from the PFK-M-deficient islets compared with wild type (Fig. 4 and Table 2). The amplitude of the insulin pulses was also reduced in PFK-M-deficient islets, whereas the period remained unchanged. Assay of the total insulin content in the islets retrieved from the columns of the experiment shown in Fig. 4C showed that the PFK-M-deficient group did not have less insulin (in fact, it had ~20% more). Therefore, the secretory defect is not due to insulin deficiency. The regularity of pulses was analyzed by measurement of approximate entropy for each group, where a relatively lower value indicates greater regularity (36). In general, pulsatile secretion from PFK-M-deficient islets was more irregular than that from wild type (approximate entropy = 0.002 ± 0.000 in PFK-M-deficient group vs. 0.0003 ± 0.0001 in wild type, n = 4; P = 0.00002). Basal (3 mM glucose) secretion was very low compared with stimulated secretion, and there was no consistent difference in basal secretion between PFK-M-deficient and wild-type islets.


Figure 4
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  		Fig. 4. Oscillatory glucose-stimulated insulin secretion from perifused islets. Groups of islets isolated from WT and PFK-M-deficient mice were perifused with basal (3 mM) glucose. A: the glucose concentration was raised to 16.7 mM at 8 min. B: the glucose concentration was raised to 8 and then to 16.7 mM at 29 min. C and D: the glucose concentration was raised to 11 mM at 6 min. Insulin concentrations at 3 and 8 mM were generally undetectable with the standard assay kit used in A and B, and the points are omitted. (A more sensitive kit was used in C and D.) {blacktriangleup}, WT; {circ}, Def.

 

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  		Table 2. Oscillatory insulin secretion from perifused islets

 
[Ca2+]i oscillations in islet cells. Slow oscillations in [Ca2+]i normally correlate with insulin oscillations (2, 18) and can be measured at the single-cell level. Oscillations in [Ca2+]i in response to glucose stimulation were observed in cell clusters from PFK-M-deficient islets (Fig. 5) in a proportion of cells similar to that of wild-type cells. Thus, of the 139 cells from three PFK-M-deficient mice, 91% responded to 8 mM glucose and 71% exhibited oscillations, whereas of the 111 cells from three wild-type mice, 92% responded to 8 mM glucose and 75% exhibited oscillations. To test the possibility that a small percentage of cells from PFK-M-deficient islets might not be deficient (corresponding to the residual 2–5% PFK-M seen in Western analysis) and might thus be serving as pacemakers in cell clusters, we also examined single cells not in clusters; however, 50% (of 10 cells) were still seen to oscillate (compared with 7 of 10 wild-type cells). Basal [Ca2+]i and initial and second-phase glucose-stimulated average [Ca2+]i appeared to be similar. The time of onset of the Ca2+ response after glucose addition was also not different (98 ± 5 s, n = 112, for PFK-M deficient vs. 110 ± 7 s, n = 87, for wild type).


Figure 5
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  		Fig. 5. Glucose-induced [Ca2+]i oscillations in islet cell aggregates. Recordings shown are of individual cells in small cell clusters from WT (A) and PFK-M-deficient (B) mouse islets, perifused initially at 3 and then 8 mM glucose at the times indicated. Oscillations were seen in 75% of the WT (n = 111) and 71% of the PFK-M-deficient (n = 139) islet cells.

 
ATP/ADP ratio. The rise in the ATP/ADP ratio in response to glucose stimulation was normal in PFK-M-deficient islets. Thus the ATP/ADP ratio was 2.5 ± 0.1 at 3 mM glucose and 4.5 ± 0.2 and 4.4 ± 0.3 after 1 and 5 min, respectively, of 11 mM glucose in PFK-M-deficient islets, compared with a rise from 2.5 ± 0.1 to 4.5 ± 0.2 and 4.5 ± 0.1 in wild-type islets, combining the data of two experiments each with 3–4 samples of islets per data point. In a third experiment with somewhat lower values, there was also no difference between PFK-M-deficient and wild-type islets.

