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. 2006 Dec;55(12):3381-6.
doi: 10.2337/db06-0531.

Sodium-coupled glucose cotransporters contribute to hypothalamic glucose sensing

Dervla O'Malley  1 , Frank Reimann, Anna K Simpson, Fiona M Gribble
Affiliations

Sodium-coupled glucose cotransporters contribute to hypothalamic glucose sensing

Dervla O'Malley et al. Diabetes. 2006 Dec.

Abstract

Specialized neurons within the hypothalamus have the ability to sense and respond to changes in ambient glucose concentrations. We investigated the mechanisms underlying glucose-triggered activity in glucose-excited neurons, using primary cultures of rat hypothalamic neurons monitored by fluorescence calcium imaging. We found that 35% (738 of 2,139) of the neurons were excited by increasing glucose from 3 to 15 mmol/l, but only 9% (6 of 64) of these glucose-excited neurons were activated by tolbutamide, suggesting the involvement of a ATP-sensitive K(+) channel-independent mechanism. alpha-Methylglucopyranoside (alphaMDG; 12 mmol/l), a nonmetabolizable substrate of sodium glucose cotransporters (SGLTs), mimicked the effect of high glucose in 67% of glucose-excited neurons, and both glucose- and alphaMDG-triggered excitation were blocked by Na(+) removal or by the SGLT inhibitor phloridzin (100 nmol/l). In the presence of 0.5 mmol/l glucose and tolbutamide, responses could also be triggered by 3.5 mmol/l alphaMDG, supporting a role for an SGLT-associated mechanism at low as well as high substrate concentrations. Using RT-PCR, we detected SGLT1, SGLT3a, and SGLT3b in both cultured neurons and adult rat hypothalamus. Our findings suggest a novel role for SGLTs in glucose sensing by hypothalamic glucose-excited neurons.

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Figures

Figure 1
Figure 1. Glucose Excited neurons respond to αMDG but not tolbutamide
A. Sample ratiometric Ca2+-imaging traces from primary hypothalamic neurons illustrating the increase in the 340/380nm fluorescence ratio in response to an increase in glucose from a background level of 3mM (3G) to 15mM but not to tolbutamide (100μM, tolb). B. Sample Ca2+-imaging trace showing the increase in [Ca2+]i in response to addition of glucose (12mM, 12G) and αMDG (12mM), to a background solution containing 3 mM glucose. C. Histogram of the pooled data obtained as in A and B, illustrating the change in 340/380nm ratio (Δ ratio) in response to an increase in glucose concentration from 3–15mM (black bars) paired with responses to αMDG (12mM, open bar), tolbutamide (100μM, open bar) and a 2nd application of glucose (12mM, open bar). All cells found to respond to the 1st increase in glucose were included in the analysis. N values are shown above the bars. Statistical significance was tested by comparing the mean change in ratio in response to αMDG, tolbutamide or a 2nd application of glucose, with the first glucose response in the same cell, using a paired t-test. ***P<0.001, ns = P>0.05.
Figure 2
Figure 2. Responses to glucose and αMDG are inhibited by phloridzin and removal of extracellular Na+
A. Sample trace showing the responses to application of glucose (12mM:G12) under control conditions (background 3mM glucose: 3G) and in the presence of the competitive SGLT inhibitor, phloridzin (100 nM, Phl). B Sample ratiometric trace illustrating the Ca2+ response to glucose (12mM) and αMDG (12mM) under control conditions and upon removal of extracellular Na+. C Histogram of pooled data showing glucose (12mM, grey bars) and αMDG (12mM, black bars) - induced Ca2+ responses in the absence (filled bars) and presence (open bars) of phloridzin (100nM), or the presence (filled bars) or absence (open bars) of Na+. The numbers of cells are shown above the bars. Statistical significance was tested by comparing the mean substrate-triggered changes in ratio in the absence or presence of phloridzin, or the absence or presence of Na+, using a paired t-test. ***P<0.001, **P<0.01.
Figure 3
Figure 3. Sodium coupled glucose transporter expression in the hypothalamus
RT-PCR detects expression of SGLT1, SGLT3A, SGLT3B and SGLT4 in primary rat hypothalamic cultures. SGLT1, SGLT3A and SGLT3B were also readily detected in cDNA generated from a hypothalamic block from adult rat brain, while the SGLT4 primers only amplified a bigger (unspliced/genomic) band from this cDNA. All primer pairs did not amplify any bands when water was used instead of cDNA in the initial PCR and gave the expected band sizes when duodenal cDNA was used, in which expression of these SGLTs has been reported previously. The expected band sizes were: SGLT1: 323 bp, SGLT3A 209 bp, SGLT3B 227 bp, SGLT4 274 bp. Band identity was confirmed by direct sequencing.
Figure 4
Figure 4. SGLT1 can facilitate Ca2+ influx in GE neurons
A. Sample Ca2+ imaging trace from a GE neuron illustrating the response to addition of glucose (12mM: 12G), 3-0-MDG (12mM) and galactose (gal, 12mM) to the control 3 mM glucose (3G) solution. B. Histogram of pooled data showing the change in ratio triggered by galactose (12mM) and 3-0-MDG (12mM) in the absence (black bars) and presence of phloridzin (200μM, Phl, open bars). The numbers of cells are shown above the bars. Statistical significance was tested by comparing the mean changes in ratio in the absence or presence of phloridzin using a paired t-test. *P<0.05 C. Histogram illustrating that responses to αMDG (12mM) and 3-O-MDG (12mM, open bars) in GE neurons are significantly greater than the matched glucose (12mM) responses (black bars), whereas mean galactose (12mM, open bar) responses are not significantly different from glucose (12mM). Only cells that responded to both substrates tested were included in the analysis. N values are shown above the bars. Statistical significance was tested by comparing the mean change in ratio in response to glucose against αMDG, 3-O-MDG and galactose in matched cells using a paired t-test. ***P<0.001, **P<0.01, ns P>0.05.
Figure 5
Figure 5. αMDG triggers Ca2+ elevation at low substrate concentrations
A. An example of a GE neuron inhibited by a decrease in glucose from 3 to 0.5mM, which was activated by tolbutamide (tolb, 100μM) and further activated by αMDG (3.5mM).
Figure 6
Figure 6. Lactate reduces the magnitude of αMDG responses
A. Representative Ca2+-imaging trace illustrating that responses to αMDG (12 mM) are reduced in the presence of lactate (10 mM). Gadolinium (100 μM) was present throughout the experiment B. Histogram of pooled data showing the magnitude of αMDG-triggered responses in the presence and absence of lactate (10 mM); n=32. Statistical significance was tested by comparing αMDG responses in the absence or presence of lactate, using a paired t-test. ***P<0.001.
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