Insulin oscillations in the presence of diazoxide and high KCl. Alternative mechanisms have been suggested for oscillations in [Ca2+]i and, hence, insulin secretion that involve Ca2+ feedback effects on KATP channels (42). If this occurs, it could hide the effects of loss of a metabolic/glycolytic oscillator on oscillations in secretion. Therefore, secretion from perifused islets was examined in the presence of diazoxide (to keep KATP channels open) and high KCl (to depolarize the cell directly and keep [Ca2+]i high). Oscillations in insulin secretion were still seen from PFK-M-deficient islets (as well as from wild-type islets; Fig. 6) of similar period and amplitude as in the absence of diazoxide/KCl.


Figure 6
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  		Fig. 6. Oscillatory insulin secretion in the presence of diazoxide and high KCl. Groups of islets were perifused in a column with basal (3 mM) followed by stimulatory (16.7 mM) glucose in the presence of 0.2 mM diazoxide and 30 mM KCl as indicated. Oscillation amplitude and period at high glucose were 4.5 ± 0.8 ng/ml and 3.4 ± 1.2 min (A) and 1.1 ± 0.4 ng/ml and 3.2 ± 1.4 min (B). Results shown are representative of 2 experiments with WT and 3 experiments with PFK-M-deficient islets.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This mouse model with a disruptive tag inserted into the PFK-M gene is particularly interesting because of the tissue-dependent deficiency in PFK-M, with muscle and heart being less or little affected compared with 99% loss in brain and 95–98% loss in pancreatic islets. The results provide further evidence for the two-promoter system of the PFK-M gene, with its alternative initiation sites of the PFK-M message. The mouse and human PFK-M genes contain two promoters: a distal promoter that is expressed in all tissues and a proximal promoter that can be regulated by muscle-specific enhancers (16, 28–30, 52). By comparing a downstream sequence provided by Lexicon Genetics with the mouse PFK-M genomic sequence, we ascertained that the OmniBank gene trap vector was inserted at position –3,273 to –3,272 relative to the ATG start site. The proximal transcript 5'-untranslated region has been mapped to –106 to –9 bp, whereas the distal transcript 5'-untranslated region has been mapped to –3,612 to –3,550, with a putative splice junction between –3,550 and –3,549 (29). Both transcripts share the –8 to –1 sequence. Thus the disrupting tag is between the two promoters and close to the distal promoter. This disrupts the functioning of the distal promoter, as shown by the RT-PCR data in Table 1, and therefore, tissues such as brain and presumably islet that rely on the distal promoter have greatly reduced PFK-M. Muscle and heart utilize the proximal promoter as well as the distal promoter. The 50–75% reduction in PFK-M protein in muscle in PFK-M-deficient mice may suggest that there is normally a substantial contribution of the distal promoter to PFK-M protein production in muscle or that the action of upstream sequences controlling the proximal promoter (16) was also affected, as indicated by the 50% decrease in proximal transcript (Table 1). Nevertheless, because of the great surplus of PFK-M in muscle (45), this limited reduction may have little physiological consequence. This may be the reason that the PFK-M-deficient mice did not show insulin resistance in contrast to that reported in some studies of humans with global loss of PFK-M (41).

Heart showed little reduction in PFK-M protein in these mice, and the relative amount of the proximal transcript was increased. Perhaps there are differences in the muscle-specific enhancers or their potency in heart vs. skeletal muscle. Conversely, there could be loss of regulatory sequences that restrict transcription and are normally more potent in heart. In addition, on a translational level, Nakajima et al. (29) noted that the 5'-untranslated region in the proximal promoter transcript contained putative regulatory sequences that might affect the translation efficiency.

The two-promoter system has also been found in the rat PFK-M gene. However, in contrast to mouse and human, in rat the proximal promoter appears to be operative in nonmuscle tissues such that brain had as much proximal transcript as distal transcript (26). Recently, a third type of PFK-M transcript has been found in mouse and human that is initiated much further upstream from the distal promoter and is specific to testis and embryo (51).

Interestingly, the PFK-M deficiency led to a deficiency in insulin secretion. The mice showed impaired glucose tolerance but were not insulin resistant, at least not to a degree detectable by the insulin tolerance test. The glucose-stimulated insulin levels in vivo were perhaps only somewhat lower than in wild-type mice, but the plasma glucose levels were considerably higher. Normalization to basal insulin showed a significantly lower percentage increase in insulin in the PFK-M-deficient mice. Furthermore, their isolated perifused islets showed diminished glucose-stimulated insulin secretion. A simple explanation for this could be reduced glycolytic flux; however, this is unlikely to be the case because of the presence of the other PFK isoforms (which were actually increased in amount in the PFK-M-deficient islets, as shown in Fig. 1D) and the considerable reserve of PFK activity compared with the metabolic flux. Most likely, glucokinase, the glucose sensor for glycolytic flux (25), remains rate limiting. Trus et al. (47) reported PFK activities in islet extracts that were 40 times those of glucokinase, and even that may have been a gross underestimate because of the reported instability of PFK in the cell extracts; a companion paper (5) showed in addition that glucose usage at 10 mM glucose was equivalent to about 50% of the assayed glucokinase activity but to 1% of the assayed PFK activity. Note that, in brain, a tissue with a similar PFK isoform mix as islet, total assayed PFK activity was decreased only 50% when PFK-M was decreased 99% in the deficient mice. That glucokinase remains the rate-limiting step in the PFK-M-deficient islets is further indicated by the great stimulation of secretion by increased glucose concentration and the fact that the impairment was not much greater at higher (16.7 mM) than lower (11 mM) stimulatory glucose (Fig. 4 and Table 1). The observation that Ca2+ responses were normal suggests that the deficiency is in amplifying or incretin effects. It has been suggested that an elevated ATP/ADP ratio may be involved in downstream effects of glucose as well as the triggering of Ca2+ influx (17). A normal glucose-stimulated rise in the ATP/ADP ratio was seen in PFK-M-deficient islets. Although it is possible that the rise was impaired in the cytosol, but not detected in our whole cell assay because of the ADP in the insulin secretion granules and perhaps mitochondria (12), nevertheless, clearly it was sufficient to give a normal [Ca2+]i response. Another possible reason for diminished insulin secretion could be deficiency in lipid molecules, which are important in secretion (8, 53) and which we have shown enhance the amplitude of oscillations in secretion from perifused islets (11). Although the PFK-M-deficient mice were not consistently of lower body weight, at least in the age range used here, it did appear that they were considerably less fatty, a topic that is being pursued elsewhere. Finally, it is possible that the decreased secretion could be due to loss of PFK-M protein, independent of its enzymatic activity, since PFK is known to interact with at least three proteins that influence exocytosis, namely tubulin, calmodulin, and caveolin (6, 22, 31, 39).

Insulin secretion in vivo and from perifused islets is oscillatory with a period of minutes (10, 38, 44). Loss or impairment of the oscillations is seen in patients with type 2 diabetes and in their near relatives (34, 37), and this may be contributory to the development of the disease because oscillations enhance the potency of insulin (24, 35). On the basis of the spontaneous oscillations of glycolysis and the ATP/ADP ratio caused by autocatalytic F16BP activation of PFK in skeletal muscle extracts, we proposed that the oscillations in [Ca2+]i and insulin secretion in the pancreatic beta-cell are due to similar oscillations in glycolysis generated by PFK-M (9, 44). Oscillations in insulin secretion were still observed in PFK-M-deficient islets but were of reduced amplitude and diminished regularity. [Ca2+]i oscillations in clusters of cells appeared normal, and there was no great decrease in the number of oscillators even among isolated cells, arguing against the presence of a small number of unimpaired cells serving as pacemakers. Hence, superficially, one might conclude that PFK-M is not the oscillator, and that may indeed be the case. However, these islets had a residual 2–5% PFK-M, which may still have been sufficient to generate metabolic oscillations. As noted above, there is a large reserve of PFK activity compared with the metabolic flux. Metabolic oscillations have been observed in dilute extracts of muscle (1, 46) and islet (7). Thus it is not clear whether the [Ca2+]i oscillations and residual insulin oscillations in this mouse model are due to residual activity of PFK-M or to other PFK isoforms present in islets or may involve another oscillator. For example, a mitochondrial oscillator (23) of unknown mechanism is another possibility. On the other hand, in studies of a human family with inherited PFK-M deficiency, normal regular insulin oscillations (under basal conditions) were lost in the brother with a homozygous PFK-M deficiency (40). The human situation may be more complicated since the point mutations involved may lead to truncated, enzymatically inactive proteins that might affect heterotetramers, not just simple loss of PFK-M expression as in this mouse model. The decreased amplitude of insulin oscillations in the PFK-M-deficient mouse islets may reflect the overall decrease in secretion, that is, diminished incretin effects as noted above. The decreased regularity may reflect a less efficient synchronization of the group of islets perifused in the column.

Some other candidate models for generation of oscillations in the pancreatic beta-cell involve feedback effects of [Ca2+]i (42). However, insulin oscillations were still observed here in the presence of diazoxide and high KCl, as we reported previously in rat islets (10). Others have also shown insulin oscillations from ob/ob mouse islets when [Ca2+]i was constant (49). Our results show that this response is not restricted to islets from the hyperglycemic ob/ob mice, as suggested by some (21). This indicates that there is an oscillating mechanism that does not require oscillations in and, hence, feedback regulation by Ca2+ directly or indirectly on the KATP channel or on other channels or enzymes. The most likely possibility at this point is the existence of (at least) a glycolytic oscillator and a calcium/ionic oscillator, which can operate separately or interact with each other; mathematical modeling based on these concepts can account for the variety and complexity of oscillatory behavior observed in islets/beta-cells (3, 4, 33). Under conditions when [Ca2+]i is oscillating (whether independently or driven by a metabolic oscillator), it may be the major factor causing the oscillatory changes in insulin secretion. However, clearly other, presumably metabolic factors can also contribute, as shown here and elsewhere (19). In the PFK-M-deficient islets, the [Ca2+]i oscillations and residual insulin oscillations could possibly be due to the calcium/ionic oscillator, but the experiments with diazoxide and high KCl indicate that a metabolic oscillator is still operative, too.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Swedish Research Council, Berth von Kantzows Foundation, the Family Stefan Persson Foundation, Novo Nordisk Foundation, funds of Karolinska Institutet, the Swedish Diabetes Association, EuroDia LSHM-CT-2006-518153, Juvenile Diabetes Research Foundation Grant 1-2002-372, and NIDDK Grants DK-53064, DK-063356-03S1, and DK-58508. A.-M. T. Richard was in part covered by a postdoctoral fellowship on the Metabolism, Endocrinology, and Obesity Training Grant DK07201-28. D.-L. Webb was supported in part by Lars Hiertas Minne and Kungliga Fysiografiska Sallskapet.


   ACKNOWLEDGMENTS
 
We thank Drs. Cory Acuff and Wendy Jarman of Lexicon Genetics and Robin MacDonald of the Transgenic Core, Boston University School of Medicine, for their helpfulness with the PFK-M-deficient mice and Drs. Odile Peroni and Barbara Kahn of the Metabolic Physiology Core of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P30-DK-57521 for instruction in the glucose tolerance tests.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Tornheim, Boston University School of Medicine, 650 Albany St., Rm. 815, Boston, MA 02118 (e-mail: tornheim{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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